Molecular mechanisms regulating expression of the oncoproteins ErbB2 and ErbB3 By Markus Dietrich Laboratory for Molecular and Cellular Cancer Research, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Norway © Markus Dietrich, 2018 Series of dissertations submitted to the Faculty of Medicine, University of Oslo ISBN 978-82-8377-161-9 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo. ACKNOWLEDGMENTS The presented work was funded by The South-Eastern Norway Regional Health Authority, to whom I am very grateful for the given opportunity. I would also like to express my gratitude to several persons that supported me during the time of this thesis. I would like to thank my supervisor Dr. Espen Stang for giving me the opportunity to work as a PhD student. Thank you for being an excellent supervisor and for the introduction to the field of molecular cancer research. I am very grateful that you were always available for discussion of results and even in challenging phases never lost the positive attitude. Your great knowledge and never ending new ideas for experiments to try really helped me in the completion of my thesis. Thank you for the attention to detail and for being very patient and supportive. A big thank you also goes to my co-supervisor Dr. Vibeke Bertelsen. I am very grateful for your feedback over the last years and also very glad that even though you left the group one year ago you were still always available for discussion of results and feedback. Also thank you for your attention to details that for sure raised the quality of the work done in the lab. Prof. Henrik Huitfeldt, thank you for being a co-supervisor and for providing good mood and enthusiasm in our meetings. Your thinking out of the box for new experimental approaches and your critical questions on results were always appreciated. All present and former members of the Stang group deserve a big thank you. Marianne Rødland, thank you for the experimental support to my projects, especially the countless microscopy stainings. Thank you to Salman Malik and Filip Nikolaysen for great experimental support, especially while I was writing my thesis. Your contribution was highly appreciated. Anne Marthe Fosdahl, thank you for being a great office mate and co-author throughout my PhD. I really appreciate your hard work and your contributions to our successful projects. Thank you to Monika Szymanska for being a great co-worker, co-author and office mate. I really appreciate all your contributions to our projects and that you always helped to solve problems even after you had already moved to a new work group. Thank you to you and Tara Sarin for always pleasant lunch and coffee breaks that helped to come back to work with full motivation. You two are really good friends and we will stay in contact also when we cannot spend lunch breaks together anymore. I am very grateful for all the co-authors that helped in my projects and a big thank you goes to all of you and I want to thank all my colleagues at the Department of Pathology at Rikshospitalet for an inspiring, friendly and very comfortable work environment. I wish to express my deep felt gratitude to my family and friends that always supported and motivated me throughout these years. Your constant encouragement helped me over the past years. I could not have done that without you. Finally, I want to thank Laura for always being there for me. Your patience, support, motivation and love helped throughout the good and bad times in the last four years. Oslo, October 2017 Markus Dietrich Table of Contents Table of Contents Abbreviations.................................................................................................. I Papers Included .......................................................................................... IV Introduction ................................................................................................... 1 Receptor Tyrosine Kinase (RTK) .............................................................................. 2 The ErbB protein family ............................................................................................... 2 Structure and function ............................................................................................................. 3 ErbB ligands ................................................................................................................................. 4 Phosphorylation and ubiquitination.................................................................................. 6 ErbB Activation and Signaling .............................................................................................. 7 Maturation and trafficking of ErbB proteins .................................................................. 9 Endocytosis ....................................................................................................................... 9 Clathrin-mediated endocytosis ......................................................................................... 10 Adaptors of clathrin-mediated endocytosis ...........................................................................13 Clathrin-independent endocytosis .................................................................................. 16 Macropinocytosis ..............................................................................................................................16 Caveolin-dependent endocytosis ................................................................................................16 Clathrin- and caveolin-independent pathways .....................................................................17 Endosomal sorting ...................................................................................................... 18 Endocytic recycling ................................................................................................................ 20 Endocytic degradation .......................................................................................................... 21 Endocytosis and endosomal sorting of EGFR ........................................................................22 Endocytosis and endosomal sorting of ErbB2.......................................................................23 Endocytosis and endosomal sorting of ErbB3.......................................................................25 ErbB proteins in cancer ............................................................................................. 26 Protein kinase C (PKC) ............................................................................................... 27 PKC structure ............................................................................................................................ 27 PKC activation........................................................................................................................... 28 Growth factor receptors and PKC signaling ................................................................ 30 Aims of the Study ....................................................................................... 33 Summary of Papers ................................................................................... 34 Paper I.............................................................................................................................. 34 Paper II ............................................................................................................................ 35 Paper III........................................................................................................................... 36 Paper IV ........................................................................................................................... 37 Table of Contents Methodological Considerations ............................................................ 38 Cell culture ...................................................................................................................................... 38 Chemical inhibitors and activators ...................................................................................... 39 Transient transfections ............................................................................................................. 40 RNA interference ......................................................................................................................... 41 High-throughput screening ..................................................................................................... 42 Immunological detection.......................................................................................................... 42 Western blotting .......................................................................................................................... 43 Immunoprecipitation (IP) and co-IP ................................................................................... 43 Wide-field fluorescent microscopy and confocal microscopy ................................. 44 Immuno-electron microscopy ................................................................................................ 45 Flow cytometry............................................................................................................................. 46 Internalization of radioactive transferrin (125I-Tf) ....................................................... 46 General Discussion .................................................................................... 47 Internalization and intracellular sorting of ErbB3.......................................... 47 Effects of PKC activity on ErbB2 and ErbB3 ....................................................... 50 Hsp90 and ErbB2 in endocytosis resistance ...................................................... 53 References .................................................................................................... 55 Abbreviations Abbreviations 17-AAG AP(s) AP-2 Arf6 ARH Cbl BAR CCP CCV CHIP CHC CHX CIE CLASP(s) CME CUL5 DAB2 DAG EEA1 EGF EGFP EGFR EHD1 ENTH Eps15 ER ERC ERAD ESCRT FEME GA 17-N-allylamino-17-demethoxygeldanamycin Accessory protein(s) Adaptor protein 2 ADP-ribosylation factor 6 Autosomal recessive hypercholesterolemia Casitas B-lineage lymphoma Bin-Amphiphysin-Rvs Clathrin-coated pit Clathrin-coated vesicle C-terminus of Hsc70-interacting protein Clathrin heavy chain Cycloheximide Clathrin- independent endocytosis Clathrin Associated Sorting Protein(s) Clathrin- mediated endocytosis Cullin-5 Disabled-2 Diacylglycerol Early endosomal antigen 1 Epidermal growth factor Enhanced green fluorescent protein Epidermal growth factor receptor Eps15-homologydomain protein Epsin N-terminal homology Epidermal growth factor receptor substrate 15 Endoplasmic reticulum Endocytic recycling compartment ER-associated degradation Endosomal sorting complexes required for transport Fast endophilin-mediated endocytosis Geldanamycin I Abbreviations GAK GPCR GPI-AP Grb2 HRG Cyclin G-associated kinase G-protein-coupled receptor Glycosyl phosphatidylinositol-anchored proteins Growth factor receptor-bound protein 2 Heregulin Hrs Horseradish peroxidase Hsc70 substrate HRP Hsp IGF1 IL-2R-β ILVs ImmunoEM IP IP3 Lamp1 MAPK mRNA mTORC2 Hepatocyte growth factor regulated tyrosine kinase Heat shock cognate 70 Heat shock protein Insulin-like growth factor 1 Interleukin-2 receptor-β-chain Intraluminal vesicles Immuno-electron microscopy Immunoprecipitation Inositol trisphosphate Lysosomal-associated membrane protein 1 Mitogen-activated protein kinase Messenger RNA Mammalian target of rapamycin complex 2 Nedd4 Multivesicular bodies Nrdp1 downregulated protein 4 MVBs PAE PDK1 PI3K PI3P PIP2 PIP3 PKC PLCγ Neural precursor cell-expressed developmentally Neuregulin receptor degradation pathway protein 1 Porcine Aortic Endothelial Phosphoinositide-dependent protein kinase 1 Phosphatidylinositol 3-kinase Phosphatidylinositol-3-phosphate Phosphatidylinositol 4,5-bisphosphate Phosphatidylinositol 3,4,5-trisphosphate Protein kinase C Phospholipase C-γ II PMA PS Rab RNAi RTK SDS-PAGE SH2 siRNA Sos STAT Tf TfR TGFα TGN Tsg101 UIM Phorbol 12-myristate 13-acetate Abbreviations Phosphatidylserine Ras-associated binding RNA interference Receptor tyrosine kinase Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Src homology 2 Short interfering RNA Son of sevenless Signal transducer and activator of transcription Transferrin Transferrin receptor Transforming growth factor alpha Trans-Golgi network Tumor susceptibility gene 101 Ubiquitin interacting motif III Papers Included Papers Included Paper I Szymanska, M., Fosdahl, A.M., Raiborg, C., Dietrich, M., Liestøl, K., Stang, E. and Bertelsen, V. Interaction with epsin 1 regulates the constitutive clathrin-dependent internalization of ErbB3. Biochim Biophys Acta. 2016 Volume 1863, Issue 6, Part A, June 2016, Pages 1179-1188. doi:10.1016/j.bbamcr.2016.03.011 Paper II Fosdahl, A.M., Dietrich, M., Schink, K.O., Malik, M.S., Skeie, M., Bertelsen, V. and Stang, E. ErbB3 interacts with Hrs and is sorted to lysosomes for degradation Biochim Biophys Acta. 2017 Volume 1864, Issue 12, December 2017, Pages 2241-2252. doi.org/10.1016/j.bbamcr.2017.08.011 Paper III Dietrich, M., Bertelsen V. and Stang, E. Protein kinase C regulates ErbB3 turnover Manuscript Paper IV Dietrich, M., Malik, M.S., Nikolaysen, F. and Stang, E. Protein kinase C induced internalization of ErbB2 is independent of clathrin, ubiquitination and Hsp90 dissociation Manuscript IV Introduction Introduction Cellular signaling is one of the basic molecular mechanisms of life. It helps cells to receive and transmit signals that control fundamental processes such as proliferation, migration and survival. Plasma membrane expressed receptors function in this context as transmitters of extracellular signals in the form of ligands to the intracellular environment. Among the variety of receptor families the epidermal growth factor receptor (EGFR) or ErbB family of receptor tyrosine kinases (RTKs) plays a very important role. 1 Introduction Receptor Tyrosine Kinase (RTK) Receptor tyrosine kinases are primary mediators of information from the extracellular environment into the cells. The family of RTKs consists of 58 members (Roskoski 2014). RTKs are involved in various cellular processes, such as proliferation and differentiation, cell survival and metabolism, as well as cell migration and cell- cycle control (Ullrich and Schlessinger 1990, Blume-Jensen and Hunter 2001). Mutations in RTKs and the resulting aberrant activation of intracellular signaling pathways are often linked to the development of among others cancer, diabetes, inflammation and arteriosclerosis. RTKs function as cell surface allosteric enzymes and their basic structure consists of a single transmembrane domain that connects the extracellular ligand-binding domain with the intracellular kinase domain. In most cases activation occurs after ligand-binding followed by homo- or heterodimerization. However, a subset of RTKs dimerizes also in the absence of ligand like the insulin receptor and insulin-like growth factor 1 (IGF1) receptor (Ward, Lawrence et al. 2007). Activation of RTKs leads to recruitment of target proteins to the intracellular domain that in turn initiates complex signaling cascades. The ErbB protein family The EGFR or ErbB protein family consists of the four members EGFR (also known as ErbB1/HER1), ErbB2 (HER2/Neu), ErbB3 and ErbB4. This family of receptors is ubiquitously expressed in epithelial, mesenchymal and neuronal cells. The EGFR family is involved in multiple complex and tightly controlled signaling pathways involved in various cellular functions such as cell proliferation and organ development (Casalini, Iorio et al. 2004, Tebbutt, Pedersen et al. 2013). 2 Introduction Structure and function The receptors in the EGFR family are composed of an extracellular domain, a single hydrophobic transmembrane segment and an intracellular protein kinase domain (Roskoski 2014). The ligand-binding extracellular domain consists of four subdomains. Domain I and III participate in ligand binding while the cysteine rich domains II and IV facilitate disulfide bond formation and especially domain II, which often is referred to as the dimerization arm, is involved in homo- and heterodimer formation (Roskoski 2014) (Figure 1). The transmembrane segment consists of 19-25 amino acids and spans the plasma membrane to connect extra- and intracellular domain. The intracellular domain is responsible for interactions with intracellular signaling molecules. It consists of a juxtamembrane segment, a protein kinase domain and a carboxyterminal tail. The four ErbB family members share approximately 40-45% sequence homology but have become functionally specialized (Stein and Staros 2000). Except for ErbB2 the receptors exist in a tethered conformation in the absence of ligand. The β-hairpin loop of domain II interacts with domain IV which sequesters the dimerization arm. The dimerization domain is therefore, not available for interaction with other ErbB family members (Baselga and Swain 2009) (Figure 1). Binding of ligand to the ectodomain at domain I and III leads to a conformational change. The receptor adopts an open conformation in which the dimerization loop gets exposed and therefore, allows for interaction of receptor ectodomains (Figure 1). The crystal structure for ErbB2 revealed that the receptor is locked in the open conformation poised to constantly interact with other receptors (Garrett, McKern et al. 2003). This conformation also explains the inability to identify a high-affinity ligand for ErbB2 as the potential ligand-binding domains I and III are fixed in close proximity. Ligand binding to the extracellular domain of ErbB proteins and dimerization subsequently induces a conformational change in the intracellular tyrosine kinase domain leading to autophosphorylation of the receptor. For activation of the tyrosine kinase the N-lobe of one tyrosine kinase domain interacts with the C-lobe of its dimerization partner (Zhang, Gureasko et al. 2006) (Figure 1). 3 Introduction Different expression patterns of the different receptors and ligands reflect cell type specific involvement of the receptors in growth mediation and differentiation (Olayioye, Neve et al. 2000). Figure 1. The ErbB protein family and their ligands. There are four members of the ErbB family. The receptors share a similar structure and are composed of an extracellular domain, a transmembrane α-helix and an intracellular tyrosine kinase domain. The extracellular subdomains I and III are important for ligand-binding, while sub-domains II and IV are facilitating dimerization. ErbB2 has no known soluble ligand but exists in a constitutively open conformation. The other ErbB family members exist in a closed conformation in the absence of ligands. ErbB3 has only a marginal kinase activity compared to the other ErbB receptors. Abbreviations: epidermal growth factor (EGF), transforming growth factor alpha (TGFα), amphiregulin (AR), epigen (EPG), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EPR), heregulin (HRG). Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Cancer (Baselga and Swain 2009). The figure and the figure legend are modified. ErbB ligands Activating ligands of the ErbB family are produced from transmembrane precursors and have an EGF-like domain for high affinity binding. Expression and processing of the precursors is strictly regulated to control activation of the receptors (reviewed in Yarden and Sliwkowski 2001). Specifically to mention in this context is ErbB2 which so far has no known ligand but due to its open conformation serves as a preferred dimerization partner for the other ErbB 4 Introduction proteins. Binding of ligands to the extracellular domain of EGFR, ErbB3 or ErbB4 induces the formation of kinase active receptor dimers. Seven different ligands bind to and activate mammalian EGFR. The ligands can be classified into high-affinity ligands including epidermal growth factor (EGF), transforming growth factor-alpha (TGFα), heparin-binding EGF-like growth factor and betacellulin and low-affinity ligands such as amphiregulin, epiregulin and epigen (Singh, Carpenter et al. 2016). All EGFR ligands are initially produced as transmembrane precursor proteins that have to undergo extracellular domain cleavage to release soluble ligands. These soluble ligands can then bind and activate EGFR. Heregulins, or also called neuregulins, are a large family of EGF-like signaling molecules that trigger activation of ErbB3 and ErbB4. They are involved in cell- cell communication during development but also in adults. Heregulins are ErbBspecific ligands which all share an EGF-like motif of 45-55 amino acids including six cysteine residues that covalently interact and form three loops. The EGF domain alone is sufficient for activation of ErbB receptor tyrosine kinases. The heregulin family comprises four family members HRG-1, HRG-2, HRG-3 and HRG-4 (Holmes, Sliwkowski et al. 1992, Carraway, Weber et al. 1997, Zhang, Sliwkowski et al. 1997, Harari, Tzahar et al. 1999). Nevertheless, the variety of ligands is much bigger due to alternative splicing and thereby, creation of at least 26 different isoforms in different species (Breuleux 2007). While heregulin isoforms encoded by the genes HRG-1 and HRG-2 activate both ErbB3 and ErbB4, isoforms encoded by HRG-3 and HRG-4 are exclusively inducing activation of ErbB4. For this study of most interest is the splice variant Type I HRG-1 hereafter generally referred to as heregulin (Hrg) as it is the main activating ligand for ErbB3. 5 Introduction Phosphorylation and ubiquitination In general, activation of receptor tyrosine kinases including the ErbB family happens via activating ligands or growth factor binding to the ectodomains of two receptors and subsequently inducing an activated dimerization state (Lemmon and Schlessinger 2010). The induced post translational modifications are mainly phosphorylation and ubiquitination. The kinase domains catalyze phosphorylation of various tyrosine residues that function as docking sites for adaptor proteins or enzymes necessary for downstream signaling. The phosphorylation happens in trans, meaning the first member of the dimer phosphorylates the second member and vice versa. Ubiquitin is a small regulatory protein and ubiquitination represents a universal and major way to affect stability, interaction with other proteins, enzymatic activity and subcellular localization of target proteins (Acconcia, Sigismund et al. 2009). Ubiquitination is facilitated by a sequential activation cascade mediated by three classes of enzymes. Ubiquitin- activating enzymes, also known as E1 enzymes, bind and activate ubiquitin and pass it on to the ubiquitin-conjugating enzymes (E2 enzymes). These enzymes conjugate the activated ubiquitin with ubiquitin ligases (E3 enzymes) that facilitate the covalent attachment of ubiqutin to the ε-amino group of a lysine residue of the target protein via an isopeptide bond (Acconcia, Sigismund et al. 2009). While there are only two E1 enzymes encoded in the human genome, there are more than 40 E2 enzymes. The number of E3 enzymes is constantly increasing and outnumbering the other two enzyme classes (Clague, Heride et al. 2015). The variety of ubiquitin-conjugating and ubiquitin-activating enzymes gives rise to a variety of ubiquitination reactions. A substrate can become both mono- and polyubiquitinated. Ubiquitin does in itself contain seven lysine residues and a C-terminal methionine residue which all can be ubiquitinated Polyubiquitination occurs by linking several ubiquitin molecules together in a chain on the same substrate protein. The function of the polyubiquitin chain depends on the lysine where it becomes linked. While linkage of ubiquitin via lysine 48 is a classical signal for proteasome-mediated degradation, linkage of ubiquitin via lysine 63 is often associated with lysosomal degradation (reviewed in Haglund and Dikic 2012). 6 Introduction ErbB Activation and Signaling Most ErbB receptors resemble a simple growth factor receptor signaling pathway characterized by binding of ligand to a monomeric receptor which promotes receptor dimerization, activation of the cytoplasmic catalytic domain and auto-phosphorylation of tyrosine residues. Nevertheless, in higher eukaryotes the simple signaling pathway has evolved to a multilayer signaling network with combinatorial expression of receptors. Furthermore, it involves a range of second messengers, protein-protein interactions, protein-lipid interactions and post translational modifications. Activation of ErbB receptors by its ligands can induce homo- or heterodimerization (Figure 2). Monomeric receptors that do not have bound ligand are under physiological conditions not capable of signaling. The ErbB proteins including splice variants of ErbB4 can form 28 different homo- and heterodimers. ErbB2 has no identified ligand and, even though there have been reports on homodimerization when ErbB2 is overexpressed (Hu, Sun et al. 2015), it most likely has to rely on heterodimerization with other ErbB proteins for activation and downstream signaling. Likewise does ErbB3, which has a very weak tyrosine kinase activity, to a high degree depend on heterodimerization for full activation and phosphorylation. Dimerization induces transphosphorylation of the dimer and subsequently activation of a variety of intracellular pathways such as the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, the phospholipase C-γ (PLCγ) pathway and the signal transducer and activator of transcription (STAT) pathway (reviewed in Yarden and Pines 2012). As MAPK and PI3K pathways are the major pathways activated downstream of ErbB proteins they will be described in more detail. Tyrosine phosphorylation of EGFR and other ErbBs create binding sites for growth factor receptor-bound protein 2 (Grb2) and Src homology 2 (SH2). This leads to recruitment of son of sevenless (Sos), which activates the MAPK pathway consisting of Ras, Raf, MEK and MAPK/ERK before induction of transcription of target proteins. With respect to the PI3K pathway ErbB3 plays a major role. Even though ErbB2 can potently activate MAPK/ERK signaling it needs ErbB3 as dimerization 7 Introduction partner for binding of the p85 regulatory subunit of PI3K. ErbB2/ErbB3 heterodimers signal predominantly via the PI3K/Akt pathway. The regulatory subunit (p85) of PI3K binds to a wide variety of phosphotyrosines in ErbB3 and lead in turn to the activation of PI3K (Roskoski 2014). PI3K further catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphat- idylinositol 3,4,5-trisphosphate (PIP3). This conversion attracts the protein- serine/threonine kinase Akt which further gets phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2). Biphosphorylated Akt catalyzes the phosphorylation and activation of mTOR and from there a variety of further downstream pathways get activated (Engelman 2009, Vanhaesebroeck, Stephens et al. 2012). In addition, ligand-independent transactivation of ErbB proteins can add another layer of signaling complexity as it has been shown that the receptors and downstream pathways can be recruited by G-protein, Wnt, integrin and other growth factor pathways. Figure 2. Activation of the ErbB proteins. Ligand binding to the extracellular domain of the receptor induces conformational changes leading to exposure of sub-domain II, which functions as dimerization arm. Subsequently, the receptors dimerize, the tyrosine kinase domain becomes activated and tyrosine residues in the C-terminal tail become phosphorylated. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Cancer (Baselga and Swain 2009). The figure and the figure legend are modified. 8 Introduction Maturation and trafficking of ErbB proteins Generally, after translation ErbB proteins translocate through the endoplasmic reticulum membrane to the Golgi apparatus where post translational modifications like glycosylation of the extracellular domain occur. From there the receptors are delivered to the plasma membrane (Sorkin and Goh 2009). All ErbB receptors are synthesized and routed to specific cellular locations in the absence of ligand. The distribution can be influenced with respect to internal and external stimuli like cell polarization. The ErbB family members have a ligand-independent turnover that strongly varies between the receptors and also partly correlates to the expression level. While the turnover of EGFR and ErbB2 is in the range of t1/2 6-10h (Beguinot, Lyall et al. 1984), the ErbB3 turnover rate was 2.4h (Cao, Wu et al. 2007). Thus, an average receptor may potentially cycle through the endocytic pathway dozens of times during its life span, without being targeted for degradation (Wiley 2003). Endocytosis Endocytosis is a basic cellular process used by cells for the internalization of a variety of molecules. Signaling receptors undergo endocytosis to downregulate them from the plasma membrane. This process limits the sensitivity of a cell to receptor-activating ligands. Once internalized the receptor can be recycled back to the cell surface or be targeted for degradation. In general there is a distinction between clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) (Figure 3). 9 Introduction Figure 3. Endocytic pathways. The variety of endocytic activities executed by cells includes phagocytosis, for uptake of large particles, macropinocytosis, for larger uptake of fluid and plasma membrane, and a variety of pinocytic mechanisms. Pinocytosis is characterized by uptake of smaller particles via invagination of the plasma membrane leading to the formation of vesicles. The various pinocytic mechanisms are classified by their cellular requirements for dynamin and actin. Cargo uptake can be clathrin- and dynamin-dependent, clathrin- and/or dynamin-independent, caveolin- and dynamin-dependent or independent of clathrin, caveolin and dynamin. The different endocytic uptake mechanisms result in the formation of intracellular vesicles that will fuse with early endosomes. Abbreviations: GEEC, glycosyl phosphatidylinositol-anchored protein enriched early endosomal compartments; CLIC, clathrinindependent carrier. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology (Mayor and Pagano 2007). The figure legend is modified. Clathrin-mediated endocytosis Clathrin-mediated endocytosis describes the mechanism by which the proteins involved in the process recruit their cargo into clathrin-coated pits (CCPs) and subsequently form clathrin-coated vesicles (CCVs). CME serves a range of different functions including the regulation of surface expression of proteins; sampling the cells environment for growth and guidance cues; nutrient uptake into the cell; control of signaling pathway activation and turnover of membrane compartments by targeting them for degradation. CME is used by all known eukaryotic cells. Clathrin coats also form on endosomes and clathrin-coated vesicles are formed at the trans-Golgi network (TGN). The term clathrin- mediated endocytosis is, however, used to refer to vesicles formed from the plasma membrane. The most common implicated cargoes for CME are receptor tyrosine kinases, G-protein-coupled receptors (GPCRs), the transferrin receptor 10 Introduction (TfR) and the anthrax toxin (McMahon and Boucrot 2011). This chapter will focus on the endocytosis of plasma membrane localized receptors. The endocytosis of cargo receptors can be stimulated by ligand binding (for example, EGFR), but it has also been shown that some receptors are constitutively internalized such as the TfR (Hopkins, Miller et al. 1985) and ErbB3 (Sak, Breen et al. 2012). The CCV cycle consists of five steps: nucleation, cargo selection, coat assembly, scission and uncoating (Figure 4). After uncoating, the vesicles fuse with endosomes where cargo is sorted and either recycled back to the plasma membrane or targeted to more mature endosomes and later compartments such as multivesicular bodies (MVBs) and lysosomes (Grant and Donaldson 2009). The main and name-giving player in CME is clathrin. It was purified and identified already in 1975 by Barbara Pearse, who named it clathrin (from the Latin clatratus, meaning “like a lattice”) (Pearse 1975, Pearse 1976). It forms a triskelion structure consisting of three clathrin heavy chains (CHCs) and three clathrin light chains. Interaction of the triskelia leads to formation of a polyhedral lattice that coats the forming vesicle. As clathrin does not bind directly to the plasma membrane or cargo receptors, the connection occurs via adaptor and accessory proteins such as adaptor protein 2 (AP-2) which will be closer described in the next chapter (Kelly, McCoy et al. 2008). A wide range of plasma membrane accessory adaptor proteins has been identified that can be cell type as well as cargo specific and bind to different receptors (reviewed in Traub 2009). When cargo is finally selected and bound by AP-2 or other cargo-specific adaptor proteins the clathrin coat needs to be assembled. Soluble clathrin triskelia, which are directly recruited from the cytosol, start polymerizing into hexagons and pentagons. Clathrin is nucleating at sites of the plasma membrane that are destined for internalization and often characterized by an accumulation of adaptor proteins. The nucleation promotes the further polymerization of clathrin and the creation of curved lattices. The subsequent displacement of cargo accessory adaptor proteins and curvature effectors, such as epidermal growth factor receptor substrate 15 (Eps15), promotes the curvature formation of the plasma membrane (Tebar, Sorkina et al. 1996, Saffarian, Cocucci et al. 2009)(Figure 4). This consequently stabilizes the deformation of the attached membrane and leads into the formation and 11 Introduction constriction of a vesicle neck. Dynamin is a GTPase that has been shown to be responsible for the pinching off of the CCP from the plasma membrane (Chen, Obar et al. 1991, van der Bliek and Meyerowitz 1991). Dynamin is recruited by Bin-Amphiphysin-Rvs (BAR) domain containing proteins such as amphiphysin or endophilin, that have a preference to accumulate at the curvature of the vesicle neck (Wigge, Kohler et al. 1997, Ferguson, Raimondi et al. 2009, Sundborger, Soderblom et al. 2011). After formation of the CCP, dynamin assembles into ringlike structures (Hinshaw and Schmid 1995) around the constricted neck of deeply invaginated CCPs and upon hydrolysis of GTP it mediates the fission of the vesicle from the plasma membrane (Praefcke and McMahon 2004) (Figure 4). Nevertheless, the exact mechanism of the scission is not yet fully understood, but it is known that the protein undergoes a GTP hydrolysis- dependent conformational change which is thought to support the scission (Sweitzer and Hinshaw 1998, Roux, Uyhazi et al. 2006). After detachment from the plasma membrane and formation of the CCV, the clathrin coat is removed from the vesicle with the help of the ATPase heat shock cognate 70 (hsc70) and its cofactor auxilin or cyclin G-associated kinase (GAK) (Schlossman, Schmid et al. 1984, Ungewickell, Ungewickell et al. 1995) (Figure 4). The uncoated vesicle can travel to and fuse with target endosomes while the released clathrin machinery can be reused for another cycle of CCP formation. Figure 4. Formation of clathrin-coated vesicles (CCV). The formation of CCVs is a multi-step process. The nucleation is mediated by clathrin-associated sorting proteins (CLASPs) and association of clathrin. The clathrin coated pit (CCP) begins to mature, more adaptor and scaffolding proteins are recruited and the clathrin coat gets assembled. The GTPase dynamin is recruited to the neck of the forming vesicle and mediates the scission of the vesicle. The formed vesicle is released into the cytoplasm and rapidly uncoated. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology (McMahon and Boucrot 2011). The figure and the figure legend are modified. 12 Introduction Adaptors of clathrin-mediated endocytosis A wide range of adaptors necessary for the formation of clathrin-coated pits and subsequently vesicles at the plasma membrane have been identified. Endocytic adaptors are divided in two main groups: multimeric adaptor proteins and monomeric non-classic adaptor proteins also known as clathrin-associated sorting proteins (CLASPs), which recognize certain motifs of target proteins (Table 1). Nevertheless, it is worth mentioning that not all the adaptors are always used or required for CME, their requirement may differ between cell types and/or different cargoes. Cargo likely plays a driving role in CCP formation as it is expected that empty CCVs will not have a useful function. A group of researchers around David Drubin (University of California, Berkeley) suggested that the wide range of more than 40 proteins involved in CME can be grouped into four different functional modules that mediate coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission (Kaksonen, Toret et al. 2005). The following paragraphs will take a closer look on some of the key adaptors relevant for this study. Table 1. Endocytic signals and adaptors Signals or domains Adaptors Adaptor subunits or domains YXXØ AP-2 µ2 [DE]XXXL[LI] AP-2 α-σ2 [YF]XNPX[YF] ARH; Dab2; Idol; SNX17, 27, and 31 PTB domain NPFX(1,2)D Sla1p SLA1 homology domain Ubiquitin Eps15, Epsin 1 and 2 UIM domain GPCR phosphorylation β-arrestin 1 and 2 Amino terminus Synaptotagmin I C2A (C2B) domain Stonin 2 μHD domain Mid2p cytosolic domain Syp1p μHD domain Alk8 cytosolic domain Fcho1 μHD domain VAMP 7 longin domain Hrb, AP-3 C-terminal unstructured domain VAMP 2, VAMP 3, VAMP 8 SNARE motifs CALM ANTH domain Abbreviations: ARH, autosomal recessive hypercholesterolemia; PTB, phosphotyrosine-binding; UIM, ubiquitin-interacting motifs; GPCR, G-protein-coupled receptor; CALM, clathrin assembly lymphoid myeloid leukemia. Adapted with permission Cold Spring Harbor Laboratory Press: CSH Perspectives in Biology (Traub and Bonifacino 2013). 13 Introduction “Assembly” or “accessory” proteins (APs) were isolated and identified (Zaremba and Keen 1983, Pearse and Robinson 1984, Ahle and Ungewickell 1986) as the connecting partner between the clathrin coat and the plasma membrane, where they act as adaptors (Vigers, Crowther et al. 1986). Among the first identified adaptors was the heterotetrameric AP-2 complex (Pearse and Robinson 1984). It was initially believed to be always involved in CME but it has been later shown that other adaptors can compensate the AP-2-mediated processes in CME. The complex consists of two large “adaptin” subunits (α and β2), one medium sized subunit (µ2), and one small subunit (σ2). For AP-2 the internalization sequences that are recognized by different protein subunits have been identified (reviewed in Traub and Bonifacino 2013). A simple tyrosine motif (YXXØ) present in cytoplasmic tails of receptors such as the TfR is recognized by the AP-2 µ2- subunit (Ohno, Stewart et al. 1995) and the acidic dileucine [DE]XXXL[LIM] sequence is recognized by the α-σ2 hemicomplex (Kelly, McCoy et al. 2008). Furthermore, AP-2 binds to the plasma membrane-specific lipid PIP2 via the αsubunit (Honing, Ricotta et al. 2005). When AP-2 gets recruited to the plasma membrane with the help of the large subunits (α and β2), it undergoes a conformational change that enables the protein to simultaneously bind to cargo- sorting signals and clathrin (Kelly, Graham et al. 2014). The interaction with the cytoplasmic tail of transmembrane receptors occurs directly via the µ-subunit and σ-subunit, and indirectly via cargo using its appendage domains to bind AP-2 (Collins, McCoy et al. 2002, Kelly, McCoy et al. 2008). Since AP-2 is not only binding clathrin but also most of the accessory proteins, it is considered to be the major hub of interactions in the maturing CCP. The CLASPs are mono- and dimeric and vary in structure and binding properties. Cargo-specific adaptors recognize a single transmembrane receptor or a small family of receptors. Known adaptors are Numb, autosomal recessive hypercholesterolemia (ARH) or disabled-2 (DAB2). These proteins contain an Nterminal phosphotyrosine binding domain that recognizes [YF]XNPX[YF] cargo- sorting motifs (Uhlik, Temple et al. 2005). DAB2 is a known adaptor involved in CME of megalin (Gallagher, Oleinikov et al. 2004). Arrestins are crucial in trafficking of signaling receptors. They regulate the inactivation, internalization and signaling of GPCRs (reviewed in Gurevich and Gurevich 2006). β-arrestins 14 Introduction help control the strength and duration of GPCR signaling by binding to phosphorylated GPCRs. This attenuates the signaling and blocks the receptor interaction with G-proteins (Lohse, Benovic et al. 1990). Furthermore, βarrestins promote GPCR endocytosis by interaction with clathrin and other adaptor proteins (Goodman, Krupnick et al. 1996). There are also a few proteins that bind cargo but do not contain any clathrin-binding motif. These proteins include Eps15 that selects ubiquitinated cargo and stonins that bind to and sort cargos of the synaptic vesicle. Stonins bind AP-2 with their N-terminal domain, while the C-terminal domain is essential for selecting cargos of the synaptic vesicle. For this study of greatest interest is the epsin family of adaptor proteins. The epsin family is a conserved family of endocytic adaptor proteins. They are essential for embryonic development in higher eukaryotes. Epsins recognize ubiquitinated cargo and contribute to membrane bending. Three members of the epsin family epsin-1, -2 and -3 have been identified as plasma membrane localized. The structure of epsin proteins is comprised of an approximately 150 amino acid long epsin N-terminal homology (ENTH) domain, which is responsible for binding to PIP2, a lipid enriched at endocytic sites in the plasma membrane (Itoh, Koshiba et al. 2001). In addition, the ENTH domain promotes membrane curvature (Ford, Mills et al. 2002). The ENTH domain is followed by ubiquitin interacting motifs (UIMs). Most epsins contain two to three UIM copies for mediating interaction with ubiquitinated cargo (Polo, Sigismund et al. 2002, Shih, Katzmann et al. 2002). The region between the UIMs and the C-terminal domain displays high divergence between species but contain some conserved motifs for binding of various components of the endocytic machinery such as clathrin and AP-2. Epsin-1 has also been suggested to be involved in CIE of EGFR when the receptor is stimulated with high concentrations of EGF (Sigismund, Woelk et al. 2005). 15 Introduction Clathrin-independent endocytosis Several endocytic pathways do not utilize clathrin for the internalization of cargo into the cell. Some of these pathways occur constitutively while others are triggered by specific signals or even hijacked by pathogens. The main differences in the clathrin-independent pathways of endocytosis are in their mechanisms and kinetics of vesicle formation, the molecular machinery involved and the final destination of the incorporated cargo. There is a variety of different pathways characterized of which some of them will be shortly explained below. Macropinocytosis Macropinocytosis does not appear to have specific cargoes or markers and is therefore only characterized by morphology. Large ruffles or blebs of plasma membrane are forming initially before collapsing back onto the plasma membrane. Thereby large irregular shaped vacuoles get formed, called macropinosomes. This leads to a nonselective engulfment of membrane portions containing, among others, activated receptors. Furthermore, this pathway can facilitate a nonspecific uptake of fluids and solutes. Macropinocytosis can be triggered by overstimulation by stimuli such as growth factors, phosphatidylserine (PS)-containing apoptotic cell remnants, viruses and bacteria (Kerr and Teasdale 2009). Due to the unspecific uptake of membrane molecules the sorting of the receptors happens after uptake. Inactive receptors are usually recycled back to the plasma membrane while activated receptors are sorted by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation. Caveolin-dependent endocytosis Endocytosis mediated by caveolae is an endocytic pathway for uptake of small molecules and belongs to the best studied CIE. Caveolae are cholesterol- and sphingolipid-rich (Simons and Ikonen 1997), 50-80nm flask-shaped invaginations of the plasma membrane whose expression is associated with a member of the caveolin protein family (reviewed in Nabi and Le 2003). Another 16 Introduction important assembly partner is glycosyl phosphatidylinositol-anchored proteins (GPI-APs) (Aboulaich, Vainonen et al. 2004). The main molecules taken up via this pathway include sphingolipids and sphingolipid binding toxins such as cholera and shiga toxin, endothelin, growth hormones and bacteria (Duncan, Shin et al. 2002, Pelkmans and Helenius 2002). Caveolae-dependent endocytosis is characterized by sensitivity to dynamin inhibition and cholesterol depletion (Henley, Krueger et al. 1998). The internalization of caveolae is facilitated by disruption of the actin cytoskeleton (reviewed in Nabi and Le 2003) and caveolin-1 functions as a stabilizer of the caveolae. The budding of the caveolae occurs in a dynamin-dependent manner and the forming caveolar vesicles are then further transported to their intracellular destination. Clathrin- and caveolin-independent pathways Among the variety of clathrin- and caveolin-independent pathways the most common ones are regulated by small GTPases that regulate internalization among them RhoA, CDC42 and ADP-ribosylation factor 6 (Arf6). RhoA-dependent endocytosis is best studied for the internalization of the β-chain of the interleukin-2 receptor (IL-2R-β). After ligand binding IL-2R-β sorts into detergent-resistant membranes, which are frequently found to be involved in CIE. RhoA and dynamin inhibition potently inhibits this endocytic route (Lamaze, Dujeancourt et al. 2001). CDC42-mediated endocytosis facilitates the main clathrin-independent and caveolin-independent endocytic route for the uptake of cholera toxin B and the plant protein ricin (Llorente, Rapak et al. 1998, Lajoie, Kojic et al. 2009). The invaginations of the plasma membrane in CDC42-regulated endocytosis are longer and have a relatively wide surface in contrast to the clathrin- and caveolin-regulated pathways (Kirkham, Fujita et al. 2005). This leads to a cointernalization of large volumes of fluid phases. In addition, a role for the GTPase Arf6 has been suggested but it is still unclear whether this can be considered an own pathway or just contributing to other internalization pathways by actin remodeling. Furthermore, most of the effects 17 Introduction of Arf6 appeared to be cell type specific (Naslavsky, Weigert et al. 2004, Kalia, Kumari et al. 2006). Some CIE events might be too fast to be recorded by classical methods to study endocytosis. However, in recent years there have been major advances revealing fast and ultrafast clathrin-independent endocytic pathways. One of the recently discovered pathways is fast endophilin-mediated endocytosis (FEME) (Boucrot, Ferreira et al. 2015). This pathway is mediated by endophilin and induces a rapid formation of endocytic vesicles upon certain stimuli. It is triggered by receptor activation and specific to certain receptors. It has so far been identified for some GPCRs, interleukin-2 receptor and certain receptor tyrosine kinases (Boucrot, Ferreira et al. 2015). Endosomal sorting After internalization of molecules through an endocytic pathway, they are sorted and traffic through a variety of tubulovesicular compartments, collectively termed endosomes (Elkin, Lakoduk et al. 2016). The endosomal network is a dynamic and interconnected system that allows trafficking and transfer of cargoes between compartments in the cell. After entering the cell the endocytic vesicles undergo several cycles of homotypic fusion to finally form early endosomes (Salzman and Maxfield 1988). Within early endosomes the fate of the internalized cargo gets determined by the sorting into different endosomes. A key protein family involved in most membrane transport processes is the Rasassociated binding (Rab) protein family. It consists of more than 60 small GTPases and they exhibit various functions in the endosomal system. Rab proteins are involved in cargo budding, selection, coating, vesicle transport, vesicle uncoating, tethering and vesicle fusion (reviewed in Seabra, Mules et al. 2002). As these processes are facilitated by different Rab family members, Rab proteins are also commonly used as microscopic markers for identification of the different intracellular sorting compartments. The initial cargo sorting station is the early endosome. Cargo can be routed from early endosomes to late endosomes and lysosomes for degradation, to recycling endosomal carriers that transport cargo directly back to the plasma membrane, to the endocytic recycling 18 Introduction compartment (ERC), or to the TGN (Figure 5). Early endosomes receive the incoming material from primary vesicles that are internalized from the plasma membrane. Rab5, early endosome antigen (EEA1) and phosphatidylinositol-3- phosphate (PI3P) mark the early endosome and are important regulators for early endosomes. The pH in the early endosomes gradually decreases during fusion events and as a consequence of the low pH in the lumen of the endosome, many ligands are released from their receptors (Maxfield and McGraw 2004). This step can already determine the further sorting of the internalized proteins for recycling or degradation. Figure 5. Endosomal compartments. After release of the CCP in the cytoplasm it fuses with early endosomes, where internalized cargo is either sorted back to the plasma membrane in recycling endosomes or further sorted into ILV of MVBs. Cargo can also undergo retrograde transport from endosomes to the Golgi and ER. MVBs may fuse with or mature to lysosomes where cargo may be degraded. MVBs can be released into the extracellular space by fusion of the MVBs with the plasma membrane as exosomes. Abbreviations: CCP, clathrin coated pit; ILV, intraluminal vesicle; MVB, multivesicular body; ER, endoplasmic reticulum. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Drug Discovery (Rajendran, Knolker et al. 2010). The figure legend is modified. 19 Introduction Endocytic recycling Endosomal recycling pathways balance the removal of internalized extracellular material by returning a large part of internalized proteins and lipids back to the plasma membrane. This helps in maintaining the composition of the plasma membrane. The recycling pathways are generally classified in a rapid recycling route and a slow recycling route. The rapid recycling route that recycle proteins directly back to the plasma membrane from early endosomes. Even an earlier stage has been documented for the TfR (Mayor, Presley et al. 1993) and glycosphingolipids (Choudhury, Sharma et al. 2004). The two Rab proteins Rab4 and Rab35 have been identified as key components in mediating the rapid recycling. The slow recycling route requires transport of cargo from the early endosome to the ERC and from there back to the plasma membrane. In many tissues the ERC, is located in close proximity to the Golgi complex and it is molecularly defined by the presence of Rab11 and/or Eps15-homology-domain protein (EHD1). It evolves from extended tubules of early endosomes that loose Rab5 and acquire Rab11 (Sonnichsen, De Renzis et al. 2000). From the ERC internalized proteins can be sorted for transport back to the plasma membrane or to the TGN. An example for the latter is sorting of the protein TGN38 which is mainly expressed in the TGN but a small percentage is also found on the plasma membrane. Membrane downregulated TGN38 can be sorted back to the TGN via the ERC (Ghosh, Mallet et al. 1998). The transport from the ERC back to the plasma membrane can probably occur by several distinct mechanisms. These include mechanisms that are dependent on Arf6, Rab11, Rab 22a or Rab8a (reviewed in Grant and Donaldson 2009). The transport from the ERC to the plasma membrane is mediated by recycling carriers that are released from the ERC and that finally fuse with the plasma membrane. Another mechanism of recycling is the formation of exosomes. Exosomes are small vesicles which are released from cells when specialized MVBs fuse back with the plasma membrane and release their internal vesicles and thus their endosomal packed content into the extracellular space. This process is mediated by Rab27a, the ESCRT-III complex and syndecans (Baietti, Zhang et al. 2012). 20 Introduction Endocytic degradation The endosomal compartments undergo a maturation process, from so called early to late endosomes/lysosomes, in which the luminal pH decreases, key phosphatidylinositol lipids get altered and different Rab-family GTPases get recruited (Elkin, Lakoduk et al. 2016). Late endosomes/lysosomes are characterized by certain markers such as lysosomal-associated membrane protein 1 (Lamp1) and CD63. Early endosomal sorting depends initially on acidification. The acidification occurs with the help of a v-type vacuolar H+ ATPase, which pumps hydrogen ions into the endosomal lumen and decreases the pH (Mellman, Fuchs et al. 1986, Fuchs, Schmid et al. 1989). Maturing early endosomes can accumulate intraluminal vesicles (ILVs) in their vacuolar portions. These ILVs increase as the endosomes mature and finally develop into late endosomes/MVBs. One of the most common sorting signals for downregulation and degradation of surface proteins is ubiquitin which gets attached to the cytoplasmic domain of the protein. The recognition of ubiquitin and sorting of cargo into ILVs is mediated by the ESCRT machinery. This machinery is composed of four different protein complexes called ESCRTs -0, -I, -II, -III. The ESCRT complexes exist as cytoplasmic complexes which get recruited to the limiting membrane of endosomes during the formation of MVBs. The different ESCRT subunits can recognize a variety of cellular components including ubiquitinated cargo, coat proteins such as clathrin and endosomal lipids including PI3P (Raiborg and Stenmark 2009). These complexes retain ubiquitinated cargo in the membrane of early endosomes and hinder receptor recycling (Raiborg, Bache et al. 2002). The ESCRT-0 complex consists of the subunits hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) and STAM. These proteins interact preferably with polyubiquitin chains of cargo via their UIMs (Polo, Sigismund et al. 2002). Hrs has the ability to bind PI3P via a FYVE domain which recruits ESCRT-0 to endosomal membranes (Raiborg, Bremnes et al. 2001). Hrs also interacts with the tumor susceptibility gene 101 (Tsg101) which is part of ESCRT-I. This leads to recruitment of ESCRT-I and subsequent recruitment of further ESCRT complexes. Vps4 is responsible to disassemble the ESCRTs and complete the process of ILV biogenesis (reviewed in Henne, Stenmark et al. 21 Introduction 2013). After fusion of the late endosomes/MVBs with lysosomes the ILVS are exposed to and degraded by lysosomal hydrolysis. Endocytosis and endosomal sorting of EGFR The endocytic downregulation of EGFR is the best studied endocytic pathway for the ErbB proteins. The protein half life of EGFR is normally between 6 and 10 hours but can be much longer when EGFR is overexpressed. The protein half life does however, strongly decrease when EGF binds to EGFR leading to an activated receptor that becomes rapidly internalized and degraded (reviewed in Sorkin and Goh 2009). Acceleration of internalization and degradation should prevent excessive signaling of activated receptors. Endocytosis via CME is the main pathway of activated EGFR under physiological conditions, although several examples of clathrin-independent EGFR endocytosis have been reported (Yamazaki, Zaal et al. 2002, Sigismund, Woelk et al. 2005, Orth, Krueger et al. 2006). Sigismund et al. as an example showed that increasing EGF concentrations for receptor stimulation shift the internalization of EGFR from CME to clathrin-independent pathways. Furthermore, they reported that low doses of EGF do not induce EGFR ubiquitination and that non-ubiquitinated EGFR is endocytosed via CME. Sigismund et al. also suggest that while low concentrations of EGF give rise to prolonged/sustained signaling, high concentrations of EGF lead to degradation (Sigismund, Argenzio et al. 2008). Nevertheless, several studies demonstrated that EGFR is ubiquitinated also at low concentrations of EGF and undergoes CME also at high doses of EGF (Kazazic, Bertelsen et al. 2009, Sorkin and Goh 2009, Sousa, Lax et al. 2012, Fortian, Dionne et al. 2015). Several mechanisms and adaptor proteins that regulate ligand-induced CME of EGFR have been identified among them interaction with AP-2 and ubiquitination (Sorkin and Carpenter 1993, Goh, Huang et al. 2010). Studies have shown that ubiquitination is sufficient for internalization of EGFR, as a truncated EGFR with ubiquitin fused to the C-terminal tail (Haglund, Sigismund et al. 2003) or a chimeric EGFR with four connected ubiquitin molecules (Bertelsen, Sak et al. 2011), were constitutively internalized. The ubiquitin ligase casitas B-lineage lymphoma (Cbl) is recruited 22 Introduction to activated EGFR either directly to pY 1045 or indirectly via the SH3 domain of Grb2 (Meisner, Conway et al. 1995, Huang and Sorkin 2005). Ubiquitination of EGFR via Cbl occurs at the plasma membrane. This leads to internalization and sorting of EGFR in ILVs of MVBs and therefore, the entry into the lysosomal degradative pathway (reviewed in Katzmann, Odorizzi et al. 2002). It has been shown that a decrease in ubiquitination of EGFR for example by mutation of critical tyrosine residues leads to degradation impaired receptors as they get not sorted to ILVs (Grovdal, Stang et al. 2004). It has also been shown that Cbl mutants displaying impaired activity block EGFR degradation but instead sort the receptors for recycling (Peschard and Park 2003). The interaction with both epsin and Eps15 which are found in CCPs (Stang, Blystad et al. 2004, Kazazic, Bertelsen et al. 2009), are important for CME of ubiquitinated EGFR (Stang, Blystad et al. 2004, Haglund and Dikic 2012, Fortian, Dionne et al. 2015). Internalized EGFR gets initially sorted in early endosomes. A known sorting signal for EGFR is ubiquitin. Furthermore, the ligand which binds to EGFR is determining the fate of the receptor. While EGF-activated EGFR gets sorted for degradation in lysosomes, binding of TGFα leads to internalization of the receptor followed by recycling. One reason for this is that, unlike EGF, TGFα dissociates from the receptor in the mildly acidic environment of the early endosomes, leading to dephosphorylation and deubiquitination of the receptor (Longva, Blystad et al. 2002, Roepstorff, Grandal et al. 2009). In addition, it has been suggested that the localization of EGFR influences the signaling mediated by EGFR. While EGFR at the plasma membrane mediates proliferative signaling, it has been shown that EGFR signaling from endosomes can induce apoptosis (Burke, Schooler et al. 2001, Hyatt and Ceresa 2008, Rush, Quinalty et al. 2012). Endocytosis and endosomal sorting of ErbB2 The behavior of ErbB2 regarding endocytic downregulation differs significantly from that of EGFR. ErbB2 is considered to be strongly endocytosis-deficient as it is not internalized. This was suggested to occur due to a lack of common 23 Introduction endocytosis signals like interaction with AP-2 (Baulida, Kraus et al. 1996), or because of possible endocytosis inhibitory signals (Sorkin, Di Fiore et al. 1993). Studies of ErbB2 localization revealed that it is strictly located to membrane protrusions at the plasma membrane, apart the newly synthesized ErbB2 which can also be found in the endoplasmic reticulum/Golgi region (Hommelgaard, Lerdrup et al. 2004). The lack of ErbB2 specific ligands makes it impossible to study ligand-induced internalization of ErbB2 homodimers, but it has been shown that heterodimerization of ErbB2 with EGFR inhibits downregulation of EGFR (Wang, Zhang et al. 1999, Haslekas, Breen et al. 2005). This supports the notion that ErbB2 is resistant to downregulation. Nevertheless, other studies suggested that EbB2 is internalized but very fast and efficiently recycled back to the plasma membrane (Harari and Yarden 2000, Austin, De Maziere et al. 2004). The best studied mechanism to induce downregulation and subsequent endosomal sorting of ErbB2 is inhibition of heat shock protein (Hsp) 90. Hsp90 is an ATPase and a chaperone which is associated with several client proteins during maturation. However, it can remain bound to clients also after maturation as this is the case for ErbB2 and Akt. ErbB2 is thus an Hsp90 client and Hsp90 has been suggested to actively stabilize ErbB2 at the plasma membrane (Austin, De Maziere et al. 2004). Hsp90 is bound to ErbB2 on a loop within the N-lobe of the kinase domain (Tikhomirov and Carpenter 2003, Xu, Yuan et al. 2005). A recent study suggested that interaction of ErbB2 with flotillins (Pust, Klokk et al. 2013) and/or Erbin (Asp, Kvalvaag et al. 2016), either directly or through Hsp90, keeps ErbB2 at the plasma membrane and it was shown that depletion of flotillins cause internalization and degradation of ErbB2. Hsp90 inhibitors, such as Geldanamycin (GA) or 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) induce ubiquitination of ErbB2 which can serve as an internalization signal and/or signal for proteasome-mediated degradation of the C-terminus (Lerdrup, Bruun et al. 2007, Pedersen, Madshus et al. 2008) and thereby potentially cleave a region containing a plasma membrane retention signal (Tikhomirov and Carpenter 2000). Inhibition of Hsp90 with GA or 17-AAG results in interference with ATP-binding and dissociation of Hsp90 accompanied by recruitment of Hsp70 (Xu, Mimnaugh et al. 2001) and the ubiquitin ligase C-terminus of Hsc70- interacting protein (CHIP) (Xu, Marcu et al. 2002, Zhou, Fernandes et al. 2003) 24 Introduction and Cullin-5 (CUL5) (Ehrlich, Wang et al. 2009, Samant, Clarke et al. 2014). Which ErbB2 lysine residues become ubiquitinated upon Hsp90 inhibition is still unknown, but it has been shown that Hsp90 inhibition results in both K48- and K63-linked polyubiquitination of ErbB2 (Marx, Held et al. 2010, Vuong, Berger et al. 2013). Therefore, ErbB2 internalization could be mediated in two different ways, where ubiqutin either serves as internalization signal itself, or mediates proteasome mediated cleavage of an intracellular region leading to ErbB2 internalization. Upon internalization of ErbB2, mediated by Hsp90 inhibitors, ErbB2 localizes to early endosomes. From there it is further sorted to ILVs of MVBs (Pedersen, Madshus et al. 2008). The involvement of the ESCRT machinery in endosomal sorting of ErbB2 is not yet known, but a possible involvement of ubiquitin in sorting of ErbB2 to ILVs has been suggested (Vuong, Berger et al. 2013). Endocytosis and endosomal sorting of ErbB3 Remarkably little has been known about the downregulation of ErbB3 compared to EGFR and ErbB2. ErbB3 was for a long time considered to be endocytosisresistant as it does not interact with AP-2 due to a lack of sorting signals (Baulida, Kraus et al. 1996), but it was shown that it undergoes a slow liganddependent internalization (Baulida, Kraus et al. 1996). A recent study also showed that ErbB3 is constitutively internalized in a clathrin-dependent manner and it is constitutively degraded (Sak, Breen et al. 2012). The ubiqutin ligases neuregulin receptor degradation protein-1 (Nrdp1) (Diamonti, Guy et al. 2002, Qiu and Goldberg 2002) and neural precursor cell-expressed developmentally downregulated protein 4 (Nedd4) (Huang, Choi et al. 2015) have been reported to be involved in ubiquitination and proteasomal degradation of ErbB3. Other studies have also shown that ErbB3 may undergo lysosomal degradation as lysosomal inhibitors reduced the ligand-induced degradation of ErbB3 (Cao, Wu et al. 2007). Furthermore, it has been shown that ErbB3 expression is regulated by a quantity control mechanism involving the ER-associated degradation (ERAD) pathway (Fry, Simion et al. 2011). 25 Introduction ErbB proteins in cancer ErbB family receptors are associated with a range of diseases like psoriasis, heart disease and Alzheimer’s disease, but are best studied for their involvement in cancer development. Cancer is often referred to as a pathophysiological condition of abnormal signaling. The potent cell proliferation signals induced by the ErbB network are therefore utilized by cancer cells in the form of oncogenic mutations leading to strong clonal expansion. EGFR was the first cell-surface receptor linked to cancer (Todaro, De Larco et al. 1976). In the 40 years since this discovery, other ErbB family members have been found dysregulated in various cancers. The oncogenic potential of ErbBs arise from mutation (Humphrey, Gangarosa et al. 1991), overexpression of the receptor (Leahy 2004), or as a downstream effect of inappropriate ligand expression (Sizeland and Burgess 1992). The central role of the ErbB network in tumor development makes it a very attractive target for pharmacological intervention. Due to the possibility of extracellular manipulation and the detailed understanding of the underlying intracellular biochemistry huge progress has been made in the development of drugs and therapeutic antibodies targeting ErbB family members, especially EGFR and ErbB2. Nevertheless, when ErbB family members are targeted with drugs the receptor blockade can be by-passed by increased expression of other family members, especially ErbB3 (Frolov, Schuller et al. 2007). Another by- passing mechanism is the activation of other receptor tyrosine kinases such as c-met. This can again lead to an increased expression of ErbB3 (Engelman, Zejnullahu et al. 2007). The biggest problem with this signaling diversion is different pathway activation and subsequently a loss of treatment efficiency. 26 Introduction Protein kinase C (PKC) Protein kinase C is a family of phospholipid- and calcium-dependent protein kinases (Takai, Kishimoto et al. 1979). The PKCs are serine/ threonine kinases which phosphorylate target proteins involved in a wide variety of fundamental physiological processes such as among many others, signal transduction, proliferation, differentiation and modulation of gene expression (Dekker and Parker 1994, Nishizuka 1995). PKC also autophosphorylates (Newton and Koshland 1987) by an intramolecular mechanism at the N-terminus, hinge and C- terminus (Flint, Paladini et al. 1990). The different PKC isozymes are involved in a variety of signal transduction pathways that respond to external stimulators, among them hormones, growth factors and other membrane receptor ligands. Importantly, PKCs also function as the main receptors for tumor-promoting phorbol esters (Castagna, Takai et al. 1982). The PKC family consists of 10 phospholipid-dependent isozymes that are subgrouped into three families: the classical PKCs which are PKCα, βI, βII, and γ, the novel PKCs (PKC δ, ε, and η) and the atypical PKCs (PKCζ, and λ/τ). The different subfamilies are classified by their activation mechanisms. Classical PKCs need Ca2+ and diacylglycerol (DAG) or phorbol esters for activation, while novel PKCs are Ca2+-independent, but need DAG or phorbol esters. Atypical PKCs are unresponsive to Ca2+, DAG and phorbol esters, but require PS. Despite their high homology, the regulation of the different PKC isoforms varies depending on phosphorylation and protein-protein interactions. PKC structure PKC isozymes consist of a regulatory and a catalytic region and cloning of the first PKC isozymes in the 1980s revealed a general structure of conserved domains termed C1-C4 (Coussens, Parker et al. 1986) (Figure 6). The domains are composed of a number of conserved regions which are interspersed with lower homology regions called variable domains. The catalytic region (approximately 20-40 kDa) which is composed of the C3 and C4 domains, contains the kinase activity and resides in the carboxy-terminal part of the protein. It consists of a conserved ATP and magnesium-binding site (C3 domain) 27 Introduction and a substrate-binding site (C4 domain) (Figure 6). The N-terminal regulatory region (approximately 20-40 kDa) is composed of a conserved tandem C1 domain and a C2 domain. The tandem C1 domain contains a Cys-rich motif that forms the DAG/phorbol ester binding site directly followed by an autoinhibitory pseudosubstrate sequence inhibiting constant activation of the enzyme (Figure 6). The C2 domain contains the recognition site for acidic lipids. For the conventional PKC isoforms domain C2 also contains the Ca2+ binding site. Novel PKCs also possess a C2 domain, but it is not a Ca2+ sensor, as the necessary aspartate residues required for interaction are missing. In atypical PKCs the second C1 domain and the C2 domain are missing, and they do thus not respond to DAG or Ca2+ (reviewed in Steinberg 2008, Mochly-Rosen, Das et al. 2012). Figure 6. Structure of PKC isozymes. Schematic sequence of PKC isozymes displaying the domain structure of PKC subfamilies and their respective activators. Abbreviations: PKC, protein kinase C; PS, pseudosubstrate; ATP, adenosine triphosphate; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; PIP3, phosphatidylinositol (3,4,5)-trisphosphate. Adapted by permission from MDPI AG: International Journal of Molecular Sciences (Cosentino-Gomes, RoccoMachado et al. 2012). The figure legend is modified. PKC activation Newly synthesized PKCs are produced in an open conformation where all membrane targeting modules and the pseudosubstrate are exposed. After binding of Hsp90 and its co-chaperon Cdc37 via a conserved PXXP motif (Gould, Kannan et al. 2009), several phosphorylation events mediated by the kinase complex mTORC2 and PDK1 occur (Guertin, Stevens et al. 2006). These 28 Introduction phosphorylation events lead to an adoption of the autoinhibited conformation, which is stable and has a half-life of approximately two days. PKC is maintained in an autoinhibited conformation due to the binding of the pseudosubstrate in the substrate-binding cavity. The complex is stabilized and locked in place by the C2 domain (Antal, Callender et al. 2015). The regulatory and the catalytic domain are connected by a hinge region. In the inactive state the regulatory domain is directly bound to the catalytic domain and the activity of the enzyme is inhibited (Figure 7). This region becomes proteolytically labile when the protein is membrane bound and thereby frees the kinase domain from inhibition by the pseudosubstrate. This creates a catalytically active enzyme (reviewed in Newton 1995) (Figure 7). Activation of conventional PKCs occurs via hydrolysis of PIP2 by a two step mechanism. At first, Ca2+ binds to the C2 domain, leading to engagement of PKCs at the plasma membrane via binding of phospholipids and PIP2 (Corbalan-Garcia, Garcia-Garcia et al. 2003, Evans, Murray et al. 2006). This results in a conformational change and binding of the membrane-bound ligand DAG (Takai, Kishimoto et al. 1977) via the C1B domain and a release of the pseudosubstrate from the substrate binding cavity, which leads to activation of the enzyme. As novel PKC isozymes bind DAG with a higher affinity than conventional PKCs they do not need a specific targeting to membranes by Ca2+ for effective DAG binding. Many PKCs are also pharmacologically activated by phorbol esters such as phorbol 12-myristate 13-acetate (PMA), which are acting as a molecular mimicry to DAG (Castagna, Takai et al. 1982). These tumor-promoting phorbol esters anchor PKCs in their active conformation to the plasma membrane (Steinberg 2008) and are often used to study effects of constant PKC activation. 29 Introduction Figure 7. Activation of protein kinase C (PKC). PKC isozymes are activated by phosphorylation of the activation segment and the hydrophobic motif by mTORC2 and PDK1, respectively. Full activation occurs by binding of the second messengers DAG and Ca2+. PKC isozymes are differentially sensitive to these second messengers. Abbreviations: C, conserved region; PH, pleckstrin homology; mTORC2, mTOR Complex 2; PROTOR1, protein observed with RICTOR 1; RICTOR, rapamycin-insensitive companion of TOR; SIN1, SAPK-interacting protein kinase 1;PDK1, pyruvate dehydrogenase lipoamide kinase isozyme 1; DAG, diacylglycerol. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology (Pearce, Komander et al. 2010). The figure and the figure legend are modified. Growth factor receptors and PKC signaling Ligand-induced activation of RTKs and phosphorylation of their cytoplasmic domains lead to the creation of high affinity docking sites for signaling proteins such as PLCγ and several growth factors like EGF, PDGF and FGF are known to activate PLCγ via their respective receptors (Nishibe, Wahl et al. 1990, Peters, Marie et al. 1992, Larose, Gish et al. 1993, Lemmon and Schlessinger 1994). Upon RTK activation, PLCγ translocates from the cytosol to the plasma membrane (Todderud, Wahl et al. 1990), binds to the activated receptor tyrosine kinases via its SH2 domains and becomes phosphorylated. The tyrosine phosphorylation increases the PLCγ activity and promotes the generation of DAG and inositol trisphosphate (IP3) from PIP2 (Figure 8). While DAG activates classical and novel PKC isozymes, IP3 leads to a release of Ca2+ from intracellular storages. Ca2+ then recruits classical PKCs to the plasma membrane for activation. 30 Introduction Figure 8. Activation of the PKC pathway by EGFR. Ligand binding to the EGFR activates the intrinsic tyrosine kinase activity and leads to phosphorylation on the cytoplasmic tail. Phosphorylation of specific tyrosines recruits PLCγ which in turn generates DAG and IP3 from PIP2. PKC activation is mediated by DAG and Ca2+ released in response to IP3 from the ER Abbreviations: PKC, protein kinase C; EGFR, epidermal growth factor receptor; PLCγ, phospholipase C-γ; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol trisphosphate; DAG, diacylglycerol; ER, endoplasmic reticulum. Adapted by permission from Discovery Medicine (Brand, Iida et al. 2011). The figure and figure legend are modified. PKC activation leads to direct phosphorylation of EGFR at Thr-654 (Hunter, Ling et al. 1984). This phosphorylation reduces tyrosine kinase activity, decreases ligand affinity and promotes internalization (Welsh, Gill et al. 1991, Jimenez de Asua and Goin 1992, Morrison, Takishima et al. 1993). Overall the EGFR Thr-654 phosphorylation through PKCs regulate processes such as mitogenesis and lamellipodial retraction (Welsh, Gill et al. 1991). It has also been shown that PKC phosphorylates and promotes the internalization of ErbB2 (Ouyang, Gulliford et al. 1998). PKCα can suppress signaling downstream of the PI3K/Akt pathway by an inhibitory phosphorylation of the PI3K catalytic subunit (Sipeki, Bander et al. 31 Introduction 2006) as well as an inactivating dephosphorylation of Akt (Tanaka, Gavrielides et al. 2003). PKCs can induce internalization for a range of plasma membrane proteins including EGFR (Liu, Idkowiak-Baldys et al. 2013) and ErbB2 (Baietti, Zhang et al. 2012). The best understood mechanism is downregulation of EGFR. After activation of PKCs and translocation to the plasma membrane, PKCs can phosphorylate EGFR on Thr-654, which may protect EGFR from degradation in lysosomes and target for endosomal recycling instead (Bao, Alroy et al. 2000). After phosphorylation PKCs and EGFR are translocated and sequestered into perinuclear recycling endosomes around pre-existing Rab11 endosomes (Becker and Hannun 2003, Idkowiak-Baldys, Baldys et al. 2009). This region is called pericentrion. PMA-mediated translocation of EGFR from the plasma membrane to the pericentrion in a PKC-dependent manner sequesters EGFR from EGF- binding (Liu, Idkowiak-Baldys et al. 2013) 32 Aims of the Study Aims of the Study The main focus of the work presented in this thesis has been to study mechanisms that are involved in the endocytic downregulation, degradation and recycling of the ErbB proteins, ErbB2 and ErbB3. ErbB2 has no known ligand and is strongly endocytosis impaired while ErbB3 has a very weak tyrosine kinase activity. Nevertheless, heterodimers consisting of ErbB2 and ErbB3 are strong activators of proliferative signaling. While ErbB2 is often found overexpressed in breast cancer, ErbB3 expression is associated with resistance to cancer therapy. The molecular mechanisms of ErbB3 turnover are not fully understood and contradictory reports exist. Recently, a connection between PKCs and ErbB proteins has been established. PKCs are involved in endocytic recycling of ErbB proteins and a better understanding of the mechanisms is of great importance for understanding how expression of ErbB proteins is regulated. • The aim of paper I was to identify adaptor proteins involved in CME of ErbB3 and to characterize the initial steps of CME of ErbB3. • The aim of paper II was to study the molecular mechanisms involved in ErbB3 degradation and to characterize the degradation pathway(s). • The aim of paper III was to investigate if and how PKCs regulate endocytic downregulation, degradation and/or recycling of ErbB3. • The aim of paper IV was to investigate the molecular mechanisms involved in PKC-induced internalization and stabilization of ErbB2. 33 Summary of Papers Summary of Papers Paper I Szymanska, M., Fosdahl, A.M., Raiborg, C., Dietrich, M., Liestøl, K., Stang, E. and Bertelsen, V. Interaction with epsin 1 regulates the constitutive clathrin-dependent internalization of ErbB3. Biochim Biophys Acta. 2016 Volume 1863, Issue 6, Part A, June 2016, Pages 1179-1188. doi:10.1016/j.bbamcr.2016.03.011 ErbB3 was reported not to interact with AP-2 due to the lack of necessary sorting signals, and ErbB3 was thus for a long time considered to be endocytosis resistant. We have, however, previously shown that ErbB3 is constitutively internalized via CME, indicating the availability of other sorting proteins than AP-2. In this study we performed a high-throughput siRNA screen to identify adaptor proteins required for constitutive internalization of ErbB3. 25 different CLASPs known to participate in CME and several components of the AP-2 complex were knocked down and their influence on CME of ErbB3 was measured. CHC and dynamin 2 served as stronger and milder positive controls to help identify a broad spectrum of involved adaptors. Measurement of effects was done by an automatic fluorescent microscope and several proteins interfered with CME of ErbB3 upon knock-down. We could confirm that depletion of the AP-2 subunits had no or minimal effect on the internalization of ErbB3. Only epsin 1 knock-down, among the 25 CLASPs, was able to inhibit ErbB3 internalization to the same extent as depletion of CHC. We further showed that ErbB3 is constitutively ubiquitinated and ligand-binding further increased ubiquitination. By the use of an epsin 1 mutant containing only membrane and ubiquitin binding domains, we could demonstrate that ErbB3 interacts with these domains. Thus, our data suggests an interaction between ErbB3 and epsin 1 in an ubiquitin-dependent manner and that both constitutive and ligandinduced CME of ErbB3 are regulated through interaction with epsin 1. 34 Summary of Papers Paper II Fosdahl, A.M., Dietrich, M., Schink, K.O., Malik, M.S., Skeie, M., Bertelsen, V. and Stang, E. ErbB3 interacts with Hrs and is sorted to lysosomes for degradation Biochim Biophys Acta. 2017 Volume 1864, Issue 12, December 2017, Pages 2241-2252. doi.org/10.1016/j.bbamcr.2017.08.011 The ErbB family mediates activation of a wide network of signaling pathways and therefore, a tight control of activation is necessary. The network is generally negatively regulated by endocytosis of the receptors, followed by their degradation. ErbB3 is, despite having a very weak kinase activity, contributing to proliferative signaling via its six docking sites for the p85 subunit of PI3K. We have previously shown that ErbB3 is constitutively internalized and degraded and it is also known that ligand binding causes increased internalization and degradation. However, the exact mechanisms of ErbB3 degradation are still unclear and several contradictory reports exist. In the current study we have investigated in more detail the constitutive and ligand-mediated trafficking and degradation of ErbB3. We found that ErbB3 internalization depends on CME both in the presence and absence of ligand, and we identified an interaction of ErbB3 with the ESCRT-0 subunit Hrs both in the presence and absence of an activating ligand. In addition, we could show that impaired ESCRT function leads to an endosomal accumulation of ErbB3 indicating an ESCRT-mediated sorting of ErbB3 to late endosomes and lysosomes. In the absence of ligand ErbB3 undergoes both lysosomal and proteasomal degradation, while ligand-induced degradation appears to be predominantly lysosomal. The proteasome mediated degradation is probably mainly representing a recently discovered ER-localized quantity control. 35 Summary of Papers Paper III Dietrich, M., Bertelsen V. and Stang, E. Protein kinase C regulates ErbB3 turnover Manuscript ErbB3 can activate a variety of signaling pathways via heterodimerization with other family members despite an own weak tyrosine kinase activity. We have previously shown that ErbB3 is constitutively internalized and degraded and this process gets accelerated after ligand binding. Several studies showed that PKC can regulate activation, localization and stability of EGFR and ErbB2. In the present study we investigated effects activation of PKC has on ErbB3 stability and intracellular trafficking. We found that PKC activation increases stability of ErbB3 but induces a downregulation from the plasma membrane. While investigating the intracellular localization we found that internalized ErbB3 under normal conditions gets sorted for degradation via EEA1-positive early endosomes to Lamp1-positive late endosomes. PKC activation sorted ErbB3 into EEA1 and Lamp1 negative compartments and protected the receptors from degradation. In addition, we found that PKC inhibition mildly increased degradation supporting that PKC regulates stability of ErbB3 by altering intracellular trafficking of ErbB3. 36 Summary of Papers Paper IV Dietrich, M., Malik, M.S., Nikolaysen, F. and Stang, E. Protein kinase C induced internalization of ErbB2 is independent of clathrin, ubiquitination and Hsp90 dissociation Manuscript A recent study suggested the involvement of PKCs in endocytic recycling of ErbB2. We have now further investigated ErbB2 trafficking after PKC activation. We found that PMA alone induced internalization of ErbB2, and that PMA had an additive effect when combined with the Hsp90 inhibitor 17-AAG. This indicates that Hsp90 inhibition and PKC activation induce internalization by different mechanisms. In line with this we found that while 17-AAG-induced downregulation is clathrin-dependent, PMA-induced internalization is clathrin- independent, but sensitive to cholesterol depletion. Also the intracellular sorting differs. While 17-AAG induced sorting of ErbB2 to MVBs followed by degradation, there was no measurable ErbB2 degradation after PKC activation. ErbB2 was instead sorted to tubulovesicular and/or cisternal organelles probably representing an endocytic recycling compartment. These differences may be explained by our finding that 17-AAG, but not PMA, induced dissociation of Hsp90 followed by ubiquitination of ErbB2. This supports previous studies suggesting that ubiquitination serves as a signal for ErbB2 internalization and degradation. 37 Methodological Considerations Methodological Considerations Precise descriptions of the methods used in the papers and manuscripts can be found in the respective materials and methods sections. The experiments in the attached papers have been done in at least three independent repetitions and statistical significance analysis was carried out where it was necessary and appropriate to do so. The following section will only provide a discussion of advantages and disadvantages of the used methods. Cell culture The studies in this thesis were carried out in human cancer cell lines and/or porcine aortic endothelial (PAE) cells. The different cell lines used in this study express EGF receptor family members in varying amounts. For studies of ErbB2 mostly SK-BR-3 cells, a human breast adenocarcinoma cell line, were used. These cells overexpress ErbB2 and moderately express ErbB3 and EGFR. The simultaneous expression of several EGFR family members must also be taken into consideration when interpreting the data. The advantage is that also possible interactions between the receptors are taken into consideration in the experimental settings which is closer to human physiology. On the other hand this complicates the interpretation of data. For studies of ErbB3 most experiments were carried out in MCF-7 cells, a human breast cancer cell line, that express moderate levels of ErbB3 but low levels of ErbB2 and EGFR (Sak, Breen et al. 2012). For experiments where the receptors should be studied alone or in specific receptor combinations, PAE cells were used that endogenously do not express any members of the EGF receptor family. The cells were stably transfected to express receptors originating from a different species. This system has the advantage that ErbB proteins can be studied separately, without the interference of other members of the receptor family. Studies for EGFR have shown that the mechanisms of internalization in this artificial model system are comparable to human cells expressing wild type EGFR (Carter and Sorkin 1998) and PAE cells have also been extensively used to study endocytic downregulation of ErbB proteins without finding unusual behavior of ErbBs expressed in PAE cells (Sak, 38 Methodological Considerations Breen et al. 2012, Sak, Szymanska et al. 2013). PAE cells do also have the advantage that they are relatively easy to transfect. The stably transfected PAE cells were grown under antibiotic selection and routinely tested for receptor expression. Cell lines are immortalized and easy to maintain in culture for longer periods of time. The reproducibility of experiments at later time points is therefore, greatly enhanced and the cells do as such serve as a good model system for analysis of molecular biological processes. Nevertheless, there are also disadvantages in the use of cell lines, as these cells have already undergone genotypic and phenotypic changes and are also likely to further acquire more modifications when kept in culture for an extended amount of time. We kept cells in culture for a limited number of passages and confluency was avoided. Cells were routinely authenticated by genotyping and also tested for infection with mycoplasma to avoid misleading experimental results. Chemical inhibitors and activators The use of chemical inhibitors allows the inhibition of certain cellular processes and proteins to study their effects on processes such as signaling and trafficking. It is a powerful tool to investigate intracellular trafficking and signaling but it certainly also has disadvantages of use. In this study a variety of chemical inhibitors were used. Among them were inhibitors of protein activation such as Ro 31-8220 and AG1478. Ro 31-8220 is a broad spectrum PKC inhibitor that mainly inhibits the activation of classical PKC isozymes. It has also been reported to inhibit other kinases such as Rsk-2 and S6K1 with similar potency as for PKC (Alessi 1997). Therefore, results should be interpreted with caution as the observed effects can be also mediated by the inhibition of other protein kinases than PKC isozymes. The kinase inhibitor AG1478 is considered to be specific for the EGFR kinase, but our previous studies have shown that it also potently inhibits ErbB3 phosphorylation in cells only expressing ErbB3 from the EGFR family (Sak, Breen et al. 2012). Other studies have also shown that AG1478 potently inhibits ErbB2 (Kurokawa, Lenferink et al. 2000) and ErbB4 (Fukazawa, Miller et al. 2003). This gave us the possibility to also use this inhibitor to study ErbB3 kinase activity. We also used inhibitors such as monensin, MG-132 and 39 Methodological Considerations concanamycin A, which block different intracellular processes. Monensin is an inhibitor of recycling by functioning as a Na+/H+ antiporter and thereby inhibiting the acidification of endosomes. Monensin has been shown to inhibit the exit of internalized receptors from sorting endosomes and the endocytic recycling compartment (Stein, Bensch et al. 1984). The proteasome inhibitor MG132 was used to study whether degradation of ErbB3 depends on proteasomal activity. One problem with the inhibition of proteasomal activity is that the level of free ubiquitin becomes reduced due to impaired ubiquitin recycling. This could have effects on other cellular processes as well. Lysosomal degradation of ErbB3 was inhibited by using concanamycin A at a concentration that is known to inhibit EGF-induced EGFR degradation. Concanamycin A inhibits vacuolar type H+-ATPases. This inhibits lysosomal degradation mostly by an increase in the pH in the lumen of lysosomes. Due to the variety of side effects different inhibitors can have, we tried to test similar inhibitors in parallel and included positive controls when possible. The PKC activator PMA was used to study effects of PKC activation on ErbB2 and ErbB3. PMA is a very potent activator of classical and novel PKC isozymes, which poses the challenge to identify specific PKC isozymes involved in the effects observed after PMA- mediated PKC activation. Prolonged exposure to phorbol esters, such as PMA, causes almost complete depletion of several PKC isozymes from the cells, most likely as a result of proteolysis (Kishimoto, Mikawa et al. 1989, Hug and Sarre 1993). After PMA treatment, the steady state level of PKC is thus decreased through an increase in proteolysis while the rate of synthesis remains constant (Young, Parker et al. 1987). When using PMA as an activator these effects should always be taken into consideration. Transient transfections Transient transfection with plasmids is a method that allows the introduction of foreign or mutated genes or to increase the expression of normally very low expressed genes of interest, without alteration of the genome. This allows studying of protein interactions in cell lines that normally do not express the proteins of interest, or where endogenous expression levels are below efficient 40 Methodological Considerations detection levels of available antibodies for methods such as western blotting or immunoprecipitation. By the use of specific mutants that carry modified versions of the protein of interest it is possible to analyze cellular processes where the protein is involved. In addition, it is possible to introduce tagged proteins into the cell, which can also increase the detection efficiency by the use of antibodies against the protein tag. The incorporated plasmids will be only transiently expressed and not incorporated into the genome. Furthermore, the plasmids can also be lost over time due to cell division and other environmental factors. There is a wide range of plasmids encoding cellular proteins available. Transient transfection was applied in paper I where a non-tagged ErbB3 and an enhanced green fluorescent protein (EGFP)-tagged epsin mutant were used. In paper II we used Vps4 and Hrs but also EGFP-tagged ErbB3 and Hrs were introduced by transfection to ease detection and precipitation efficiency for detection of interactions of ErbB3 with Hrs. Endogenous protein expression was not sufficient enough to detect the interaction by co-IP. It should be generally taken care that transient transfection can lead to overexpression of the protein of interest which can alter the intracellular behavior and distribution of the protein of interest and proteins that interact and/or regulate the protein of interest. RNA interference RNA interference (RNAi) is a post-transcriptional process where a specific mRNA is degraded to control and in that way inhibit or limit protein expression. It was discovered by Fire and Mello (Fire, Xu et al. 1998) and it is used by different organisms. There are several methods for RNAi but the most common way is the introduction of small interfering RNA (siRNA). A standard siRNA is a synthetic oligonucleotide and is composed of 19 complementary base pairs and 2 nucleotide 3’ overhangs. In the siRNA screen (Paper I), a variety of human cell lines was initially tested for transfection efficiency with different siRNAs and different lipid reagents to assess the specificity of the protein knock down as well as the cell viability and cytotoxicity. This testing was necessary as siRNA usage is limited to certain cell lines that are easy to transfect and robust enough to withstand the treatment. It should also be kept in mind that effects of siRNA treatments are transient. 41 Methodological Considerations A common problem with usage of siRNAs is the appearance of off-target effects, which can occur due to unspecific binding of the siRNA besides the target sequence. This leads to reduction of messenger RNA (mRNA) levels of proteins that are not supposed to be targeted and therefore, to unwanted side-effects. To minimize off-target effects different siRNA concentrations where tested to find the lowest concentration that gives an efficient knockdown. Nevertheless, it should be noted that mammalian cells actively respond to the introduction of double-stranded RNA by triggering the interferon response pathway. This can also lead to unwanted side effects and should be taken into consideration when interpreting results. For all siRNA experiments in this thesis, at least two different sequences for knock down of the protein of interest were tested and a non targeting (random sequence that is not present in the target genome) sequence to detect off target effects was always included High-throughput screening High-throughput screening was carried out in Paper I to assess the influence on internalization of ErbB3 of siRNA-mediated knockdown of 31 selected proteins known to be involved in CME. In the siRNA screen we have used 4 individual sequences for each protein of interest and in addition, 4 individual non-targeting siRNA sequences. Also siRNA to housekeeping genes was used to ensure the observed effects are due to knock-down of the particular protein. Automatic cell recognition for image acquisition occurred on the basis of Hoechst stained nuclei. The cell phenotype was also taken into consideration to identify “hits”. Appropriate statistical measures to avoid false positive results were applied to prevent an influence of small variations in the experimental procedures. Immunological detection Immunological detection depends on the interaction between an antigen epitope and the corresponding antibody. This interaction is depending on specificity of the antibody, the status of the protein, meaning being in native or denatured state, concentration of the antibody and optimal incubation time and temperature. Detection of the interaction can be facilitated by different mechanisms. Enzymes or fluorescent dyes can be bound directly to the primary 42 Methodological Considerations antibody (direct detection) or be bound to a secondary antibody that targets the primary antibody (indirect detection). While direct detection may be more specific as there is no possible cross reactivity of the secondary antibody, indirect detection can have increased signal amplification as more than one antibody can bind to the primary antibody. We also routinely checked the specificity of primary antibodies against EGFR family members for cross reactivity by testing them in PAE cells carrying other EGFR family members than the ErbB protein of interest. Western blotting Western blotting is a technique for detection of proteins of interest in cell lysates. It also relies on immunological detection of antigen antibody interactions. Western blotting is a powerful method to detect and quantify amounts of protein in lysates after different kinds of treatment such as the use of chemical inhibitors or RNA interference. Proteins in the lysates are denatured and afterwards separated by molecular weight by the use of sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Proteins are then transferred to a nitrocellulose or PVDF membrane on which they can be detected by specific antibodies. Detection of proteins usually occurred by the use of secondary antibodies conjugated to horseradish peroxidase (HRP), which create chemiluminescence detected by a camera. Images were acquired below saturation level and processed using Image Lab 4.1 software (Bio-Rad Laboratories). Quantification of western blots was based on pixel intensity of the bands of interest and normalized against a loading control, usually tubulin run on the same gel. This was done to account for variations between samples. Nevertheless, quantification of western blots can be inaccurate and affected by varying size of the bands and background noise (Mollica, Oakhill et al. 2009). Immunoprecipitation (IP) and co-IP To study protein modifications like ubiquitination and phosphorylation or particular protein-protein interactions it becomes very important to exclude all non-interacting proteins from the sample. This can be achieved by precipitating proteins of interest from lysates by using antibodies to the specific protein 43 Methodological Considerations coupled to magnetic beads coated with Protein A or Protein G. The precipitated proteins are then eluted in SDS-buffer to ensure denaturation of proteins and dissociation of antibodies from the beads. The eluted samples can then undergo western blotting. To ensure that the obtained results do not occur due to unspecific binding of antibodies or proteins, a control bead sample without pre- bound antibody was routinely added in every experiment. For analysis of post- translational modifications such as tyrosine phosphorylation or ubiquitination the samples underwent hot (96°C) SDS-based lysis. This method causes protein denaturation without breaking covalent bonds between the post-translational modifications and the protein. Interaction between proteins was analyzed by co-IP. A lysis buffer without SDS was used to avoid disruption of protein complexes and incubations happened on ice in presence of inhibitor cocktails. The precipitated protein complexes were eluted using SDS buffer and underwent western blotting for detection of proteins. Nevertheless, this method needs a rather strong interaction of the proteins or a high expression level of the proteins of interest to detect interaction. Therefore, we had to overexpress proteins and/or mutants of interest to increase the chance for detection. It should also be noted, that co-immunoprecipitation does not prove a direct interaction of the proteins of interest; it only reveals that the proteins exist in the same protein complex indicating an interaction. Wide-field fluorescent microscopy and confocal microscopy Wide-field fluorescent microscopy is an imaging technique where the whole sample is illuminated simultaneously. This technique was applied in paper I. We have used an automatic wide-field fluorescent microscope so that data from thousands of cells could be collected. In addition, this allows/increase the possibility of a non-biased interpretation. Confocal microscopy was used as a qualitative method to analyze subcellular localization and co-localization of proteins of interest, using fluorochromeconjugated secondary antibodies or ligands. With the use of an antibody-based assay we were also able to visualize the endocytic ability of ErbB proteins after various treatments. Confocal microscopes take advantage of the spatial pinhole and point illuminations to increase optical resolution and contrast by decreasing 44 Methodological Considerations out-of-focus light. Therefore, confocal microscopy allows optical sectioning. In each confocal microscopy experiment a variety of cells were analyzed to exclude artifacts from individual cells. A problem in fluorescent microscopy techniques can be autofluorescence from among others the fixation reagent. Therefore, samples were treated with ammonium chloride to quench background fluorescence. By adjusting the laser intensities below saturation levels the risk of bleaching was minimized. Pictures were acquired sequentially to minimize the risk of fluorescence bleed through from other laser wavelengths. The fluorescent dye with the longest emission spectra was scanned first and the dye with the shortest last. Images were processed using ImageJ FIJI and Adobe Photoshop CS4 and if image adjustments were necessary they were applied on the whole image. Detection of intracellular localization of ErbB proteins was done by microscopy and the use of fluorescent dyes coupled to secondary antibodies. To determine subcellular localization in more detail, double or triple staining including markers of intracellular compartments was applied. To avoid cross reactivity in this approach we used antibodies from different species and for avoiding fluorescence overlap we choose fluorochromes with non-overlapping emissions spectra. To test for unspecific binding of the antibodies, we carried out controls where one primary antibody was incubated with secondary antibodies of a different species. Immuno-electron microscopy In paper IV we used immuno-electron microscopy (immuno-EM) to localize ErbB2 and characterize the intracellular compartments into which ErbB2 was sorted. The advantages of immuno-EM over light-based microscopy include a higher resolution than confocal microscopes and that certain cellular organelles, but not all, can be identified based on morphology without the need of specific markers. Since immuno-EM is an antibody based technique it is depending on antibody specificity as well as labeling efficiency. Due to this, and the lack of antibodies that give specific and sufficient labeling efficiency, immuno-EM was not included in the ErbB3 studies. A limitation of immuno-EM is the use of ultrathin sections which limits the three dimensional interpretation of compartments. Due to this it was not possible to decide whether the ErbB2 positive structures 45 Methodological Considerations seen upon incubation with PMA represent tubules or cisterns, or a mixture of these and small vesicles. Flow cytometry Flow cytometry was used to measure plasma membrane expression of ErbB proteins in fixed non permeabilized cells by the use of fluorescent antibodies. It is a powerful tool to measure downregulation of proteins from the plasma membrane. Flow cytometry allows the fast, quantitative analysis of huge numbers of cells in a short period of time. We used fluorescently-conjugated primary antibodies to the extracellular part of either ErbB2 or ErbB3 and the antibodies where used at, by titration, optimized concentrations where the antigen was saturated to avoid pipetting variation. A viability dye was routinely used for the exclusion of dead cells and cellular gating strategies where put in place for duplet discrimination. Internalization of radioactive transferrin (125I-Tf) Transferrin (Tf) is internalized in a clathrin-dependent manner only. Due to this, internalization of radioactive labeled transferrin was used to quantitatively determine the specificity of siRNA silencing of major components involved in clathrin-mediated endocytosis. The advantage of the use of radioactive labeled transferrin is the high sensitivity of detection of even small amounts of internalized ligand. However, the use of radioactive solutions needs to be handled with great caution and proper routines in place. 46 General Discussion General Discussion Internalization and intracellular sorting of ErbB3 ErbB3 has been found expressed in several cancers but evidence for gene amplification and overexpression is limited. It is an important activator of PI3K, and due to its potent heterodimerization capability with ErbB2, studies suggest a paracrine loop in some prostate cancer types involving heregulin and the ErbB2ErbB3 heterodimer (Lyne, Melhem et al. 1997). AP-2 is considered to be the major hub for mediating CME, and as ErbB3 does not have sorting signals recognized by AP-2 (Baulida, Kraus et al. 1996), it was for a long time considered endocytosis resistant. We did however, recently show that ErbB3 is internalized in absence of ligand in a clathrin-dependent manner (Sak, Breen et al. 2012). Therefore, other internalization signals and adaptor proteins must be involved in CME of ErbB3. In recent years several other internalization signals apart from the motifs recognized by AP-2 have been identified (reviewed in Traub and Bonifacino 2013). Lately, a lot of research has focused on the role of ubiquitin in mediating internalization. For EGFR it has been shown that ubiquitination can serve as a signal for both CME and CIE (Sigismund, Woelk et al. 2005, Madshus and Stang 2009, Sorkin and Goh 2009). In paper I we show that ErbB3 is constitutively ubiquitinated and confirmed that ubiquitination increases with ligand binding (Cao, Wu et al. 2007, Huang, Choi et al. 2015). Furthermore, did we show that ErbB3 interacts with the endocytic adaptor protein epsin 1. Knock-down of epsin 1 inhibited internalization of ErbB3 to the same extent as knock-down of CHC. To show that this was not a result of a general inhibition of CME, we confirmed that epsin 1 knock-down does not affect the constitutive clathrin-dependent endocytosis of TfR. By immunoprecipitation we showed that ErbB3 interacted with an epsin mutant that contains the UIM domains, but lacks the clathrin and AP-2 binding regions. When this mutant was expressed, it inhibited the internalization of ErbB3. We have previously shown similar data for ubiquitinated EGFR (Kazazic, Bertelsen et al. 2009, Bertelsen, Sak et al. 2011) and ErbB2 (Vuong, Berger et al. 2013). Altogether this shows that epsin 1 is required for CME of ErbB3, and it suggests that ubiquitin can serve as a signal for internalization of ErbB3. It has been 47 General Discussion previously shown that the ubiquitin ligase Nrdp1 constitutively ubiquitinates ErbB3 (Diamonti, Guy et al. 2002, Qiu and Goldberg 2002). This could indicate that Nrdp1-mediated ubiquitination regulates ErbB3 internalization. In paper II, however, we showed that although knock-down of Nrdp1 increased plasma membrane expression of ErbB3, the knock-down did not inhibit its constitutive internalization. This suggests that Nrdp1-mediated ubiquitination could be involved in the quantity control but not internalization of ErbB3. The ubiqutin ligase Nedd4 was recently reported to negatively regulate ErbB3 level and signaling (Huang, Choi et al. 2015). Therefore, Nedd4 could potentially have an effect on ErbB3 internalization as different ligases may induce different ubiquitination patterns and/or mediate ubiquitination at different lysines within ErbB3. We have thus initiated studies on the role of Nedd4 in ErbB3 internalization in absence and presence of ligand. With respect to endocytosis, ubiquitination does not only serve as a signal for internalization. Ubiquitination also plays an important role in endosomal sorting, as ubiquitinated proteins are recognized by the ESCRT-machinery, where cargo gets sorted for lysosomal degradation (reviewed in Raiborg and Stenmark 2009). It has been shown for EGFR that impaired ubiquitination of the receptor, by using a mutant that is unable to interact directly with the ubiquitin ligase Cbl, leads to internalization followed by recycling instead of lysosomal degradation (Grovdal, Stang et al. 2004). On the other hand it was shown that a TfR mutant with fused ubiquitin chains was sorted for lysosomal degradation instead of endocytic recycling (Raiborg, Bache et al. 2002). Not a lot is known so far about the involvement of the ESCRT machinery in endosomal sorting of ErbB3. In paper II we identified that the ESCRT-0 subunit Hrs interacts with ErbB3. Hrs is involved in the trafficking of ubiquitinated cargo into late endosomes. It has been shown for EGFR that the Hrs EGFR interaction is necessary for sorting of EGFR to internal vesicles of MVBs (Bache, Raiborg et al. 2003, Myromslien, Grovdal et al. 2006). We now demonstrate that Hrs and ErbB3 colocalize on endosomes. We further identified that Hrs and ErbB3 interact even in absence of ligand, but that this interaction increased upon ligand binding and increased ErbB3 ubiquitination. It has previously been demonstrated that Hrs overexpression blocked the degradation of EGFR (Raiborg, Bache et al. 2001, Urbe, Sachse et al. 48 General Discussion 2003). In line with this we observed that Hrs overexpression also blocked degradation of ErbB3. We showed that heregulin induced phosphorylation of Hrs which is in line with findings that EGF induces phosphorylation of Hrs (Bache, Raiborg et al. 2002, Urbe, Sachse et al. 2003). In addition, we showed that overexpression of a dominant negative mutant of the ESCRT-III component Vps4 impaired the trafficking of ErbB3. If aside of Hrs and Vps4, other components of the ESCRT-machinery are directly involved in endosomal sorting of ErbB3 remains to be further investigated, but our results clearly demonstrate the need of functional ESCRTs. The mechanisms how ErbB3 is degraded are still debated. While the involvement of the ESCRT-machinery indicates that ErbB3 is degraded in lysosomes, other studies concluded that ErbB3 is degraded both by proteasomes (Diamonti, Guy et al. 2002, Qiu and Goldberg 2002, Fry, Simion et al. 2011, Huang, Choi et al. 2015) and in lysosomes (Cao, Wu et al. 2007). In paper II we show that lysosomal inhibition caused accumulation of constitutively internalized ErbB3 in late endosomes, and correspondingly, that the constitutive degradation of ErbB3 then was slightly reduced. This indicates that constitutive degradation of ErbB3 can occur in lysosomes However, also inhibition of proteasomes showed a clear effect on constitutive degradation. A likely explanation for this is inhibition of the ERAD-mediated quantity control which is known to regulate the overall ErbB3 expression. It has, however, been shown for other ErbB proteins, such as EGFR and ErbB2, that proteasomal activity is important also for lysosomal degradation, even though the receptors are not directly targeted (Longva, Blystad et al. 2002, Pedersen, Madshus et al. 2008). Proteins like Hrs and Eps15, involved in sorting and degradation, are regulated by proteasomal activity and also are degraded in proteasomes (Klapisz, Sorokina et al. 2002, Polo, Sigismund et al. 2002, Haglund and Dikic 2012). Therefore, the observed effects on ErbB3 degradation upon inhibition of proteasomes may not be caused by direct proteasomal degradation of the receptor, but by impaired availability or activity of proteins that are important for sorting and lysosomal degradation of ErbB3. When analyzing the ligand-induced internalization and degradation the effect of lysosomal inhibition was prominent, while proteasomal inhibition showed only a minor effect. This indicates that ligand-induced degradation mainly is lysosomal. 49 General Discussion Overall these results indicate that internalized ErbB3, both constitutive and ligand-induced, is degraded in lysosomes, but that proteasomes, in addition to being involved in ERAD mediated degradation, might also, as for EGFR and ErbB2, regulate sorting of ErbB3 into late endosomes. Effects of PKC activity on ErbB2 and ErbB3 It has been previously shown that aside of constitutive and ligand-induced turnover of ErbB proteins, also other RTKs, GPCRs and kinases influence expression and localization of ErbB proteins. PKC is among the kinases that have been shown to influence plasma membrane expression and degradation of EGFR (Bao, Alroy et al. 2000, Llado, Timpson et al. 2008, Liu, Idkowiak-Baldys et al. 2013) and ErbB2 (Bailey, Luan et al. 2014). As the PKC family has several critical roles in regulating physiological processes as well as disease pathogenesis, a link to ErbB proteins opens new possibilities of oncogenic deregulation of cellular processes. PKCα has been shown to be involved in regulation of proliferation, migration and resistance to anti-estrogens in studies of breast cancer (Urtreger, Kazanietz et al. 2012). In addition, clinical trials have shown a connection between PKCα overexpression and poor prognosis in breast cancer (Tan, Li et al. 2006, Lonne, Cornmark et al. 2010, Tonetti, Gao et al. 2012). The novel PKC isozyme PKCδ is also known to be overexpressed in a subset of breast cancers and is, like PKCα, connected with a poor survival in ErbB2-positive breast cancer (Allen-Petersen, Carter et al. 2014). The mechanisms by which PKCα and PKCδ play a role in ErbB2-positive breast cancers remain unclear to a large extent. ErbB2 can be found overexpressed in up to 30% of human invasive ductal breast cancers (Slamon, Clark et al. 1987). These cancers are often correlated with more aggressive tumors and a reduced overall survival time (Leahy 2004). ErbB2 is considered to be endocytosis resistant, and if internalized it is rapidly recycled back to the plasma membrane (reviewed in Bertelsen and Stang 2014). The molecular mechanisms regulating ErbB2 recycling are still to a large extent unexplored. A variety of studies have suggested a pro-oncogenic role of PKCα and PKCδ in breast cancer. PKCα has been shown to be activated in ErbB2 overexpressing breast cancer cell lines and Hrg-induced apoptosis of SKBR3 cells 50 General Discussion was potentiated when PKCα activity was inhibited (Le, Marcelli et al. 2001). Even though cellular, animal and clinical studies propose a strongly pro-oncogenic role of PKCs in breast cancer, the mechanisms that are involved in ErbB2-driven breast cancer are still less certain. A recent study (Bailey, Luan et al. 2014) did, however, identify that PKCs can promote downregulation of ErbB2 from the plasma membrane and sort it to what probably represent an ERC and thereby protects internalized ErbB2 from degradation. In paper IV we confirmed that ErbB2 is downregulated from the plasma membrane, but not measurably degraded after PMA-mediated PKC activation, and in paper III we demonstrate that PKC activation in a similar way regulates ErbB3 localization and stability. The molecular mechanisms involved in PKC-mediated ErbB2 and ErbB3 downregulation, are not known but for EGFR it has been shown that a PKCdependent juxtamembrane threonine phosphorylation (Thr-654) regulates the endocytic trafficking away from lysosomal degradation towards the ERC (Bao, Alroy et al. 2000). The PKC activator phorbol dibutyrate has been shown to induce phosphorylation of the corresponding Thr-686 in ErbB2 (Ouyang, Gulliford et al. 1996, Ouyang, Gulliford et al. 1998). It is worth noting that in ErbB3 the corresponding amino acid is alanine and that ErbB3, therefore, lacks the corresponding threonine phosphorylation site (Ouyang, Gulliford et al. 1998). We could not definitely determine whether the observed downregulation of ErbB3 was a result of induced internalization, inhibited recycling, or if it inhibited transport of newly synthesized ErbB3 to the plasma membrane. However, as we observed that PMA increased the downregulation of ErbB3 when protein synthesis was inhibited by treatment with cycloheximide (CHX), we clearly favor the notion that PKC alter internalization and intracellular sorting of ErbB3. Contrary to Bailey et al. we were not able to identify the intracellular localization of ErbB2 based on anti-ErbB2 antibody internalization after PMA stimulation. This could be due to different experimental setups and microscopy procedures. Immuno-EM did, however, indicate that ErbB2 is sequestered within a complex network of tubulovesicular or cisternal structures which show morphological similarities to previously identified ERCs. The internalization of anti-ErbB3 antibody could, however, be identified by confocal microscopy and contrary to 51 General Discussion the early and late endosomal localization found in control cells, incubation with PMA caused the localization of internalized anti-ErB3 antibody in LAMP1 negative compartments. This indicates that PKC activation sequesters also ErbB3 in some sort of recycling compartment. It was previously shown that the downregulation of ErbB2 from the plasma membrane after GA treatment is clathrin-dependent (Pedersen, Madshus et al. 2008), and we also confirmed this finding for the GA derivative 17-AAG. The PKC-mediated downregulation was not altered by depletion of CHC with siRNA. This indicates that the mechanisms of induction of ErbB2 downregulation for 17AAG and PMA are generally different. ErbB2 has been shown to interact with raft localized proteins like flotillins and the finding that cholesterol depletion was sufficient to inhibit PMA-mediated internalization of ErbB2 supports a role of lipid rafts in PKC-induced internalization. The uptake of shiga toxin and the plant protein ricin has been shown to be cholesterol-independent, but otherwise most endocytic pathways, including CME, are to varying degrees sensitive to cholesterol depletion (reviewed in Sandvig, Pust et al. 2011). The exact pathway(s) involved do thus remain to be investigated. We have so far not investigated the PMA-mediated internalization pathways for ErbB3, but as for ErbB2 we have initiated studies to test clathrin and cholesterol dependency. To further limit the number of possible pathways, we will study to what extent PKCinduced internalization of the two receptors depends on the GTP-ase activity of dynamin. Ubiquitination has been suggested to serve as a signal for ErbB2 internalization and degradation (Vuong, Berger et al. 2013), and as discussed above it is also involved in constitutive and ligand-induced downregulation of ErbB3. We observed a strong ubiquitination of ErbB2 after treatment with 17-AAG leading to a subsequent degradation of ErbB2. Similar effects were seen for ErbB3 after Hrg stimulation. In cells stimulated with PMA we could not observe ubiquitination or degradation of ErbB2 or ErbB3, strengthening the suggested role of ubiquitination in ErbB2 and ErbB3 internalization and degradation. Furthermore, in line with an important role of ubiquitination, PKC activation did not inhibit 17-AAG induced degradation of ErbB2 or Hrg-induced degradation of ErbB3, indicating that ubiquitination is superior to PKC-induced modifications 52 General Discussion when it comes to endosomal sorting. Combinatorial treatment with 17-AAG and PMA showed a nearly additive effect on downregulation of ErbB2. These results differ slightly from previous studies that showed no additional effect of 17-AAG and PMA (Bailey, Luan et al. 2014). This difference could potentially be explained by the increased 17-AAG concentration used in our study. Why PMA which otherwise stabilize ErbB2, increased the degradative effect of 17-AAG is unclear, but in support of ubiquitination as a degradative signal, we found that PMA increased the 17-AAG induced ubiquitination of ErbB2. Hsp90 and ErbB2 in endocytosis resistance It is well established that ErbB2 is stabilized through interaction with Hsp90 and that Hsp90 inhibitors induce internalization and degradation of the otherwise endocytosis resistant ErbB2. However, it is still unclear whether Hsp90 actively retains ErbB2 at the plasma membrane and that this interaction has to be inhibited for ErbB2 to be internalized, or if Hsp90 inhibition leads to recruitment of other molecular factors that induce downregulation of ErbB2. Such factors could be a conformational change of the receptor that leads to uncovering of otherwise hidden internalization signals or ubiquitination which is induced by recruitment of Hsp70 and ubiquitin ligases like CHIP and CUL5 (reviewed in Bertelsen and Stang 2014). The data in paper IV indicate that the ErbB2 Hsp90 interaction does not have to be disrupted for ErbB2 to be internalized, but suggests that the interaction regulates the pathway by which ErbB2 gets internalized. Based on this it is tempting to speculate that Hsp90 retains ErbB2 in lipid rafts and that Hsp90 inhibitors release ErbB2 from rafts at the same time as ubiquitination induces clathrin-mediated internalization of ErbB2. The cholesterol dependence of PMA-induced internalization may on the other hand indicate that PKCs induce internalization of lipid rafts containing ErbB2 in complex with Hsp90 and other raft localized proteins like the flotillins and ERM proteins. 53 General Discussion 54 References References Aboulaich, N., J. P. Vainonen, P. Stralfors and A. V. Vener (2004). "Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes." Biochem J 383(Pt 2): 237-248. Acconcia, F., S. Sigismund and S. Polo (2009). "Ubiquitin in trafficking: the network at work." Exp Cell Res 315(9): 1610-1618. Ahle, S. and E. Ungewickell (1986). "Purification and properties of a new clathrin assembly protein." EMBO J 5(12): 3143-3149. Alessi, D. R. (1997). "The protein kinase C inhibitors Ro 318220 and GF 109203X are equally potent inhibitors of MAPKAP kinase-1beta (Rsk-2) and p70 S6 kinase." FEBS Lett 402(2-3): 121-123. Allen-Petersen, B. L., C. J. Carter, A. M. Ohm and M. E. Reyland (2014). "Protein kinase Cdelta is required for ErbB2-driven mammary gland tumorigenesis and negatively correlates with prognosis in human breast cancer." Oncogene 33(10): 1306-1315. Antal, C. E., J. A. Callender, A. P. Kornev, S. S. Taylor and A. C. Newton (2015). "Intramolecular C2 Domain-Mediated Autoinhibition of Protein Kinase C betaII." Cell Rep 12(8): 1252-1260. Asp, N., A. Kvalvaag, K. Sandvig and S. Pust (2016). "Regulation of ErbB2 localization and function in breast cancer cells by ERM proteins." Oncotarget 7(18): 25443-25460. Austin, C. D., A. M. De Maziere, P. I. Pisacane, S. M. van Dijk, C. Eigenbrot, M. X. Sliwkowski, J. Klumperman and R. H. Scheller (2004). "Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin." Mol Biol Cell 15(12): 5268-5282. Bache, K. G., C. Raiborg, A. Mehlum, I. H. Madshus and H. Stenmark (2002). "Phosphorylation of Hrs downstream of the epidermal growth factor receptor." Eur J Biochem 269(16): 3881-3887. Bache, K. G., C. Raiborg, A. Mehlum and H. Stenmark (2003). "STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes." J Biol Chem 278(14): 12513-12521. Baietti, M. F., Z. Zhang, E. Mortier, A. Melchior, G. Degeest, A. Geeraerts, Y. Ivarsson, F. Depoortere, C. Coomans, E. Vermeiren, P. Zimmermann and G. David (2012). "Syndecan-syntenin-ALIX regulates the biogenesis of exosomes." Nat Cell Biol 14(7): 677-685. Bailey, T. A., H. Luan, E. Tom, T. A. Bielecki, B. Mohapatra, G. Ahmad, M. George, D. L. Kelly, A. Natarajan, S. M. Raja, V. Band and H. Band (2014). "A kinase inhibitor screen reveals protein kinase C-dependent endocytic recycling of ErbB2 in breast cancer cells." J Biol Chem 289(44): 30443-30458. Bao, J., I. Alroy, H. Waterman, E. D. Schejter, C. Brodie, J. Gruenberg and Y. Yarden (2000). "Threonine phosphorylation diverts internalized epidermal growth factor receptors from a degradative pathway to the recycling endosome." J Biol Chem 275(34): 26178-26186. Baselga, J. and S. M. Swain (2009). "Novel anticancer targets: revisiting ERBB2 and discovering ERBB3." Nat Rev Cancer 9(7): 463-475. Baulida, J., M. H. Kraus, M. Alimandi, P. P. Di Fiore and G. Carpenter (1996). "All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired." J Biol Chem 271(9): 52515257. Becker, K. P. and Y. A. Hannun (2003). "cPKC-dependent sequestration of membrane-recycling components in a subset of recycling endosomes." J Biol Chem 278(52): 52747-52754. Beguinot, L., R. M. Lyall, M. C. Willingham and I. Pastan (1984). "Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes." Proc Natl Acad Sci U S A 81(8): 2384-2388. Bertelsen, V., M. M. Sak, K. Breen, M. S. Rodland, L. E. Johannessen, L. M. Traub, E. Stang and I. H. Madshus (2011). "A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner." Traffic 12(4): 507-520. Bertelsen, V. and E. Stang (2014). "The Mysterious Ways of ErbB2/HER2 Trafficking." Membranes 4(3): 424-446. Blume-Jensen, P. and T. Hunter (2001). "Oncogenic kinase signalling." Nature 411(6835): 355-365. Boucrot, E., A. P. Ferreira, L. Almeida-Souza, S. Debard, Y. Vallis, G. Howard, L. Bertot, N. Sauvonnet and H. T. McMahon (2015). "Endophilin marks and controls a clathrin-independent endocytic pathway." Nature 517(7535): 460-465. Brand, T. M., M. Iida, C. Li and D. L. Wheeler (2011). "The nuclear epidermal growth factor receptor signaling network and its role in cancer." Discov Med 12(66): 419-432. Breuleux, M. (2007). "Role of heregulin in human cancer." Cell Mol Life Sci 64(18): 2358-2377. Burke, P., K. Schooler and H. S. Wiley (2001). "Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking." Mol Biol Cell 12(6): 1897-1910. 55 References Cao, Z., X. Wu, L. Yen, C. Sweeney and K. L. Carraway, 3rd (2007). "Neuregulin-induced ErbB3 downregulation is mediated by a protein stability cascade involving the E3 ubiquitin ligase Nrdp1." Mol Cell Biol 27(6): 2180-2188. Carraway, K. L., 3rd, J. L. Weber, M. J. Unger, J. Ledesma, N. Yu, M. Gassmann and C. Lai (1997). "Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine kinases." Nature 387(6632): 512-516. Carter, R. E. and A. Sorkin (1998). "Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera." J Biol Chem 273(52): 35000-35007. Casalini, P., M. V. Iorio, E. Galmozzi and S. Menard (2004). "Role of HER receptors family in development and differentiation." J Cell Physiol 200(3): 343-350. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa and Y. Nishizuka (1982). "Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters." J Biol Chem 257(13): 7847-7851. Chen, M. S., R. A. Obar, C. C. Schroeder, T. W. Austin, C. A. Poodry, S. C. Wadsworth and R. B. Vallee (1991). "Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis." Nature 351(6327): 583-586. Choudhury, A., D. K. Sharma, D. L. Marks and R. E. Pagano (2004). "Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit rab4 and perturb membrane recycling." Mol Biol Cell 15(10): 4500-4511. Clague, M. J., C. Heride and S. Urbe (2015). "The demographics of the ubiquitin system." Trends Cell Biol 25(7): 417-426. Collins, B. M., A. J. McCoy, H. M. Kent, P. R. Evans and D. J. Owen (2002). "Molecular architecture and functional model of the endocytic AP2 complex." Cell 109(4): 523-535. Corbalan-Garcia, S., J. Garcia-Garcia, J. A. Rodriguez-Alfaro and J. C. Gomez-Fernandez (2003). "A new phosphatidylinositol 4,5-bisphosphate-binding site located in the C2 domain of protein kinase Calpha." J Biol Chem 278(7): 4972-4980. Cosentino-Gomes, D., N. Rocco-Machado and J. R. Meyer-Fernandes (2012). "Cell signaling through protein kinase C oxidation and activation." Int J Mol Sci 13(9): 10697-10721. Coussens, L., P. J. Parker, L. Rhee, T. L. Yang-Feng, E. Chen, M. D. Waterfield, U. Francke and A. Ullrich (1986). "Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways." Science 233(4766): 859-866. Dekker, L. V. and P. J. Parker (1994). "Protein kinase C--a question of specificity." Trends Biochem Sci 19(2): 73-77. Diamonti, A. J., P. M. Guy, C. Ivanof, K. Wong, C. Sweeney and K. L. Carraway, 3rd (2002). "An RBCC protein implicated in maintenance of steady-state neuregulin receptor levels." Proc Natl Acad Sci U S A 99(5): 2866-2871. Duncan, M. J., J. S. Shin and S. N. Abraham (2002). "Microbial entry through caveolae: variations on a theme." Cell Microbiol 4(12): 783-791. Ehrlich, E. S., T. Wang, K. Luo, Z. Xiao, A. M. Niewiadomska, T. Martinez, W. Xu, L. Neckers and X. F. Yu (2009). "Regulation of Hsp90 client proteins by a Cullin5-RING E3 ubiquitin ligase." Proc Natl Acad Sci U S A 106(48): 20330-20335. Elkin, S. R., A. M. Lakoduk and S. L. Schmid (2016). "Endocytic pathways and endosomal trafficking: a primer." Wien Med Wochenschr 166(7-8): 196-204. Engelman, J. A. (2009). "Targeting PI3K signalling in cancer: opportunities, challenges and limitations." Nat Rev Cancer 9(8): 550-562. Engelman, J. A., K. Zejnullahu, T. Mitsudomi, Y. Song, C. Hyland, J. O. Park, N. Lindeman, C. M. Gale, X. Zhao, J. Christensen, T. Kosaka, A. J. Holmes, A. M. Rogers, F. Cappuzzo, T. Mok, C. Lee, B. E. Johnson, L. C. Cantley and P. A. Janne (2007). "MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling." Science 316(5827): 1039-1043. Evans, J. H., D. Murray, C. C. Leslie and J. J. Falke (2006). "Specific translocation of protein kinase Calpha to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain." Mol Biol Cell 17(1): 56-66. Ferguson, S. M., A. Raimondi, S. Paradise, H. Shen, K. Mesaki, A. Ferguson, O. Destaing, G. Ko, J. Takasaki, O. Cremona, O. T. E and P. De Camilli (2009). "Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits." Dev Cell 17(6): 811-822. Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C. Mello (1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans." Nature 391(6669): 806-811. Flint, A. J., R. D. Paladini and D. E. Koshland, Jr. (1990). "Autophosphorylation of protein kinase C at three separated regions of its primary sequence." Science 249(4967): 408-411. 56 References Ford, M. G., I. G. Mills, B. J. Peter, Y. Vallis, G. J. Praefcke, P. R. Evans and H. T. McMahon (2002). "Curvature of clathrin-coated pits driven by epsin." Nature 419(6905): 361-366. Fortian, A., L. K. Dionne, S. H. Hong, W. Kim, S. P. Gygi, S. C. Watkins and A. Sorkin (2015). "Endocytosis of Ubiquitylation-Deficient EGFR Mutants via Clathrin-Coated Pits is Mediated by Ubiquitylation." Traffic 16(11): 1137-1154. Frolov, A., K. Schuller, C. W. Tzeng, E. E. Cannon, B. C. Ku, J. H. Howard, S. M. Vickers, M. J. Heslin, D. J. Buchsbaum and J. P. Arnoletti (2007). "ErbB3 expression and dimerization with EGFR influence pancreatic cancer cell sensitivity to erlotinib." Cancer Biol Ther 6(4): 548-554. Fry, W. H., C. Simion, C. Sweeney and K. L. Carraway, 3rd (2011). "Quantity control of the ErbB3 receptor tyrosine kinase at the endoplasmic reticulum." Mol Cell Biol 31(14): 3009-3018. Fuchs, R., S. Schmid and I. Mellman (1989). "A possible role for Na+,K+-ATPase in regulating ATPdependent endosome acidification." Proc Natl Acad Sci U S A 86(2): 539-543. Fukazawa, R., T. A. Miller, Y. Kuramochi, S. Frantz, Y.-D. Kim, M. A. Marchionni, R. A. Kelly and D. B. Sawyer (2003). "Neuregulin-1 protects ventricular myocytes from anthracycline-induced apoptosis via erbB4-dependent activation of PI3-kinase/Akt." Journal of Molecular and Cellular Cardiology 35(12): 1473-1479. Gallagher, H., A. V. Oleinikov, C. Fenske and D. J. Newman (2004). "The adaptor disabled-2 binds to the third psi xNPxY sequence on the cytoplasmic tail of megalin." Biochimie 86(3): 179-182. Garrett, T. P., N. M. McKern, M. Lou, T. C. Elleman, T. E. Adams, G. O. Lovrecz, M. Kofler, R. N. Jorissen, E. C. Nice, A. W. Burgess and C. W. Ward (2003). "The crystal structure of a truncated ErbB2 ectodomain reveals an active conformation, poised to interact with other ErbB receptors." Mol Cell 11(2): 495-505. Ghosh, R. N., W. G. Mallet, T. T. Soe, T. E. McGraw and F. R. Maxfield (1998). "An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells." J Cell Biol 142(4): 923-936. Goh, L. K., F. Huang, W. Kim, S. Gygi and A. Sorkin (2010). "Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor." J Cell Biol 189(5): 871-883. Goodman, O. B., Jr., J. G. Krupnick, F. Santini, V. V. Gurevich, R. B. Penn, A. W. Gagnon, J. H. Keen and J. L. Benovic (1996). "Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2adrenergic receptor." Nature 383(6599): 447-450. Gould, C. M., N. Kannan, S. S. Taylor and A. C. Newton (2009). "The chaperones Hsp90 and Cdc37 mediate the maturation and stabilization of protein kinase C through a conserved PXXP motif in the Cterminal tail." J Biol Chem 284(8): 4921-4935. Grant, B. D. and J. G. Donaldson (2009). "Pathways and mechanisms of endocytic recycling." Nat Rev Mol Cell Biol 10(9): 597-608. Grovdal, L. M., E. Stang, A. Sorkin and I. H. Madshus (2004). "Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation." Exp Cell Res 300(2): 388-395. Guertin, D. A., D. M. Stevens, C. C. Thoreen, A. A. Burds, N. Y. Kalaany, J. Moffat, M. Brown, K. J. Fitzgerald and D. M. Sabatini (2006). "Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1." Dev Cell 11(6): 859-871. Gurevich, V. V. and E. V. Gurevich (2006). "The structural basis of arrestin-mediated regulation of Gprotein-coupled receptors." Pharmacol Ther 110(3): 465-502. Haglund, K. and I. Dikic (2012). "The role of ubiquitylation in receptor endocytosis and endosomal sorting." J Cell Sci 125(Pt 2): 265-275. Haglund, K., S. Sigismund, S. Polo, I. Szymkiewicz, P. P. Di Fiore and I. Dikic (2003). "Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation." Nat Cell Biol 5(5): 461-466. Harari, D., E. Tzahar, J. Romano, M. Shelly, J. H. Pierce, G. C. Andrews and Y. Yarden (1999). "Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase." Oncogene 18(17): 2681-2689. Harari, D. and Y. Yarden (2000). "Molecular mechanisms underlying ErbB2/HER2 action in breast cancer." Oncogene 19(53): 6102-6114. Haslekas, C., K. Breen, K. W. Pedersen, L. E. Johannessen, E. Stang and I. H. Madshus (2005). "The inhibitory effect of ErbB2 on epidermal growth factor-induced formation of clathrin-coated pits correlates with retention of epidermal growth factor receptor-ErbB2 oligomeric complexes at the plasma membrane." Mol Biol Cell 16(12): 5832-5842. 57 References Henley, J. R., E. W. Krueger, B. J. Oswald and M. A. McNiven (1998). "Dynamin-mediated internalization of caveolae." J Cell Biol 141(1): 85-99. Henne, W. M., H. Stenmark and S. D. Emr (2013). "Molecular mechanisms of the membrane sculpting ESCRT pathway." Cold Spring Harb Perspect Biol 5(9). Hinshaw, J. E. and S. L. Schmid (1995). "Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding." Nature 374(6518): 190-192. Holmes, W. E., M. X. Sliwkowski, R. W. Akita, W. J. Henzel, J. Lee, J. W. Park, D. Yansura, N. Abadi, H. Raab, G. D. Lewis and et al. (1992). "Identification of heregulin, a specific activator of p185erbB2." Science 256(5060): 1205-1210. Hommelgaard, A. M., M. Lerdrup and B. van Deurs (2004). "Association with membrane protrusions makes ErbB2 an internalization-resistant receptor." Mol Biol Cell 15(4): 1557-1567. Honing, S., D. Ricotta, M. Krauss, K. Spate, B. Spolaore, A. Motley, M. Robinson, C. Robinson, V. Haucke and D. J. Owen (2005). "Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2." Mol Cell 18(5): 519-531. Hopkins, C. R., K. Miller and J. M. Beardmore (1985). "Receptor-mediated endocytosis of transferrin and epidermal growth factor receptors: a comparison of constitutive and ligand-induced uptake." J Cell Sci Suppl 3: 173-186. Hu, S., Y. Sun, Y. Meng, X. Wang, W. Yang, W. Fu, H. Guo, W. Qian, S. Hou, B. Li, Z. Rao, Z. Lou and Y. Guo (2015). "Molecular architecture of the ErbB2 extracellular domain homodimer." Oncotarget 6(3): 1695-1706. Huang, F. and A. Sorkin (2005). "Growth factor receptor binding protein 2-mediated recruitment of the RING domain of Cbl to the epidermal growth factor receptor is essential and sufficient to support receptor endocytosis." Mol Biol Cell 16(3): 1268-1281. Huang, Z., B. K. Choi, K. Mujoo, X. Fan, M. Fa, S. Mukherjee, N. Owiti, N. Zhang and Z. An (2015). "The E3 ubiquitin ligase NEDD4 negatively regulates HER3/ErbB3 level and signaling." Oncogene 34(9): 1105-1115. Hug, H. and T. F. Sarre (1993). "Protein kinase C isoenzymes: divergence in signal transduction?" Biochemical Journal 291(2): 329-343. Humphrey, P. A., L. M. Gangarosa, A. J. Wong, G. E. Archer, M. Lund-Johansen, R. Bjerkvig, O. D. Laerum, H. S. Friedman and D. D. Bigner (1991). "Deletion-mutant epidermal growth factor receptor in human gliomas: effects of type II mutation on receptor function." Biochem Biophys Res Commun 178(3): 1413-1420. Hunter, T., N. Ling and J. A. Cooper (1984). "Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane." Nature 311(5985): 480483. Hyatt, D. C. and B. P. Ceresa (2008). "Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells." Exp Cell Res 314(18): 3415-3425. Idkowiak-Baldys, J., A. Baldys, J. R. Raymond and Y. A. Hannun (2009). "Sustained receptor stimulation leads to sequestration of recycling endosomes in a classical protein kinase C- and phospholipase D-dependent manner." J Biol Chem 284(33): 22322-22331. Itoh, T., S. Koshiba, T. Kigawa, A. Kikuchi, S. Yokoyama and T. Takenawa (2001). "Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis." Science 291(5506): 1047-1051. Jimenez de Asua, L. and M. Goin (1992). "Prostaglandin F2 alpha decreases the affinity of epidermal growth factor receptors in Swiss mouse 3T3 cells via protein kinase C activation." FEBS Lett 299(3): 235-238. Kaksonen, M., C. P. Toret and D. G. Drubin (2005). "A modular design for the clathrin- and actinmediated endocytosis machinery." Cell 123(2): 305-320. Kalia, M., S. Kumari, R. Chadda, M. M. Hill, R. G. Parton and S. Mayor (2006). "Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery." Mol Biol Cell 17(8): 3689-3704. Katzmann, D. J., G. Odorizzi and S. D. Emr (2002). "Receptor downregulation and multivesicularbody sorting." Nat Rev Mol Cell Biol 3(12): 893-905. Kazazic, M., V. Bertelsen, K. W. Pedersen, T. T. Vuong, M. V. Grandal, M. S. Rodland, L. M. Traub, E. Stang and I. H. Madshus (2009). "Epsin 1 is involved in recruitment of ubiquitinated EGF receptors into clathrin-coated pits." Traffic 10(2): 235-245. Kelly, B. T., S. C. Graham, N. Liska, P. N. Dannhauser, S. Honing, E. J. Ungewickell and D. J. Owen (2014). "Clathrin adaptors. AP2 controls clathrin polymerization with a membrane-activated switch." Science 345(6195): 459-463. 58 References Kelly, B. T., A. J. McCoy, K. Spate, S. E. Miller, P. R. Evans, S. Honing and D. J. Owen (2008). "A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex." Nature 456(7224): 976-979. Kerr, M. C. and R. D. Teasdale (2009). "Defining Macropinocytosis." Traffic 10(4): 364-371. Kirkham, M., A. Fujita, R. Chadda, S. J. Nixon, T. V. Kurzchalia, D. K. Sharma, R. E. Pagano, J. F. Hancock, S. Mayor and R. G. Parton (2005). "Ultrastructural identification of uncoated caveolinindependent early endocytic vehicles." J Cell Biol 168(3): 465-476. Kishimoto, A., K. Mikawa, K. Hashimoto, I. Yasuda, S. Tanaka, M. Tominaga, T. Kuroda and Y. Nishizuka (1989). "Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain)." J Biol Chem 264(7): 4088-4092. Klapisz, E., I. Sorokina, S. Lemeer, M. Pijnenburg, A. J. Verkleij and P. M. van Bergen en Henegouwen (2002). "A ubiquitin-interacting motif (UIM) is essential for Eps15 and Eps15R ubiquitination." J Biol Chem 277(34): 30746-30753. Kurokawa, H., A. E. Lenferink, J. F. Simpson, P. I. Pisacane, M. X. Sliwkowski, J. T. Forbes and C. L. Arteaga (2000). "Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells." Cancer Res 60(20): 5887-5894. Lajoie, P., L. D. Kojic, S. Nim, L. Li, J. W. Dennis and I. R. Nabi (2009). "Caveolin-1 regulation of dynamin-dependent, raft-mediated endocytosis of cholera toxin-B sub-unit occurs independently of caveolae." J Cell Mol Med 13(9B): 3218-3225. Lamaze, C., A. Dujeancourt, T. Baba, C. G. Lo, A. Benmerah and A. Dautry-Varsat (2001). "Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway." Mol Cell 7(3): 661-671. Larose, L., G. Gish, S. Shoelson and T. Pawson (1993). "Identification of residues in the beta plateletderived growth factor receptor that confer specificity for binding to phospholipase C-gamma 1." Oncogene 8(9): 2493-2499. Le, X. F., M. Marcelli, A. McWatters, B. Nan, G. B. Mills, C. A. O'Brian and R. C. Bast, Jr. (2001). "Heregulin-induced apoptosis is mediated by down-regulation of Bcl-2 and activation of caspase-7 and is potentiated by impairment of protein kinase C alpha activity." Oncogene 20(57): 8258-8269. Leahy, D. J. (2004). "Structure and function of the epidermal growth factor (EGF/ErbB) family of receptors." Adv Protein Chem 68: 1-27. Lemmon, M. A. and J. Schlessinger (1994). "Regulation of signal transduction and signal diversity by receptor oligomerization." Trends Biochem Sci 19(11): 459-463. Lemmon, M. A. and J. Schlessinger (2010). "Cell signaling by receptor tyrosine kinases." Cell 141(7): 1117-1134. Lerdrup, M., S. Bruun, M. V. Grandal, K. Roepstorff, M. M. Kristensen, A. M. Hommelgaard and B. van Deurs (2007). "Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus." Mol Biol Cell 18(9): 3656-3666. Liu, M., J. Idkowiak-Baldys, P. L. Roddy, A. Baldys, J. Raymond, C. J. Clarke and Y. A. Hannun (2013). "Sustained activation of protein kinase C induces delayed phosphorylation and regulates the fate of epidermal growth factor receptor." PLoS One 8(11): e80721. Llado, A., P. Timpson, S. Vila de Muga, J. Moreto, A. Pol, T. Grewal, R. J. Daly, C. Enrich and F. Tebar (2008). "Protein kinase Cdelta and calmodulin regulate epidermal growth factor receptor recycling from early endosomes through Arp2/3 complex and cortactin." Mol Biol Cell 19(1): 17-29. Llorente, A., A. Rapak, S. L. Schmid, B. van Deurs and K. Sandvig (1998). "Expression of mutant dynamin inhibits toxicity and transport of endocytosed ricin to the Golgi apparatus." J Cell Biol 140(3): 553-563. Lohse, M. J., J. L. Benovic, J. Codina, M. G. Caron and R. J. Lefkowitz (1990). "beta-Arrestin: a protein that regulates beta-adrenergic receptor function." Science 248(4962): 1547-1550. Longva, K. E., F. D. Blystad, E. Stang, A. M. Larsen, L. E. Johannessen and I. H. Madshus (2002). "Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies." J Cell Biol 156(5): 843-854. Lonne, G. K., L. Cornmark, I. O. Zahirovic, G. Landberg, K. Jirstrom and C. Larsson (2010). "PKCalpha expression is a marker for breast cancer aggressiveness." Mol Cancer 9: 76. Lyne, J. C., M. F. Melhem, G. G. Finley, D. Wen, N. Liu, D. H. Deng and R. Salup (1997). "Tissue expression of neu differentiation factor/heregulin and its receptor complex in prostate cancer and its biologic effects on prostate cancer cells in vitro." Cancer J Sci Am 3(1): 21-30. Madshus, I. H. and E. Stang (2009). "Internalization and intracellular sorting of the EGF receptor: a model for understanding the mechanisms of receptor trafficking." J Cell Sci 122(Pt 19): 3433-3439. 59 References Marx, C., J. M. Held, B. W. Gibson and C. C. Benz (2010). "ErbB2 trafficking and degradation associated with K48 and K63 polyubiquitination." Cancer Res 70(9): 3709-3717. Maxfield, F. R. and T. E. McGraw (2004). "Endocytic recycling." Nat Rev Mol Cell Biol 5(2): 121132. Mayor, S. and R. E. Pagano (2007). "Pathways of clathrin-independent endocytosis." Nat Rev Mol Cell Biol 8(8): 603-612. Mayor, S., J. F. Presley and F. R. Maxfield (1993). "Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process." The Journal of Cell Biology 121(6): 1257-1269. McMahon, H. T. and E. Boucrot (2011). "Molecular mechanism and physiological functions of clathrin-mediated endocytosis." Nat Rev Mol Cell Biol 12(8): 517-533. Meisner, H., B. R. Conway, D. Hartley and M. P. Czech (1995). "Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells." Mol Cell Biol 15(7): 3571-3578. Mellman, I., R. Fuchs and A. Helenius (1986). "Acidification of the endocytic and exocytic pathways." Annu Rev Biochem 55: 663-700. Mochly-Rosen, D., K. Das and K. V. Grimes (2012). "Protein kinase C, an elusive therapeutic target?" Nat Rev Drug Discov 11(12): 937-957. Mollica, J. P., J. S. Oakhill, G. D. Lamb and R. M. Murphy (2009). "Are genuine changes in protein expression being overlooked? Reassessing Western blotting." Analytical Biochemistry 386(2): 270275. Morrison, P., K. Takishima and M. R. Rosner (1993). "Role of threonine residues in regulation of the epidermal growth factor receptor by protein kinase C and mitogen-activated protein kinase." J Biol Chem 268(21): 15536-15543. Myromslien, F. D., L. M. Grovdal, C. Raiborg, H. Stenmark, I. H. Madshus and E. Stang (2006). "Both clathrin-positive and -negative coats are involved in endosomal sorting of the EGF receptor." Exp Cell Res 312(16): 3036-3048. Nabi, I. R. and P. U. Le (2003). "Caveolae/raft-dependent endocytosis." The Journal of Cell Biology 161(4): 673-677. Naslavsky, N., R. Weigert and J. G. Donaldson (2004). "Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements." Molecular Biology of the Cell 15(8): 3542-3552. Newton, A. C. (1995). "Protein Kinase C: Structure, Function, and Regulation." Journal of Biological Chemistry 270(48): 28495-28498. Newton, A. C. and D. E. Koshland, Jr. (1987). "Protein kinase C autophosphorylates by an intrapeptide reaction." J Biol Chem 262(21): 10185-10188. Nishibe, S., M. I. Wahl, P. B. Wedegaertner, J. W. Kim, S. G. Rhee and G. Carpenter (1990). "Selectivity of phospholipase C phosphorylation by the epidermal growth factor receptor, the insulin receptor, and their cytoplasmic domains." Proc Natl Acad Sci U S A 87(1): 424-428. Nishizuka, Y. (1995). "Protein kinase C and lipid signaling for sustained cellular responses." FASEB J 9(7): 484-496. Ohno, H., J. Stewart, M. C. Fournier, H. Bosshart, I. Rhee, S. Miyatake, T. Saito, A. Gallusser, T. Kirchhausen and J. S. Bonifacino (1995). "Interaction of tyrosine-based sorting signals with clathrinassociated proteins." Science 269(5232): 1872-1875. Olayioye, M. A., R. M. Neve, H. A. Lane and N. E. Hynes (2000). "The ErbB signaling network: receptor heterodimerization in development and cancer." EMBO J 19(13): 3159-3167. Orth, J. D., E. W. Krueger, S. G. Weller and M. A. McNiven (2006). "A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization." Cancer Res 66(7): 3603-3610. Ouyang, X., T. Gulliford and R. J. Epstein (1998). "The duration of phorbol-inducible ErbB2 tyrosine dephosphorylation parallels that of receptor endocytosis rather than threonine-686 phosphorylation: implications for the physiological role of protein kinase C in growth factor receptor signalling." Carcinogenesis 19(11): 2013-2019. Ouyang, X., T. Gulliford, H. Zhang, G. C. Huang and R. Epstein (1996). "Human Cancer Cells Exhibit Protein Kinase C-dependent c-erbB-2 Transmodulation That Correlates with Phosphatase Sensitivity and Kinase Activity." Journal of Biological Chemistry 271(36): 21786-21792. Pearce, L. R., D. Komander and D. R. Alessi (2010). "The nuts and bolts of AGC protein kinases." Nat Rev Mol Cell Biol 11(1): 9-22. Pearse, B. M. (1975). "Coated vesicles from pig brain: purification and biochemical characterization." J Mol Biol 97(1): 93-98. Pearse, B. M. (1976). "Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles." Proc Natl Acad Sci U S A 73(4): 1255-1259. 60 References Pearse, B. M. and M. S. Robinson (1984). "Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin." EMBO J 3(9): 1951-1957. Pedersen, N. M., I. H. Madshus, C. Haslekas and E. Stang (2008). "Geldanamycin-induced downregulation of ErbB2 from the plasma membrane is clathrin dependent but proteasomal activity independent." Mol Cancer Res 6(3): 491-500. Pelkmans, L. and A. Helenius (2002). "Endocytosis via caveolae." Traffic 3(5): 311-320. Peschard, P. and M. Park (2003). "Escape from Cbl-mediated downregulation: a recurrent theme for oncogenic deregulation of receptor tyrosine kinases." Cancer Cell 3(6): 519-523. Peters, K. G., J. Marie, E. Wilson, H. E. Ives, J. Escobedo, M. Del Rosario, D. Mirda and L. T. Williams (1992). "Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis." Nature 358(6388): 678-681. Polo, S., S. Sigismund, M. Faretta, M. Guidi, M. R. Capua, G. Bossi, H. Chen, P. De Camilli and P. P. Di Fiore (2002). "A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins." Nature 416(6879): 451-455. Praefcke, G. J. and H. T. McMahon (2004). "The dynamin superfamily: universal membrane tubulation and fission molecules?" Nat Rev Mol Cell Biol 5(2): 133-147. Pust, S., T. I. Klokk, N. Musa, M. Jenstad, B. Risberg, B. Erikstein, L. Tcatchoff, K. Liestol, H. E. Danielsen, B. van Deurs and K. Sandvig (2013). "Flotillins as regulators of ErbB2 levels in breast cancer." Oncogene 32(29): 3443-3451. Qiu, X. B. and A. L. Goldberg (2002). "Nrdp1/FLRF is a ubiquitin ligase promoting ubiquitination and degradation of the epidermal growth factor receptor family member, ErbB3." Proc Natl Acad Sci U S A 99(23): 14843-14848. Raiborg, C., K. G. Bache, D. J. Gillooly, I. H. Madshus, E. Stang and H. Stenmark (2002). "Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes." Nat Cell Biol 4(5): 394398. Raiborg, C., K. G. Bache, A. Mehlum, E. Stang and H. Stenmark (2001). "Hrs recruits clathrin to early endosomes." EMBO J 20(17): 5008-5021. Raiborg, C., B. Bremnes, A. Mehlum, D. J. Gillooly, A. D'Arrigo, E. Stang and H. Stenmark (2001). "FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes." J Cell Sci 114(Pt 12): 2255-2263. Raiborg, C. and H. Stenmark (2009). "The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins." Nature 458(7237): 445-452. Rajendran, L., H. J. Knolker and K. Simons (2010). "Subcellular targeting strategies for drug design and delivery." Nat Rev Drug Discov 9(1): 29-42. Roepstorff, K., M. V. Grandal, L. Henriksen, S. L. Knudsen, M. Lerdrup, L. Grovdal, B. M. Willumsen and B. van Deurs (2009). "Differential effects of EGFR ligands on endocytic sorting of the receptor." Traffic 10(8): 1115-1127. Roskoski, R., Jr. (2014). "The ErbB/HER family of protein-tyrosine kinases and cancer." Pharmacol Res 79: 34-74. Roux, A., K. Uyhazi, A. Frost and P. De Camilli (2006). "GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission." Nature 441(7092): 528-531. Rush, J. S., L. M. Quinalty, L. Engelman, D. M. Sherry and B. P. Ceresa (2012). "Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis." J Biol Chem 287(1): 712-722. Saffarian, S., E. Cocucci and T. Kirchhausen (2009). "Distinct dynamics of endocytic clathrin-coated pits and coated plaques." PLoS Biol 7(9): e1000191. Sak, M. M., K. Breen, S. B. Ronning, N. M. Pedersen, V. Bertelsen, E. Stang and I. H. Madshus (2012). "The oncoprotein ErbB3 is endocytosed in the absence of added ligand in a clathrin-dependent manner." Carcinogenesis 33(5): 1031-1039. Sak, M. M., M. Szymanska, V. Bertelsen, M. Hasmann, I. H. Madshus and E. Stang (2013). "Pertuzumab counteracts the inhibitory effect of ErbB2 on degradation of ErbB3." Carcinogenesis 34(9): 2031-2038. Salzman, N. H. and F. R. Maxfield (1988). "Intracellular fusion of sequentially formed endocytic compartments." J Cell Biol 106(4): 1083-1091. Samant, R. S., P. A. Clarke and P. Workman (2014). "E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells." Proc Natl Acad Sci U S A 111(18): 6834-6839. Sandvig, K., S. Pust, T. Skotland and B. van Deurs (2011). "Clathrin-independent endocytosis: mechanisms and function." Curr Opin Cell Biol 23(4): 413-420. 61 References Schlossman, D. M., S. L. Schmid, W. A. Braell and J. E. Rothman (1984). "An enzyme that removes clathrin coats: purification of an uncoating ATPase." J Cell Biol 99(2): 723-733. Seabra, M. C., E. H. Mules and A. N. Hume (2002). "Rab GTPases, intracellular traffic and disease." Trends in Molecular Medicine 8(1): 23-30. Shih, S. C., D. J. Katzmann, J. D. Schnell, M. Sutanto, S. D. Emr and L. Hicke (2002). "Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis." Nat Cell Biol 4(5): 389-393. Sigismund, S., E. Argenzio, D. Tosoni, E. Cavallaro, S. Polo and P. P. Di Fiore (2008). "Clathrinmediated internalization is essential for sustained EGFR signaling but dispensable for degradation." Dev Cell 15(2): 209-219. Sigismund, S., T. Woelk, C. Puri, E. Maspero, C. Tacchetti, P. Transidico, P. P. Di Fiore and S. Polo (2005). "Clathrin-independent endocytosis of ubiquitinated cargos." Proc Natl Acad Sci U S A 102(8): 2760-2765. Simons, K. and E. Ikonen (1997). "Functional rafts in cell membranes." Nature 387(6633): 569-572. Singh, B., G. Carpenter and R. J. Coffey (2016). "EGF receptor ligands: recent advances." F1000Res 5. Sipeki, S., E. Bander, P. J. Parker and A. Farago (2006). "PKCalpha reduces the lipid kinase activity of the p110alpha/p85alpha PI3K through the phosphorylation of the catalytic subunit." Biochem Biophys Res Commun 339(1): 122-125. Sizeland, A. M. and A. W. Burgess (1992). "Anti-sense transforming growth factor alpha oligonucleotides inhibit autocrine stimulated proliferation of a colon carcinoma cell line." Mol Biol Cell 3(11): 1235-1243. Slamon, D. J., G. M. Clark, S. G. Wong, W. J. Levin, A. Ullrich and W. L. McGuire (1987). "Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene." Science 235(4785): 177-182. Sonnichsen, B., S. De Renzis, E. Nielsen, J. Rietdorf and M. Zerial (2000). "Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11." J Cell Biol 149(4): 901-914. Sorkin, A. and G. Carpenter (1993). "Interaction of activated EGF receptors with coated pit adaptins." Science 261(5121): 612-615. Sorkin, A., P. P. Di Fiore and G. Carpenter (1993). "The carboxyl terminus of epidermal growth factor receptor/erbB-2 chimerae is internalization impaired." Oncogene 8(11): 3021-3028. Sorkin, A. and L. K. Goh (2009). "Endocytosis and intracellular trafficking of ErbBs." Experimental Cell Research 315(4): 683-696. Sousa, L. P., I. Lax, H. Shen, S. M. Ferguson, P. De Camilli and J. Schlessinger (2012). "Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane." Proc Natl Acad Sci U S A 109(12): 4419-4424. Stang, E., F. D. Blystad, M. Kazazic, V. Bertelsen, T. Brodahl, C. Raiborg, H. Stenmark and I. H. Madshus (2004). "Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits." Mol Biol Cell 15(8): 3591-3604. Stein, B. S., K. G. Bensch and H. H. Sussman (1984). "Complete inhibition of transferrin recycling by monensin in K562 cells." J Biol Chem 259(23): 14762-14772. Stein, R. A. and J. V. Staros (2000). "Evolutionary analysis of the ErbB receptor and ligand families." J Mol Evol 50(5): 397-412. Steinberg, S. F. (2008). "Structural basis of protein kinase C isoform function." Physiol Rev 88(4): 1341-1378. Sundborger, A., C. Soderblom, O. Vorontsova, E. Evergren, J. E. Hinshaw and O. Shupliakov (2011). "An endophilin-dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling." J Cell Sci 124(Pt 1): 133-143. Sweitzer, S. M. and J. E. Hinshaw (1998). "Dynamin undergoes a GTP-dependent conformational change causing vesiculation." Cell 93(6): 1021-1029. Takai, Y., A. Kishimoto, M. Inoue and Y. Nishizuka (1977). "Studies on a cyclic nucleotideindependent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum." J Biol Chem 252(21): 7603-7609. Takai, Y., A. Kishimoto, Y. Iwasa, Y. Kawahara, T. Mori and Y. Nishizuka (1979). "Calciumdependent activation of a multifunctional protein kinase by membrane phospholipids." J Biol Chem 254(10): 3692-3695. Tan, M., P. Li, M. Sun, G. Yin and D. Yu (2006). "Upregulation and activation of PKC alpha by ErbB2 through Src promotes breast cancer cell invasion that can be blocked by combined treatment with PKC alpha and Src inhibitors." Oncogene 25(23): 3286-3295. 62 References Tanaka, Y., M. V. Gavrielides, Y. Mitsuuchi, T. Fujii and M. G. Kazanietz (2003). "Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway." J Biol Chem 278(36): 33753-33762. Tebar, F., T. Sorkina, A. Sorkin, M. Ericsson and T. Kirchhausen (1996). "Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits." J Biol Chem 271(46): 2872728730. Tebbutt, N., M. W. Pedersen and T. G. Johns (2013). "Targeting the ERBB family in cancer: couples therapy." Nat Rev Cancer 13(9): 663-673. Tikhomirov, O. and G. Carpenter (2000). "Geldanamycin induces ErbB-2 degradation by proteolytic fragmentation." J Biol Chem 275(34): 26625-26631. Tikhomirov, O. and G. Carpenter (2003). "Identification of ErbB-2 kinase domain motifs required for geldanamycin-induced degradation." Cancer Res 63(1): 39-43. Todaro, G. J., J. E. De Larco and S. Cohen (1976). "Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells." Nature 264(5581): 26-31. Todderud, G., M. I. Wahl, S. G. Rhee and G. Carpenter (1990). "Stimulation of phospholipase Cgamma 1 membrane association by epidermal growth factor." Science 249(4966): 296-298. Tonetti, D. A., W. Gao, D. Escarzaga, K. Walters, A. Szafran and J. S. Coon (2012). "PKCalpha and ERbeta Are Associated with Triple-Negative Breast Cancers in African American and Caucasian Patients." Int J Breast Cancer 2012: 740353. Traub, L. M. (2009). "Tickets to ride: selecting cargo for clathrin-regulated internalization." Nat Rev Mol Cell Biol 10(9): 583-596. Traub, L. M. and J. S. Bonifacino (2013). "Cargo recognition in clathrin-mediated endocytosis." Cold Spring Harb Perspect Biol 5(11): a016790. Uhlik, M. T., B. Temple, S. Bencharit, A. J. Kimple, D. P. Siderovski and G. L. Johnson (2005). "Structural and evolutionary division of phosphotyrosine binding (PTB) domains." J Mol Biol 345(1): 1-20. Ullrich, A. and J. Schlessinger (1990). "Signal transduction by receptors with tyrosine kinase activity." Cell 61(2): 203-212. Ungewickell, E., H. Ungewickell, S. E. Holstein, R. Lindner, K. Prasad, W. Barouch, B. Martin, L. E. Greene and E. Eisenberg (1995). "Role of auxilin in uncoating clathrin-coated vesicles." Nature 378(6557): 632-635. Urbe, S., M. Sachse, P. E. Row, C. Preisinger, F. A. Barr, G. Strous, J. Klumperman and M. J. Clague (2003). "The UIM domain of Hrs couples receptor sorting to vesicle formation." J Cell Sci 116(Pt 20): 4169-4179. Urtreger, A. J., M. G. Kazanietz and E. D. Bal de Kier Joffe (2012). "Contribution of individual PKC isoforms to breast cancer progression." IUBMB Life 64(1): 18-26. van der Bliek, A. M. and E. M. Meyerowitz (1991). "Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic." Nature 351(6325): 411-414. Vanhaesebroeck, B., L. Stephens and P. Hawkins (2012). "PI3K signalling: the path to discovery and understanding." Nat Rev Mol Cell Biol 13(3): 195-203. Vigers, G. P., R. A. Crowther and B. M. Pearse (1986). "Location of the 100 kd-50 kd accessory proteins in clathrin coats." EMBO J 5(9): 2079-2085. Vuong, T. T., C. Berger, V. Bertelsen, M. S. Rodland, E. Stang and I. H. Madshus (2013). "Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes." Exp Cell Res 319(3): 32-45. Wang, Z., L. Zhang, T. K. Yeung and X. Chen (1999). "Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation." Mol Biol Cell 10(5): 16211636. Ward, C. W., M. C. Lawrence, V. A. Streltsov, T. E. Adams and N. M. McKern (2007). "The insulin and EGF receptor structures: new insights into ligand-induced receptor activation." Trends Biochem Sci 32(3): 129-137. Welsh, J. B., G. N. Gill, M. G. Rosenfeld and A. Wells (1991). "A negative feedback loop attenuates EGF-induced morphological changes." J Cell Biol 114(3): 533-543. Wigge, P., K. Kohler, Y. Vallis, C. A. Doyle, D. Owen, S. P. Hunt and H. T. McMahon (1997). "Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis." Mol Biol Cell 8(10): 2003-2015. Wiley, H. S. (2003). "Trafficking of the ErbB receptors and its influence on signaling." Exp Cell Res 284(1): 78-88. 63 References Xu, W., M. Marcu, X. Yuan, E. Mimnaugh, C. Patterson and L. Neckers (2002). "Chaperonedependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu." Proc Natl Acad Sci U S A 99(20): 12847-12852. Xu, W., E. Mimnaugh, M. F. Rosser, C. Nicchitta, M. Marcu, Y. Yarden and L. Neckers (2001). "Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90." J Biol Chem 276(5): 3702-3708. Xu, W., X. Yuan, Z. Xiang, E. Mimnaugh, M. Marcu and L. Neckers (2005). "Surface charge and hydrophobicity determine ErbB2 binding to the Hsp90 chaperone complex." Nat Struct Mol Biol 12(2): 120-126. Yamazaki, T., K. Zaal, D. Hailey, J. Presley, J. Lippincott-Schwartz and L. E. Samelson (2002). "Role of Grb2 in EGF-stimulated EGFR internalization." J Cell Sci 115(Pt 9): 1791-1802. Yarden, Y. and G. Pines (2012). "The ERBB network: at last, cancer therapy meets systems biology." Nat Rev Cancer 12(8): 553-563. Yarden, Y. and M. X. Sliwkowski (2001). "Untangling the ErbB signalling network." Nat Rev Mol Cell Biol 2(2): 127-137. Young, S., P. J. Parker, A. Ullrich and S. Stabel (1987). "Down-regulation of protein kinase C is due to an increased rate of degradation." Biochem J 244(3): 775-779. Zaremba, S. and J. H. Keen (1983). "Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats." J Cell Biol 97(5 Pt 1): 1339-1347. Zhang, D., M. X. Sliwkowski, M. Mark, G. Frantz, R. Akita, Y. Sun, K. Hillan, C. Crowley, J. Brush and P. J. Godowski (1997). "Neuregulin-3 (NRG3): a novel neural tissue-enriched protein that binds and activates ErbB4." Proc Natl Acad Sci U S A 94(18): 9562-9567. Zhang, X., J. Gureasko, K. Shen, P. A. Cole and J. Kuriyan (2006). "An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor." Cell 125(6): 1137-1149. Zhou, P., N. Fernandes, I. L. Dodge, A. L. Reddi, N. Rao, H. Safran, T. A. DiPetrillo, D. E. Wazer, V. Band and H. Band (2003). "ErbB2 degradation mediated by the co-chaperone protein CHIP." J Biol Chem 278(16): 13829-13837. 64