Tyrosine phosphorylation in semaphorin signalling: shifting into overdrive

review review Tyrosine phosphorylation in semaphorin signalling: shifting into overdrive Mélanie Franco & Luca Tamagnone+ Institute for Cancer Research and Treatment (IRCC), University of Torino, Candiolo, Italy The semaphorins constitute a large family of molecular signals with regulatory functions in neuronal development, angiogenesis, cancer progression and immune responses. Accumulating data indicate that semaphorins might trigger multiple signalling pathways, and mediate different and sometimes opposing effects, depending on the cellular context and the particular plexin-associated subunits of the receptor complex, which can include receptor-type or cytoplasmic tyrosine kinases such as MET, ERBB2, VEGFR2, FYN, FES, PYK2 and SRC. It has also been shown that a specific plexin can alternatively associate with different tyrosine kinase receptors, eliciting divergent signalling pathways and functional outcomes. Tyrosine phosphorylation is a pivotal post-translational protein modification that regulates intracellular signalling. Therefore, phosphorylation of tyrosines in the intracellular domain of plexins could determine or modify their interactions with additional signal transducers. Here, we discuss the potential relevance of tyrosine phosphorylation in semaphorin-induced signalling, with an emphasis on its probable role in dictating the choice between multiple pathways and functional outcomes. The identification of implicated tyrosine kinases will pave the way to target individual semaphorin-mediated functions. Keywords: kinase; plexin; receptor; semaphorin; tyrosine EMBO reports (2008) 9, 865–871. doi:10.1038/embor.2008.139 See Glossary for abbreviations used in this article. Introduction The semaphorins constitute a wide family of membrane-bound and secreted proteins that provide guidance cues for axon pathfinding and cell migration (Tamagnone & Comoglio, 2000; Zhou et al, 2008). Moreover, semaphorins are implicated in the regulation of many biological processes, such as neural development and organ morphogenesis in the embryo, and immune response, angiogenesis and invasive tumour growth in the adult. Semaphorin signalling affects cytoskeletal remodelling and integrin-dependent adhesion, Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), University of Torino, S.P. 142, 10060 Candiolo, Torino, Italy *Corresponding author. Tel: +39 (0)11 993 3204; Fax: +39 (0)11 993 3225; E-mail: luca.tamagnone@ircc.it Submitted 25 January 2008; accepted 24 June 2008; published online 25 July 2008 ©2008 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION consequently impinging on cell migration; however, it has also been implicated in the regulation of cell proliferation and apoptosis, and, recently, in cell differentiation (for reviews, see Casazza et al, 2007; Kruger et al, 2005; Tamagnone & Giordano, 2006). The main functional receptors for semaphorins are members of the plexin family (Takahashi et al, 1999; Tamagnone et al, 1999); notably, a subset of secreted semaphorins require obligate co-receptors associated with the plexins—which are known as neuropilins—although their potential role in intracellular signal transduction remains controversial. In mammals, nine plexins have been identified and divided into four subfamilies: plexin A1 to plexin A4, plexin B1 to plexin B3, plexin C1 and plexin D1. Plexins are large single-pass transmembrane molecules, the extracellular moiety of which contains conserved protein motifs, such as the ‘sema domain’ that presents a ‘β-propeller’ structure (for a comprehensive review, see Gherardi et al, 2004), the cysteine-rich MET-related-sequences or PSI motifs, and the IPT domains (Artigiani et al, 1999). These structural domains are thought to mediate protein–protein interactions, although their specific functional relevance has not yet been elucidated. The cytoplasmic region of plexins is highly conserved among the family members, and yet it is unique, owing to its lack of apparent homology with other proteins (Maestrini et al, 1996). This domain has been shown to associate with several intracellular signal transducers, eliciting multiple signalling pathways in response to semaphorin stimulation (Kruger et al, 2005; Fig 1). For instance, the intracellular domains of plexins contain GTPaseactivating protein (GAP)-like motifs that are able to interact with— and downregulate—the monomeric G-protein R-Ras (Oinuma et al, 2004). Another region in the intracellular domain of plexins has been shown to interact with Rho family GTPases such as Rnd1 and Rac (Rohm et al, 2000; Tong et al, 2007). Current data indicate that the association of these monomeric G-proteins with plexins could induce the conformational changes required to elicit their GAP activity or to allow for the recruitment of additional signal transducers. Plexins have also been found to interact with GTPaseexchanger factors (GEFs), such as the Rac-GEF FARP2 (Toyofuku et al, 2005) and PDZ-Rho-GEF (Driessens et al, 2002; Perrot et al, 2002; Swiercz et al, 2002). By contrast, the cytoplasmic domain of plexin A1 and plexin B1 can interact with p190 Rho-GAP (Barberis et al, 2005); therefore, regulation of RhoA might vary in response to semaphorin signals in different cells (Swiercz et al, 2008). This is in agreement with the common observation that plexins have EMBO reports VOL 9 | NO 9 | 2008 8 6 5 reviews Tyrosine phosphorylation in semaphorin signalling M. Franco & L. Tamagnone Glossary CDK CRMP DAP12 EGF ERBB2 FAK FES MAPK PI(3)K PLCγ PTB PYK2 RhoA Rnd1 Sema4A SH2 TREM2 VEGFR2 cyclin-dependent kinase collapsin response-mediator protein DNAX-activating protein of 12 kDa epidermal growth factor erythroblastic leukaemia viral oncogene homologue 2 focal adhesion kinase feline sarcoma oncogene mitogen-activated protein kinase phosphatidylinositol-3-kinase phospholipase-Cγ phosphotyrosine-binding proline-rich tyrosine kinase 2 Ras homologue gene family member A Rho family GTPase 1 semaphorin 4A SRC homology 2 triggering receptor expressed on myeloid cells 2 vascular endothelial growth factor receptor 2 A been found to trigger multiple intracellular pathways, sometimes leading to opposing functional effects. In fact, semaphorin signals are often mediated by multimeric receptor complexes that contain additional transmembrane subunits associated with plexins. In particular, receptor tyrosine kinases (RTKs) might associate with plexins on the cell surface, and have been shown to have a pivotal role in semaphorin signalling pathways (Conrotto et al, 2004; Giordano et al, 2002; Swiercz et al, 2004, 2008; Toyofuku et al, 2004a; Winberg et al, 2001). Moreover, in dendritic cells and osteoclast precursors, plexin A1 can be found in a complex with the transmembrane protein known as TREM2, which is, in turn, associated with the immunoreceptor tyrosine-based activation motif (ITAM) transducer, DAP12 (Takegahara et al, 2006), responsible for activating the intracellular tyrosine kinase SYK. Notably, certain semaphorins might also signal through receptors that are distinct from plexins. For instance, in the immune system, Sema4A and Sema4D might use the alternative low-affinity receptors TIM2 and CD72, respectively (Kikutani & Kumanogoh, 2003; Kumanogoh & Kikutani, 2001). CD72 controls tyrosine phosphorylation cascades in B cells B Semaphorin Semaphorin Plexin Plexin RTK RTK Integrin Integrin α β TK β R-Ras P P GA TK α β R-Ras P Integrin TK P P -GT Rac -GTP Rnd GTPase regulators GTPase regulators P GA α P -GT Rac -GTP Rnd P TK CDK5 PI(3)K PI(3)K PI(3)K AKT AKT AKT Rho/Rac PI(3)K AKT Rho/Rac CRMP Fig 1 | Tyrosine kinases at the crossroads of semaphorin signalling pathways. Semaphorin receptors can elicit multiple intracellular signalling cascades to control axon guidance, cell migration and invasive growth (reviewed by Kruger et al, 2005; Zhou et al, 2008). Notably, the cytoplasmic tail of plexins might become tyrosine phosphorylated by either RTKs or cytoplasmic tyrosine kinases, indicating further regulatory mechanisms that have not been characterized (see text and Table 1 for details and specific references). (A) Semaphorin receptor complexes often include plexin-associated RTKs. For example, plexin B1 and plexin A1 have been found in association with tyrosine kinases such as MET, RON, ERBB2, VEGFR2 or OTK (off-track kinase) in a cell-specific manner. On semaphorin binding, these RTKs become activated, resulting in a differential regulation of cell migration, invasive growth and morphogenesis. (B) The intracellular domain of plexins might also associate with cytoplasmic tyrosine kinases implicated in signal transduction. For example, on Sema4D stimulation, endothelial cell chemotaxis might require integrin-dependent activation of the kinases PYK2 and SRC, triggering the PI(3)K/AKT pathway. Moreover, in different neuronal populations, the tyrosine kinases FAK, FYN and FER/FPS regulate neurite outgrowth in response to Sema3A and Sema3B. Therefore, semaphorin signals control cytoskeletal dynamics, integrin function, axon guidance, cell migration and invasive growth, leading sometimes to opposing functional effects, due to the activation of distinct pathways in a cell-specific manner. CDK5, cyclin-dependent kinase 5; CRMP, collapsin response-mediator protein; ERBB2, erythroblastic leukaemia viral oncogene homologue 2; FAK, focal adhesion kinase; FER, feline sarcoma oncogene; GAP, GTPase-activating protein; PI(3)K, phosphatidylinositol-3-kinase; PYK2, proline-rich tyrosine kinase 2; RND, Rho family GTPase 1; RTK, receptor tyrosine kinase; Sema4D, semaphorin 4D; SRC, sarcoma viral oncogene homologue; TK, tyrosine kinase; VEGFR2, vascular endothelial growth factor receptor 2. 8 6 6 EMBO reports VOL 9 | NO 9 | 2008 ©2008 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION reviews Tyrosine phosphorylation in semaphorin signalling M. Franco & L. Tamagnone Table 1 | Semaphorin signalling pathways that implicate associated tyrosine kinases Semaphorin Receptor complex Cell type Activity References Sema3A Plexin A1*/NRP1/FES-FPS or FER DRG neurons, COS Growth-cone collapse Mitsui et al, 2002; Shapovalova et al, 2007 Plexin A2*/NRP1/SRC family kinases DRG neurons, HEK293 cells Growth-cone collapse Sasaki et al, 2002 Plexin/SRC family kinases Cortical neurons Neurite formation, dendritic branching Morita et al, 2006 NRP1/L1/FAK kinases Cortical neurons Growth-cone collapse Bechara et al, 2008 Sema3B NRP2/NrCAM (?)/FAK–SRC family kinases Commissural neurons Axon outgrowth attraction (or repulsion/collapse) Falk et al, 2005 Sema4D Plexin B1*/MET or RON Epithelial cells and carcinoma cells, COS, NIH3T3 Increased cell migration, invasive growth Giordano et al, 2002; Conrotto et al, 2004 Plexin B1*/SRC/PYK2 HUVEC Increased migration Basile et al, 2005 Plexin B1/MET HUVEC Increased migration, angiogenesis Conrotto et al, 2005 Plexin B1*/MET MDA-MB 468 RhoA inhibition, inhibited migration Swiercz et al, 2008 Plexin B1*/ERBB2 MCF-7 RhoA activation, increased migration Swiercz et al, 2008 Plexin B1*/ERBB2 HEK293 cells, PC12, hippocampal neurons RhoA activation, growth-cone collapse Swiercz et al, 2004 Sema5A Plexin B3/MET NIH3T3, HUVEC Increased cell migration (or cell collapse) Artigiani et al, 2004 Sema6B Reverse signalling through Sema6B cyto-tail/SRC COS Unknown Eckhardt et al, 1997 Sema6D Plexin A1/OTK Endocardiac cells of the ventricle region Inhibition of migration, cell repulsion Toyofuku et al, 2004a Plexin A1/VEGFR2 Endocardiac cells of the CT segment Increased cell migration, invasive growth Toyofuku et al, 2004a Plexin A1–TREM2–DAP12/SYK Dendritic cells Cell differentiation Takegahara et al, 2006 Reverse signalling through Sema6D cyto-tail/ABL (plexin A1 ectodomain) Myocardiac cells Increased cell migration, invasive growth Toyofuku et al, 2004b Asterisks indicate plexins reported to become tyrosine phosphorylated. CT, conotruncal; DAP12, DNAX-activating protein of 12 kDa; DRG, dorsal root ganglion; ERBB2, erythroblastic leukaemia viral oncogene homologue 2; FAK, focal adhesion kinase; Fes, feline sarcoma oncogene; HUVEC, human umbilical vein endothelial cell; NrCAM, neuron-glia-related cell-adhesion molecule; NRP, neuropilin; OTK, off-track kinase; PYK2, proline-rich tyrosine kinase 2; RhoA, Ras homologue gene family member A; Sema, semaphorin; Trem2, triggering receptor expressed on myeloid cells 2; Src, sarcoma viral oncogene homologue; VEGFR2, vascular endothelial growth factor receptor 2. through the regulation of the associated tyrosine phosphatase SHP1. Finally, Sema7A might interact in trans with β1-integrin and trigger the activation of FAK and MAPK signalling cascades (Pasterkamp et al, 2003; Suzuki et al, 2007). Tyrosine kinases at the crossroads of semaphorin pathways Tyrosine phosphorylation is a pivotal post-translational protein modification that regulates intracellular signalling in response to several extracellular signals, which are either receptor ligands or extracellular matrix components. It is mediated by tyrosine kinases, which can be subdivided into receptor type (transmembrane) and non-receptor type (cytoplasmic and commonly membrane associated). Numerous cytoplasmic and receptor-type tyrosine kinases are implicated in controlling integrin-mediated adhesion, cytoskeletal dynamics, cell ©2008 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION migration and axon guidance, which are the main processes regulated by semaphorins. Notably, tyrosine kinase inhibitors have been shown to inhibit Sema3A-mediated and Sema3B-mediated functions in neurons (Falk et al, 2005; Morita et al, 2006; Sasaki et al, 2002). Moreover, the crucial involvement of tyrosine kinases in the signalling pathways mediated by Sema4D and Sema6D in non-neuronal cells has been shown using small-molecule inhibitors, the expression of dominant-negative constructs and RNA interference-based genesilencing approaches (Conrotto et al, 2004, 2005; Giordano et al, 2002; Swiercz et al, 2004, 2008; Toyofuku et al, 2004a,b; Table 1). As mentioned above, semaphorins might be able to trigger the activation of RTKs associated with plexins in receptor complexes on the cell surface (Fig 1A). For example, Sema4D stimulation can activate and induce tyrosine phosphorylation of MET, EMBO reports VOL 9 | NO 9 | 2008 8 6 7 reviews Sidebar A | In need of answers (i) How is the association of tyrosine kinases with plexins regulated in different cells? (ii) What are the relevant tyrosine phosphorylation sites in the cytoplasmic domain of plexins? (iii) What are the functional role(s) of plexin tyrosine phosphorylation— for example, to regulate protein conformation, as docking sites for adaptors/ transducers, or in receptor trafficking? RON and ERBB2 RTKs in different cell types (Conrotto et al, 2004; Giordano et al, 2002; Swiercz et al, 2008). Notably, it has been shown that the pattern of ERBB2 tyrosine phosphorylation induced by Sema4D is not the same as that seen upon EGF stimulation, which could point to the specific recruitment of different downstream effectors in response to these signals (Swiercz et al, 2004). Moreover, this cross-talk might be responsible for switching between different signalling pathways. For instance, Swiercz and co-workers found that the pro-migratory and anti-migratory effects observed in response to Sema4D in different epithelial cells seem to correlate with the ability of distinctive receptor complexes to regulate RhoA activity (Swiercz et al, 2008). In this regard, the activation of the plexin B1–ERBB2 receptor complex elicits RhoA activation and directional cell migration through the involvement of a plexin-associated PDZ-Rho-GEF, whereas the plexin B1–MET complex seems to mediate Rho inhibition and migration block. However, considering that MET activation in response to Sema4D might also lead to increased migration and invasive growth in other cell types (Giordano et al, 2002), the role of MET signalling in response to semaphorins remains controversial, and might be strongly dependent on the cellular context. As another example, Sema6D-mediated signals in different developing cardiac cells can induce opposing migratory effects, due to the involvement of different receptor complexes. In particular, in endocardiac cells of the conotruncal segment, the plexin A1– VEGFR2 complex mediates cell migration and invasive growth in response to Sema6D. By contrast, the migration of cardiac cells of the ventricle region, which express the plexin A1–OTK (off-track kinase) receptor complex, is inhibited in response to Sema6D (Toyofuku et al, 2004a). Notably, although OTK is an unusual RTK that is devoid of catalytic activity, VEGFR2 is known to activate intracellular signalling pathways, including PLCγ and PI(3)K/AKT (Olsson et al, 2006). The mechanisms that mediate the activation of plexin-associated tyrosine kinases in response to semaphorin stimulation have not been clearly elucidated (Sidebar A). As the association of RTKs with plexins seems to pre-date ligand binding, it has been proposed— although still not shown—that semaphorins can cluster in large receptor complexes on the cell surface, which is a process that is known to activate RTKs. Cytoplasmic tyrosine kinases have also been implicated in semaphorin signalling, and have been found to be associated with semaphorin receptors (Fig 1B). The mechanism mediating the recruitment and functional activation of these plexin-associated non-receptor tyrosine kinases is less obvious, and might be direct or indirect. The intracellular domain of plexins might contain motifs that recognize conserved protein domains such as SH2 and SH3, which are frequently found in cytoplasmic tyrosine kinases; 8 6 8 EMBO reports VOL 9 | NO 9 | 2008 Tyrosine phosphorylation in semaphorin signalling M. Franco & L. Tamagnone the conformational change after recruitment to the plexin might be sufficient to disturb the auto-inhibitory intramolecular interaction, leading to its functional activation (Moarefi et al, 1997). For example, plexin A1 and plexin A2 include proline-rich putative SH3-domain-binding sequences (Sasaki et al, 2002; M.F., unpublished observations). Notably, independent studies have shown that the intracellular tyrosine kinases FYN and FES/FPS can associate with plexins of the A-subfamily in a ligand-independent manner (Mitsui et al, 2002; Sasaki et al, 2002), and that PYK2 and SRC are recruited to the plexin B1 receptor complex, and are functionally activated in endothelial cells on Sema4D stimulation (Basile et al, 2005). Moreover, additional transmembrane molecules within semaphorin-receptor complexes might control the association and activation of intracellular tyrosine kinases; for example, the Sema3A co-receptor neuropilin 1 has been implicated in the recruitment of FES and FAK tyrosine kinases (Bechara et al, 2008; Mitsui et al, 2002). The functional role of FYN in response to Sema3A in neurons seems to implicate the activation of the CDK5 phosphorylation cascade—known to regulate cytoskeletal rearrangement and membrane endocytosis (Sasaki et al, 2002; Yamashita et al, 2007). Moreover, FES and the related kinase FER were reported to phosphorylate CRMP-associated molecules, which are substrates of CDK5 (Mitsui et al, 2002; Yamashita et al, 2007). It was shown recently that Sema3A stimulation of neurons expressing neuropilin-1/L1 receptor complexes also leads to the recruitment and activation of FAK, which is a pathway that is implicated in the turnover of integrinbased adhesions and the inhibition of axonal outgrowth (Bechara et al, 2008). Intriguingly, the same group had shown previously that FAK and SRC kinases are activated in response to Sema3B in anterior commissural neurons, the axons of which are attracted by the semaphorin (Falk et al, 2005). By contrast, FAK and SRC activation are not observed in posterior commissural axons, which are repelled by Sema3B. It seems that the addition of a specific SRC family kinase inhibitor not only abolishes the attractive response to Sema3B, but also converts it into axon repulsion (Falk et al, 2005). Therefore, FAK/SRC signalling induced by Sema3B is a crucial component of the attractive response in commissural neurons, and the kinase activity seems to tightly control the switch between opposing outcomes. Sema7A-dependent axonal outgrowth was further explained by integrin engagement, as well as activation of the FAK and MAPK signalling cascades (Pasterkamp et al, 2003). In endothelial cells, Sema4D stimulation leads to the recruitment and activation of the integrin-associated kinase PYK2, thereby eliciting the PI(3)K/AKT pathway, which is implicated in cell motility and angiogenesis (Basile et al, 2005, 2007). Therefore, the association of semaphorin signals with the activation of tyrosine kinases that regulate integrin functions, such as SRC-family kinases FAK and PYK2, as well as MET and ERBB2 (Guo et al, 2006; Trusolino et al, 2001), might represent another mechanism of controlling integrin-mediated adhesion, in addition to the inactivation of monomeric GTPases. Cytoplasmic tyrosine kinases have also been implicated in the so-called ‘reverse’ signalling pathways mediated by the intracellular domain of transmembrane semaphorins. In particular, Toyofuku and co-workers used the ABL kinase inhibitor STI-571 to show that this tyrosine kinase is required for Sema6D/plexin A1-induced migration of endocardiac cells during heart development (Toyofuku et al, 2004b). The intracellular domain of Sema6D associates with the ABL substrate Enabled, the tyrosine phosphorylation of which is required ©2008 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION reviews Tyrosine phosphorylation in semaphorin signalling M. Franco & L. 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<..09('<<.*,5409496'4'017+/$(,65$+7'6/17/9$/+4/<4<74.<<'(,,1$/(('3$$4.04/$)5/44,$$$/(1.97'/ <.6:9(5<<$',$.03$,6'4'06$</$(465/+/64)1606$/+(,<6<,7.<.'(,/$$/(.'(4$5545/56./(499'70$/66 <.6:9(5<<5',$.0$6,6'4'0'$</9(465/+$6')69/6$/1(/<)<97.<54(,/7$/'5'$6&5.+./54./(4,,6/966'6 <.1:9(5<<$',$./3$,6'4'01$</$(465/+$7()10/6$/1(,<6<96.<6((/,*$/(4'(4$5545/$<.9(+/,1$06,(6 <.1:9(5<<6',*.03$,6'4'01$</$(4650+01()1706$/6(,)6<9*.<6((,/*3/'+''4&*.4./$<./(49,7/06/'6 <5.,945<<.4,4'073/6(4(01$+/$((65.<41()1719$0$(,<.<$.5<534,0$$/($137$5574/4+.)(499$/0('1,<(&<6($ <.((9.6<<.$,5'/33/666(0(()/74(6..+(1()1((9$/7(,<.<,9.<)'(,/1./(5(5*/(($4.4//+9.9/)'(...&.:0            Fig 2 | Conserved tyrosine residues in the cytoplasmic domain of plexin family members. Alignment of protein sequences of human plexin intracellular domains. In the consensus line (shown under the sequences), amino-acid identity is marked with asterisks and similarity is indicated by dots or columns, as assigned by the CLUSTALW algorithm. Background colours highlight residues or domains of particular interest. In total, 13 tyrosine residues (Y) are conserved in all plexins (or in all but one family member) and are shown in red; moreover, three of these residues are included in highly conserved amino-acid stretches (blue background). The positions of highly conserved tyrosines are indicated on top of the sequences, with reference to the amino-acid coordinates in plexin A1 (accession number NP-115618.2). Notably, the residues diverging from this consensus in plexin B3 and plexin A4 (underlined) are conserved between the human and mouse genes. Tyrosine residues that are conserved in at least one entire plexin subfamily are shown in blue. The three conserved arginines found to be functionally required in the GTPase-activating protein-like motifs are shown in green (Vikis et al, 2002). A grey background highlights the presumptive RhoGTPase-binding domain (Tong et al, 2008). ©2008 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 9 | NO 9 | 2008 8 6 9 reviews for semaphorin-dependent myocardial cell motility. Notably, other transmembrane semaphorins of subclass 6 (such as Sema6B) contain proline-rich SH3-domain-binding motifs that are potentially implicated in the interaction with cytoplasmic SRC-like tyrosine kinases and in their functional activation (Comoglio et al, 2004; Eckhardt et al, 1997). Potential role of plexin tyrosine phosphorylation Initial experiments had shown that plexins might become phosphorylated in tyrosine residues when overexpressed (Tamagnone et al, 1999). There is now consistent evidence that plexins might be substrates for associated tyrosine kinases, both receptor type and cytoplasmic (Table 1). Moreover, in the presence of dominant-negative mutants of ERBB2, MET and FYN that lack kinase activity, the tyrosine phosphorylation of associated plexins is strongly reduced (Giordano et al, 2002; Sasaki et al, 2002; Swiercz et al, 2004). Although the tyrosine phosphorylation of plexins is thought to exert a regulatory role in semaphorin signalling, this has not yet been specifically investigated. Tyrosine phosphorylation of plexins could provide docking sites for SH2 or PTB domains (Yaffe, 2002), which are contained in several adaptors and transducer molecules that are implicated in plexin signalling, such as SRClike tyrosine kinases (SRC, ABL, FAK and PYK2) and the regulatory subunit of PI(3)K, p85. Moreover, the tyrosine phosphorylation of plexins could modulate the conformational state of the cytoplasmic domain and allow for the recruitment of specific signal transducers. Recently, oncogenic mutations found in the cytoplasmic domain of plexin B1—and linked to loss of association with Rac1 and Rnd1—have been found to have a strong impact on plexin B1 structure and function (Tong et al, 2008; Wong et al, 2007). Therefore, plexin tyrosine phosphorylation by specific RTKs might elicit relevant conformational changes in the cytoplasmic tail, differentially regulating the accessibility of specific protein modules. Along the same lines, as plexin B1 has been previously reported to differentially regulate Rho activity through the recruitment of either a RhoGEF or a RhoGAP, one could speculate that plexin B1 tyrosine phosphorylation mediated by ERBB2 might elicit RhoA activation by favouring the accessibility of its PDZ-domain-binding sequence. However, the interaction of plexin B1 with MET could also result in Rho inhibition by promoting the association of p190 RhoGAP with the cytoplasmic tail of plexin B1. The specific tyrosine residues phosphorylated in the cytoplasmic domain of plexins in response to semaphorin stimulation have not yet been identified, although this region contains numerous conserved sites that could be targets of tyrosine kinases. Sequence alignment reveals the presence of 13 conserved tyrosine residues (Fig 2). Notably, some of these tyrosines have been found, by using high-throughput screening experiments, to be phosphorylated in living cells (www. phosphosite.org). Using the ScanSite algorithm (scansite.mit.edu), we found that certain conserved tyrosines might be substrates for known kinases (for example, SRC for Tyr 1833 in plexin A1 and LCK for Tyr 2053 in plexin B1). Moreover, some of these residues, when phosphorylated, might become docking sites for SH2- or PTB-domaincontaining proteins. For example, phosphorylated Tyr 1833 in plexin A1 and the corresponding tyrosine in plexin B1 might bind the SH2 domains found in kinases of the SRC family. Therefore, tyrosine phosphorylation of plexins triggered by semaphorin signals could mediate the recruitment of distinctive transducers through specific phosphotyrosine docking sites and/or important conformational changes in the 8 7 0 EMBO reports VOL 9 | NO 9 | 2008 Tyrosine phosphorylation in semaphorin signalling M. Franco & L. Tamagnone plexin cytoplasmic tail, thereby allowing for a switch between multiple intracellular signalling routes. Clearly, these speculative hypotheses await experimental validation. In conclusion, tyrosine kinases associated with semaphorins or semaphorin receptors—and subsequent tyrosine phosphorylation cascades—have an important role in semaphorin functions. They seem to control the switch between multiple signalling routes that mediate diverse functional outcomes. Although the tyrosine phosphorylation of plexins has been observed on semaphorin stimulation, site-directed mutagenesis experiments are required to characterize its function. Eventually, the identification of distinctive tyrosine kinases implicated in semaphorin signalling pathways might allow us to test pharmacological inhibitors that target individual semaphorin functions, and could potentially be used to modulate processes such as nerve regeneration, immune response and cancer progression. ACKNOWLEDGEMENTS The authors acknowledge all of the members of the Tamagnone laboratory for helpful discussions. Research activity carried out by M.F. and L.T. is supported by the Italian Association for Cancer Research (AIRC). REFERENCES Artigiani S, Comoglio PM, Tamagnone L (1999) Plexins, semaphorins, and scatter factor receptors: a common root for cell guidance signals? 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