A displaced PAG enhances proximal signaling and SDF-1-induced T cell migration

250 Eur. J. Immunol. 2008. 38: 250–259 Anita Posevitz-Fejfµr et al. A displaced PAG enhances proximal signaling and SDF-1-induced T cell migration Anita Posevitz-Fejf
r, Michal Šmda, Stefanie Kliche, Roland Hartig, Burkhart Schraven and Jonathan A. Lindquist Institute of Immunology, Otto-von-Guericke University, Magdeburg, Germany PAG, the phosphoprotein associated with glycosphingolipid-enriched microdomains (GEM), negatively regulates Src family kinases by recruiting C-terminal Src kinase (Csk) to the membrane, where Csk phosphorylates the inhibitory tyrosine of the Src kinases. S-acylation of a CxxC motif juxtaposed to the transmembrane domain within PAG has been proposed to be responsible for targeting PAG to the lipid rafts. Here, we present the characterization of a mutant PAG molecule lacking the palmitoylation motif. We demonstrate that the mutant protein is expressed at the plasma membrane, but does not localize within the GEM. Despite being displaced, the mutant PAG molecule still binds the Src kinase Fyn and the cytoskeletal adaptor ezrin-radixin-moesin-binding phosphoprotein of 50 kDa, becomes tyrosine-phosphorylated, and recruits Csk to the membrane. Functional characterization of the mutant shows that, unlike WT PAG, it does not block proximal TCR signaling, and surprisingly enhances stromal cell-derived factor 1 (CXCL12)-induced migration. The mutant functions by depleting Csk from the GEM fractions, as apparent by changes in the phosphorylation of the inhibitory tyrosines within the Src kinases. Indeed this mechanism is supported by RNA interference of PAG, which results in enhanced migration and Src kinase activity. Our results therefore support a functional role for the compartmentalization of Src kinases within the membrane. Introduction The fluid mosaic model [1] proposed that the plasma membrane consists of proteins floating in a sea of lipids that are arranged within a bilayer. Today, we know that Correspondence: Jonathan A. Lindquist, Institute of Immunology, Otto-von-Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany Fax. +49-391-671-5852 e-mail: Jon.Lindquist@medizin.uni-magdeburg.de Abbreviations: Csk: C-terminal Src kinase
EBP50: ezrinradixin-moesin-binding phosphoprotein of 50 kDa
GEM: glycosphingolipid-enriched microdomains
LAT: linker for activation of T cells
LAX: linker for activation of X cells
Lck: lymphocyte-specific cytoplasmic protein tyrosine kinase
MW: molecular weight
PAG: phosphoprotein associated with GEM
PLC: phospholipase C, PP2: pyrazolopyrimidin
SDF-1: stromal cell-derived factor 1
TRAP: transmembrane adaptor protein f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Received 1/9/06 Revised 30/7/07 Accepted 2/11/07 [DOI 10.1002/eji.200636664] Key words: Adaptor proteins
Chemokine receptor signaling
Lipid rafts
Negative regulation
T cell signaling although this describes the plasma membrane, it is a somewhat over-simplified description. The plasma membrane is a dynamic structure, but instead of existing as a homogeneous mixture of lipids and proteins as Singer and Nicolson [1] envisioned, it is rather heterogeneous with the composition of the inner leaflet differing from that of the outer leaflet, and even within the membrane, the lipids and proteins segregate to form microdomains. These microdomains were first identified by their resistance to solubilization in certain detergents as well as their ability to float upon sucrose gradients after centrifugation and they were found to be enriched in glycosphingolipids, cholesterol, and in certain proteins, such as Src-family kinases, small G-proteins and some transmembrane adaptors, all of which are themselves lipid-modified. They have therefore been named glycosphingolipid-enriched microdomains (GEM) or lipid rafts [2]. In T cells, these microdomains are required for www.eji-journal.eu Eur. J. Immunol. 2008. 38: 250–259 activation via the T cell receptor (TCR), as their disruption (e.g. by cholesterol depletion) renders the cells refractory to stimulation [3]. The TCR is a multimeric protein complex consisting of two polymorphic antigen-binding subunits (either ab or cd) that non-covalently associate with the nonpolymorphic CD3 complex (CD3cde2) in addition to a homodimer of f-chains [4]. These associated proteins propagate the signal of ligand binding (i.e. peptide-MHC complexes) into the cell, since the antigen-binding subunits themselves lack this ability. Signaling via the TCR is primarily mediated by immunoreceptor tyrosinebased activation motifs (ITAM), which possess the consensus sequence Yxx(L/I)x(6–8)Yxx(L/I) (where x represents any amino acid) [5]. Within the TCR complex, there exist ten ITAM that may become phosphorylated by members of the Src family of protein tyrosine kinases upon activation. Indeed, ligand binding is associated with a dramatic increase in tyrosine phosphorylation. In T cells, the predominant Src kinases are p56lck and p59fyn. Src kinases are present within the GEM and there lymphocyte-specific cytoplasmic protein tyrosine kinase (Lck) associates with the coreceptors CD4 and CD8 [6–8] while Fyn binds to the TCR itself [9]. Recently it has been proposed that dimerization of the co-receptor activates Lck, which in turn activates Fyn [10]. Phosphorylation of the ITAM then recruits the f-associated protein of 70 kDa (ZAP70), a member of the Syk family of protein tyrosine kinases, through its tandem SH2 domains, where ZAP70 is itself activated by Src kinases [11, 12]. Activated ZAP70 then phosphorylates tyrosine residues within the linker for activation of T cells (LAT), a molecule essential for T cell activation [13, 14]. LAT is a transmembrane adaptor protein (TRAP) that localizes to GEM and serves as a scaffold for the assembly of the calcium initiation complex (pLAT/ Gads/SLP-76/PLCc1/Itk/Vav) as well as binding Grb2SOS [15]. In addition to phosphorylating the ITAM and ZAP70, activated Src kinases also phosphorylate other substrates, including the phosphoprotein associated with GEM (PAG)/C-terminal Src kinase (Csk)-binding protein (Cbp)hereafter referred to as PAG) [16, 17]. Although both Lck and Fyn are able to phosphorylate PAG in vitro [16], it appears that Fyn is primarily responsible for PAG phosphorylation in vivo [18]. PAG functions as a negative regulator of Src kinases by recruiting Csk to the plasma membrane, where Csk phosphorylates the inhibitory tyrosine of the Src kinases, and thus PAG acts via a feedback mechanism that terminates T cell activation [19]. The overexpression of PAG blocks antigen receptor signaling [16, 20, 21]. This inhibitory effect is mediated via Csk recruitment, as over-expression of a PAG molecule f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Molecular immunology lacking the Csk binding site enhances TCR signaling [21]. Since PAG is one of the four TRAP known to become palmitoylated [22], we set out to investigate whether palmitoylation is required for GEM localization and/or function. To do this, we generated a mutant of PAG in which both cysteines within the palmitoylation motif (CxxC) were changed to alanine. Results Mutation of the palmitoylation motif (CxxC) does not affect PAG expression S-acylation of cysteine residues located juxtaposed to the transmembrane region of PAG is proposed to be responsible for the targeting of PAG to the GEM (Fig. 1A) [16]. To study the effect of mutation at this site, it was necessary to incorporate an epitope tag at the C terminus to distinguish the mutant PAG molecule from the endogenous protein, since PAG-deficient cell lines do not exist at present. As seen in Fig. 1, neither the inclusion of an epitope tag nor mutation of the palmitoylation motif affected PAG expression or its membrane localization. Fig. 1B demonstrates that the expression levels of both the wild-type (WT) construct and CxxC mutant are comparable to one another. Interestingly, both proteins are expressed in multiple forms with the higher-molecular-weight (MW) forms being more abundant in whole cell lysate. This pattern is also seen for endogenous PAG (Fig. 1C), suggesting that palmitoylation is not responsible for the difference in the apparent MW of the protein. Fig. 1D shows that mutation of the conserved cysteines does not affect the ability of PAG to localize to the plasma membrane. Staining of endogenous PAG shows that the bulk of the protein is present in the plasma membrane. Confocal microscopy also shows some reticular staining (lower panel), which is to be expected for membrane proteins as they are synthesized in the endoplasmic reticulum. Cells transfected with vector alone show no staining, indicating that antibody staining for the epitope tag is specific. FTRIM was included as an additional positive control, as TRIM is a non-GEM-associated member of the TRAP family that is also abundant in the plasma membrane. Both FPAG and the CxxC mutant show similar patterns of staining compared to either FTRIM or endogenous PAG. Additionally, to confirm the results obtained by microscopy, we performed membrane fractionation using differential centrifugation. The results of this separation clearly demonstrated that the bulk of either WT FPAG or the CxxC mutant was present in the membrane fraction along with endogenous LAT, whereas soluble proteins such as ERK and Grb2 were www.eji-journal.eu 251 252 Anita Posevitz-Fejfµr et al. Eur. J. Immunol. 2008. 38: 250–259 Figure 1. Expression of FPAG WT and CxxC. (A) Alignment of the first 60 amino acids of PAG is presented; dots represent conserved amino acids and hyphens indicate gaps. The sequences are human (NP_060910), mouse (NP_444412), and rat (NP_071589). Domains were predicted by SMART [43]. (B, D–G) Jurkat T cells were transfected with vector, WT FPAG, or the CxxC mutant. (B) Blots were probed with rabbit anti-FLAG to visualize expression. (C) Lysates from primary human T cells or Jurkat T cells were stained with mouse anti-PAG. (D) Fluorescence microscopy of transfected Jurkat T cells. Endogenous PAG was stained with mouse antiPAG. Confocal images are presented below. (E) Fractions 2–4 and 8–9 from sucrose density gradients represent GEM and non-GEM, respectively. The CxxC mutant shows non-GEM localization. LAT staining indicates that GEM architecture was intact. (F) Upper panel: FLAG immunoprecipitates blotted with rabbit anti-FLAG and mouse anti-phosphotyrosine-HRP are presented. Lower panel: Cells were PP2-treated prior to lysis and immunoprecipitation. Subsequent blotting with rabbit anti-FLAG and mouse antiphosphotyrosine shows the levels of expression and phosphorylation. (G) Lysates and immunoprecipitates were stained for associated proteins. Data represent at least three experiments. BOS, Vector pEF-BOS; FPAG, flag-tagged wt PAG in the pEF-BOS vector; FTRIM, flag-tagged wt TRIM in the pEF-BOS vector. f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2008. 38: 250–259 primarily found in the cytoplasmic fraction (data not shown). Mutant PAG does not localize to the GEM Having established that the mutant PAG molecule is expressed and localizes within the plasma membrane, it was then necessary to determine whether the mutated protein was still capable of being targeted to the GEM. For this purpose, cell lysates were first fractionated upon sucrose density gradients. CD59, a glycosylphosphatidylinositol-anchored protein, was used as a marker to identify the GEM fractions (data not shown). Fig. 1E clearly shows that while the WT protein does localize to the GEM fractions, mutation of the palmitoylation motif clearly ablates the targeting of PAG into these microdomains. Protein over-expression was not responsible for the lack of GEM localization, as both the positive control FPAG and endogenous LAT were not excluded from the GEM fractions; endogenous PAG was also not affected (data not shown). Therefore, we can conclude that palmitoylation of the CxxC motif is required for the targeting of PAG into the GEM. It is worth noting that as both FPAG and the CxxC mutant show two species similar to endogenous PAG [16], the higher-MW form must differ from lower by a post-translational modification other than palmitoylation. The PAG CxxC mutant is tyrosine-phosphorylated Given that the CxxC mutant of PAG is correctly targeted to the plasma membrane but does not localize within the GEM fractions, we next looked to see whether this mutant becomes tyrosine-phosphorylated, as PAG is one of the most abundant phosphoproteins in a resting T cell [16]. Fig. 1F clearly shows that both FPAG and the CxxC mutant are tyrosine-phosphorylated. Also, it appears that the 85-kDa form is more highly phosphorylated than the 66-kDa form, which suggests that tyrosine phosphorylation of PAG contributes to the observed difference in the apparent MW. The Src kinase inhibitor pyrazolopyrimidin (PP2) was included in the lysis buffer to exclude the possibility that the observed phosphorylation occurred post-lysis. To further investigate the nature of this phosphorylation, we pretreated the cells with PP2. These results demonstrated that the phosphorylation was indeed PP2-sensitive, indicating that an Src kinase was involved (Fig. 1F, lower panel). The PAG CxxC mutant recruits Csk, Fyn, and EBP50 Since the mutant PAG molecule becomes tyrosinephosphorylated, we asked whether it still binds Csk, as this interaction is known to be phosphorylationf 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Molecular immunology dependent [16, 23]. Fig. 1G clearly shows that both the WT and the CxxC mutant are capable of recruiting Csk. Binding of Csk to the epitope tag can be excluded, as FTRIM does not recruit Csk (data not shown). Additionally, we looked to see whether these molecules could also bind Fyn. PAG was first identified via its interaction with Fyn [24], Fyn binding is independent of tyrosine phosphorylation [16], and Fyn is responsible for the phosphorylation of PAG [18, 25]. Since the CxxC mutant is phosphorylated outside of the GEM, it is not surprising to find that Fyn is also associated (Fig. 1G) as there exists a pool of Fyn located outside of the GEM [10]. Since Fyn is not the only Src family kinase capable of phosphorylating PAG, we next looked to see whether we could detect Lck association, as Lck resides primarily outside of the GEM and is capable of phosphorylating PAG [16]. However, no Lck association was detected (data not shown). Additionally, an interaction between PAG and the cytoskeleton has been described that is mediated by the adaptor ezrin-radixin-moesin binding phosphoprotein of 50 kDa (EBP50) [26, 27]. Probing of FLAG-immunoprecipitates with anti-EBP50 sera demonstrated that displacement of PAG from the GEM had not disrupted this interaction. The PAG CxxC mutant does not block TCR proximal signaling Since PAG exerts its negative regulatory function by recruiting Csk to the GEM, we therefore looked to see whether the CxxC mutant was also capable of suppressing cellular activation, since it could also recruit Csk to the membrane. Since Src kinase activation occurs immediately upon TCR cross-linking, we began our analysis of receptor proximal events using phosphospecific reagents (Fig. 2A). Since the global phosphotyrosine staining appears quite similar, we hypothesized that an effect of PAG upon Src kinase activity might be more visible if we examined specific substrates. Therefore, we reprobed the blot with a phospho-specific reagent for ZAP70. Whereas WT PAG clearly possesses the ability to suppress ZAP70 activation, the CxxC mutant does not. However, this did not appear to influence LAT phosphorylation (Fig. 2A). Since LAT functions as a scaffold to build complexes that are necessary to propagate signaling, we next looked to see whether the activation of phospholipase C (PLC)c was affected. Indeed, staining with phospho-PLCc showed a reduced phosphorylation upon over-expression of WT FPAG (Fig. 2A). Interestingly, although the level of PLCc activation appears comparable between the CxxC mutant and the control, the kinetic appears quite truncated. www.eji-journal.eu 253 254 Anita Posevitz-Fejfµr et al. To see whether the altered kinetic had an effect, we next looked at intracellular calcium flux (Fig. 2B). Our results demonstrate that upon TCR stimulation, WT FPAG can partially suppress the release of calcium, whereas the CxxC mutant neither suppresses nor enhances the calcium response, leaving us to conclude that although we had displaced PAG from the GEM, we had not greatly altered TCR signaling. Figure 2. The PAG CxxC mutant enhances specific TCR proximal events. (A) Analysis of TCR proximal signaling events. Cells were stimulated for the time indicated and lysates probed with the appropriate antibodies. MW standards are indicated. LAT is indicated by the arrow. Actin staining serves as the loading control. (B) Intracellular calcium flux was measured in Jurkat T cells transfected with either vector alone (light grey line), WT FPAG (dark grey line), or the CxxC mutant (black line). Addition of anti-TCR antibody (C305) is indicated by the filled triangle and the addition of ionomycin (the positive control) is indicated by the white triangle. One experiment of at least three is shown. f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2008. 38: 250–259 The PAG CxxC mutant enhances stromal cellderived factor 1-induced migration Since Src kinases also play an important role in adhesion and migration, we next tested our mutant using a stromal cell-derived factor 1 (SDF-1)-based migration assay, as SDF-1 is known to preferentially signal from within the GEM [28]. Initial results indicate that while WT FPAG is capable of mildly suppressing SDF-1induced T cell migration (compare WT FPAG with vector alone), the CxxC mutant enhanced cellular migration (Fig. 3A). The expression levels of the TCR, chemokine receptor CXCR4, LFA-1 (CD18) and VLA-4 (CD49d) were measured by FACS and no observable differences in surface expression were detected (data not shown), thereby excluding the possibility that the enhanced migration observed was due to changes in protein expression. Analysis of the proximal signaling events following chemokine receptor stimulation further supports this observation (Fig. 3B). Cells expressing WT FPAG show a strong suppression of Src kinase activation, while cells expressing the CxxC mutant show an enhancement both in the level of basal phosphorylation (at time 0) and in response to stimulation, with the level increasing during the measurement (compare the 10 min time points). Not surprisingly, there was little effect of either construct upon ERK phosphorylation (Fig. 3B) as ERK activation is mediated via bc following its dissociation from Gai [29]. The suppression of calcium signaling observed with WT FPAG (Fig. 3C) was partially reverted by the CxxC mutation, suggesting cross-talk between the Src kinases and PLC during chemokine receptor signaling. According to the current model of T cell activation [10], Lck is first activated in the non-GEM compartment by co-receptor dimerization and then translocates into the GEM to activate Fyn. To explain the effects of the CxxC mutant, we hypothesized that over-expression of WT FPAG increases the level of Csk within the GEM, thereby creating an irreversible block in activation. The CxxC mutant could recruit Csk out of the GEM and thereby relocate the block in Src kinase activation to the non-GEM compartment. For processes involving both the GEM and non-GEM, such as TCR-mediated signaling [10], the translocation of this block may not be easily distinguished; however, for processes occurring solely within the GEM, such as SDF-1-mediated signaling, the relocation of Csk away from the GEM would have a more profound effect, since it would allow for an enhanced activation of Src kinases within the GEM. To test this hypothesis, we reprobed the GEM fractions (Fig. 1E) for Csk content. Probing of whole lysates indicated that the levels of total Csk were equivalent (Fig. 4A, left panel). Indeed, we found that www.eji-journal.eu Eur. J. Immunol. 2008. 38: 250–259 after fractionation the level of Csk within the GEM was drastically reduced by over-expression of the CxxC mutant (Fig. 4A, middle panel). Additionally, one can see that over-expression of the WT PAG construct has nearly depleted Csk from the cytosol or heavy fractions (Fig. 4A, right panel). Together these results indicate that the mechanism of action appears to be effected by a redistribution of Csk within the cell. Figure 3. PAG CxxC mutant enhances SDF-1-induced migration. (A) SDF-1-induced migration of Jurkat T cells overexpressing either WT FPAG or the CxxC mutant were measured by FACS. The migration of cells is shown relative to the stimulated control. The data shown here represent five independent experiments. A single asterisk indicates a statistical significance (p=0.04) using a paired Student's t-test. (B) Analysis of CXCR4 proximal signaling. Blots of cell lysates were stained as indicated. Actin staining serves as the loading control. (C) Intracellular calcium flux was measured in Jurkat T cells transfected with either vector alone (light grey line), WT FPAG (dark grey line) or the CxxC mutant (black line). Addition of SDF-1 is indicated by the filled triangle and the addition of ionomycin (the positive control) is indicated by the white triangle. An enlargement of the y axis is included in the inset box. Data represent at least three experiments. f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Molecular immunology If this mechanism is correct, then this should also be reflected by changes in the phosphorylation of the inhibitory tyrosines of Src kinases within the GEM and non-GEM compartments. Therefore to confirm these results, we reprobed the fractions using phospho- Figure 4. PAG CxxC mutant recruits Csk out of the GEM. Sucrose density gradient fractions 2–4, 5–7 and 8–9 were pooled. Fractions 2–4 and 8–9 represent the GEM and non-GEM fractions, respectively. Cell lysate from 1106 cells, not-fractionated, was loaded as positive controls. The samples were immunoblotted with mouse anti-Csk (A), rabbit anti-phosphoY529 or rabbit anti-phospho-Y418 and mouse anti-Fyn (B). (C) The ratio of pY529 staining to total Fyn or pY418 staining to total Fyn for both the GEM and non-GEM fractions; vector (grey), FPAG (black) and CxxC (white). Data represent averages from three experiments  SEM; *p<0.05 analyzed by ANOVA. www.eji-journal.eu 255 256 Anita Posevitz-Fejfµr et al. specific antisera for either the inhibitory tyrosine of Fyn (pY529) or the activatory tyrosine (pY418). While Fig. 4B shows no change of inhibitory tyrosine phosphorylation within the GEM fractions for cells expressing the PAG CxxC mutant, there is a marked increase of inhibitory tyrosine phosphorylation in the non-GEM fractions, whereas over-expression of WT FPAG has nearly depleted the non-GEM fractions of inhibitory tyrosine phosphorylation. Total Fyn staining shows that over-expression of the mutants has slightly altered the distribution of Fyn, which is to be expected as Fyn constitutively associates with PAG [16]. To demonstrate this more clearly, we also present the quantification of the inhibitory tyrosine phosphorylation normalized to the level of total Fyn (Fig. 4C, top panel). Here one can see that for the non-GEM fractions the ratio decreases slightly in cells expressing WT FPAG, indicative of a decrease in Csk activity, whereas cells expressing the CxxC mutant, which recruits more Csk to this fraction, show a markedly increased ratio. As expected, a corresponding trend is seen for the phosphorylation of the activatory tyrosine (Y418). In cells expressing the CxxC mutant, the autophosphorylation of Fyn is enhanced within the GEM fractions and is suppressed in the non-GEM fractions (Fig. 4C, bottom panel). To better demonstrate that the loss of PAG from the GEM is responsible for the enhanced migration observed, we turned to RNA interference. Fig. 5A clearly shows that the loss of PAG expression results in both an enhanced basal migration and a significantly enhanced specific migration to SDF-1. Indeed, when one analyzes the effect of PAG supression upon these cells; one clearly sees that the basal Src kinase activity is not only enhanced, but it is also more sustained upon SDF-1 stimulation (Fig. 5B). These results clearly indicate the importance of PAG as a negative regulator. Discussion The importance of GEM localization for proper function was demonstrated for LAT, in which mutation of cysteine residues within the palmitoylation motif completely abrogated LAT function [30, 31]. However, these results have recently been brought into question, as a linker for activation of X cells (LAX)/LAT chimera was able to restore complete function to LAT-deficient T cells [32]. LAX is one of the three transmembrane adaptors that do not localize within the GEM [22] and therefore the LAX/LAT chimera was expected to function similar to the LAT palmitoylation mutants. Here we present the characterization of a PAG palmitoylation motif mutant. Similar to the LAT mutants, this protein was correctly targeted to the f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2008. 38: 250–259 Figure 5. PAG down-regulation enhances both migration and Src kinase activation. Jurkat T cells were transfected with the plasmid pCMS3-EGFP containing PAG shRNA. (A) The downregulation of PAG protein expression compared to the control is shown in the inset box. Actin is included to show equal loading. The migration of cells is shown relative to the stimulated control. Values represent the means  SD of four independent experiments. Data were analyzed using the Student's t-test (*p=0.01). (B) Analysis of proximal signaling in response to SDF-1 stimulation is presented. Cells were stimulated with SDF-1 for the time indicated, and lysates blotted with the corresponding antibodies. One representative experiment of three is shown. membrane and did not localize within the GEM fractions. However, the PAG CxxC mutant still bound Fyn, became tyrosine-phosphorylated, and recruited Csk and EBP50 to the membrane. To determine the effect of displacing PAG from the GEM, we measured TCR-mediated proximal signaling events. Here one sees that over-expression of WT FPAG had an inhibitory effect upon the ability of ZAP70 to become activated (Fig. 2A), which is in full agreement with previously published results [16, 20, 21]. Additionally, FPAG also suppresses PLCc activation, most likely by influencing Itk activation, which is in turn reflected in a decreased calcium flux. Contrary to this, displacing PAG from the GEM appears to abrogate its inhibitory effect upon proximal signaling and calcium flux (Fig. 2). However, in the chemokine-induced migration assay the functional difference between these molecules becomes evident (Fig. 3A). While the WT FPAG suppresses activation (i.e. chemokine-induced migrawww.eji-journal.eu Eur. J. Immunol. 2008. 38: 250–259 tion), the CxxC mutant now enhances the ability of these cells to migrate. It should be noted that enhanced migration was observed only in response to the stimulus (SDF-1) and no increase in spontaneous migration was seen, indicating that it is specifically receptor-mediated events that are enhanced. The receptor for SDF-1 (CXCL12) on T cells is CXCR4, a seven-transmembrane G-protein-coupled receptor that requires GEM localization for optimal signaling [28, 33, 34]. Additionally, the signaling pathways induced by this receptor upon ligand binding are mediated in part by Src kinases [35, 36]. The enhanced migration observed in the CxxC mutant is most likely due to the enhancement of Src kinase activity that results from the recruitment of Csk to the plasma membrane, but away from the GEM where PAG normally exerts its function. In this respect, we demonstrate that the CxxC mutant steals Csk from endogenous PAG. Probing of the GEM fractions for endogenous Csk showed that the level of Csk within the cell was distributed relatively equally between the GEM and non-GEM fractions (Fig. 4A, vector control); however, over-expression of WT FPAG depleted the Csk levels in the non-GEM fractions, whereas the PAG CxxC mutant had the opposite effect by recruiting Csk into the non-GEM fractions and thereby depleting Csk from within the GEM. The changes in Csk localization were mirrored by changes in the phosphorylation of both the inhibitory and activatory tyrosines within the Src kinases (Fig. 4B). Indeed, the suppresion of PAG by RNA interference would agree with this mechanism, as Fig. 5 clearly demonstrates that in the absence of PAG, Src kinase activity is dramatically enhanced, as is the migratory capacity of these cells. Additionally, we see a slight enhancement of ERK activation upon the displacement of PAG in response to SDF-1 stimulation (Fig. 3B). This could result from an enhanced activation of ZAP70 via the adaptor SLP76 to influence ERK, although a direct influence of Src kinases upon PI3K that affects ERK cannot be excluded [37, 38]. Our proposed mechanism of action is in agreement with published results demonstrating that Csk inhibits the ability of cells to migrate in response to SDF-1 [35]. Furthermore, our results build upon previous work demonstrating the importance of Csk recruitment to the membrane [39] to show that the localization of Csk within the GEM is also critical for its negative regulatory function. Emerging from our studies is a model in which the proper segregation of signaling events within the membrane is important for cell function. By recruiting Csk to the non-GEM compartment, we are either enhancing the basal activation state of the Src kinases within the GEM or allowing a prolonged activation by removing a component of the feedback inhibition loop [19], or perhaps both. f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Molecular immunology Materials and methods Antibodies and reagents Rabbit anti-FLAG and mouse anti-FLAG (M2) were purchased from Sigma. Rabbit anti-Csk, rabbit anti-phospho-PLCc (pY783) and rabbit anti-PLCc were from Santa Cruz, mouse anti-Csk (clone 52) and mouse anti-ZAP70 from BD Bioscience, mouse anti-phosphotyrosine-HRP (PY20) from Southern Biotechnology Associates, rabbit anti-EBP50 from Abcam, rabbit anti-phospho-ZAP70 (pY319) from Cell Signaling, and rabbit anti-phospho-Y418 and rabbit anti-phosphoY529 from Biosource. Mouse anti-phosphotyrosine (4G10) was produced in our institute. Mouse anti-PAG (MEM 255), mouse anti-CD59 (MEM 43/5), mouse anti-Fyn and rabbit anti-LAT were kind gifts from Dr. Vaclav Horejsi. Rabbit anti-Fyn was kindly provided by Dr. Paul Burn and rabbit anti-Lck by Dr. Anthony Magee. Goat anti-mouse-HRP, goat anti-rabbitHRP, donkey anti-mouse-FITC and donkey anti-rabbit-FITC were obtained from Dianova. RPMI 1640 and PBS were purchased from BioChrom, FCS was from PAN Biotech, Tween-20 and TEMED were from Roth, and Igepal (NP-40), glutaraldehyde, paraformaldehyde and DabcoTM were obtained from Sigma. Acrylamide/BIS was from Bio-Rad, Page Ruler Prestained Protein Ladder from Fermentas, ECL detection reagents and nitrocellulose membrane from Amersham, HEPES from Serva, Brij 58 from Pierce, and human SDF-1 from TEBU. Constructs The following constructs were used in this study: pEF-BOS [40], FLAG-TRIM/pEF-BOS [41], FLAG-PAG/pEF-BOS [16], and FLAG-C37,40DA-PAG/pEF-BOS. The PAG mutant was generated using the QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Integrity of the construct was confirmed by DNA sequencing. Cell culture Jurkat T cells (E6.1) were maintained in RPMI 1640 medium supplemented with 10% FCS at 37 C, 5% CO2 in a humidified atmosphere. Transfection Transfection of Jurkat T cells and RNA inhibition were performed as previously described [16, 42]. Microscopy Cell pellets from 1.4105 cells were resuspended in RPMI 1640 medium to a density of 7106 cells/mL. Cell suspension was pipetted at 20 lL per spot onto a 12-spot slide (Marienfeld) and incubated at 37 C for 30 min in a humid chamber. The slide was then washed and the cells fixed (1% paraformaldehyde, 0.05% glutaraldehyde in PBS) and permeabilized (0.1% Triton X-100 in PBS) for 10 min each. Before staining the slide was blocked with 1% BSA/PBS. Cells were stained with primary antibody for 60 min in a wet chamber. www.eji-journal.eu 257 258 Eur. J. Immunol. 2008. 38: 250–259 Anita Posevitz-Fejfµr et al. After washing and additional blocking, the cells were stained with FITC-labeled secondary antibody in a wet chamber. Following additional washing, the samples were embedded in mounting media (Vectashield, Glycerol, 2% DabcoTM (1:1:1)) and the cover slip fixed to the slide. The cells were visualized using a Leica DMRE-7 white-field fluorescence microscope with a 1.4/63 objective using a Spot RT camera (Diagnostic Instruments) and the Spot RT acquisition software. Deconvolution was performed using Metamorph (version 6.1; Universal Imaging). Images were processed using the IrfanView software (version 3.95). Confocal images were acquired using a Leica DMIRE2 microscope and processed with the LSM Image browser software (Zeiss). Cell lysis After washing with cold PBS, 1106 cells were lysed in 30 lL lysis buffer (500 mM HEPES, 100 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 10 mM Na4P2O7, 1% NP-40, 1% laurylmaltoside, 1 mM PMSF) for 30 min on ice. Samples were centrifuged at 13 000 rpm for 15 min at 4 C. The post-nuclear supernatant was then heated for 5 min at 95 C with 5 reducing sample buffer. Immunoprecipitation Cells (5106) were lysed in ice-cold lysis buffer for 30 min on ice. After centrifugation, post-nuclear supernatant was taken and 60 lL anti-FLAG M2 agarose (Sigma) was added; agarose was prepared according to manufacturer's instructions. To reduce non-specific binding, 1/10 volume of 10 mg/mL BSA (Sigma) was added. The samples were incubated with rotation for 2–16 h at 4 C. After washing, the FLAG-tagged constructs were eluted by incubating with 30 lL FLAG peptide (100 lg/mL; Sigma) for 30 min at 37 C. The agarose was pelleted by centrifugation and the supernatant heated with 5 reducing sample buffer for 5 min at 95 C. Gel electrophoresis and Western blotting Lysates and immunoprecipitates were separated on 10% SDSPAGE and transferred onto nitrocellulose membranes (Hybond-C Extra, Amersham). After blocking, membranes were probed with primary antibody and the appropriate HRPconjugated secondary antibody. Samples were visualized using an ECL detection system according to the manufacturer's instructions (Amersham). GEM fractionation 37 C. The cells were washed briefly and incubated for an additional 30 min before measuring the FL4 (510/20 nm) vs. FL5 (400/40 nm) ratio on a flow cytometer (BD LSR1); measurement was gated upon the GFP-positive population. The cells were stimulated first with either anti-TCR (C305, 1:50) or SDF-1 (150 ng/mL) and finally with ionomycin (2 lg/mL). Migration assay The cells were washed in migration media (RPMI 1640, 1% BSA, 20 mM HEPES pH 7.4) and resuspended at 5106/mL. Migration medium (600 lL) was pipetted into the wells of a 24-well plate. Transwell inserts of 5 lm pore size (3421; Costar) containing 100 lL (5105 cells) of cell suspension were placed into the medium containing wells and equilibrated for 1 h at 37 C. The transwell inserts were carefully removed and chemokine added to the lower chamber to 100 ng/mL final concentration. The plate was incubated for 4 h at 37 C. Migrated cells were collected from the lower chamber and counted with a FACSCalibur (60 s). Acknowledgements: The authors would like to thank Drs. S. Lindquist and L. Simeoni for critical reading of the manuscript, Drs. V. Horejsi, P. Burn, and A. Magee for generously contributing reagents, V. Posevitz for helping with the graphics, and K. Ehrecke for technical assistance. 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