Recruitment and activation of PLCγ1 in T cells: a new insight into old domains
2006; Springer Nature; Volume: 25; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7600978
ISSN1460-2075
AutoresAlex Braiman, Mira Barda‐Saad, Connie L. Sommers, Lawrence E. Samelson,
Tópico(s)CAR-T cell therapy research
ResumoArticle9 February 2006free access Recruitment and activation of PLCγ1 in T cells: a new insight into old domains Alex Braiman Alex Braiman Search for more papers by this author Mira Barda-Saad Mira Barda-Saad Search for more papers by this author Connie L Sommers Connie L Sommers Search for more papers by this author Lawrence E Samelson Corresponding Author Lawrence E Samelson Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Alex Braiman Alex Braiman Search for more papers by this author Mira Barda-Saad Mira Barda-Saad Search for more papers by this author Connie L Sommers Connie L Sommers Search for more papers by this author Lawrence E Samelson Corresponding Author Lawrence E Samelson Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Author Information Alex Braiman, Mira Barda-Saad, Connie L Sommers and Lawrence E Samelson 1 1Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA *Corresponding author. Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 37, Room 2066, Bethesda, MD 20892-4254, USA. Tel.: +1 301 496 9683; Fax: +1 301 496 8479; E-mail: [email protected] The EMBO Journal (2006)25:774-784https://doi.org/10.1038/sj.emboj.7600978 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Engagement of the T-cell antigen receptor leads to recruitment of phospholipase Cγ1 (PLCγ1) to the LAT-nucleated signaling complex and to PLCγ1 activation in a tyrosine phosphorylation-dependent manner. The mechanism of PLCγ1 recruitment and the role of PLCγ1 Src homology (SH) domains in this process remain incompletely understood. Using a combination of biochemical methods and real-time fluorescent imaging, we show here that the N-terminal SH2 domain of PLCγ1 is necessary but not sufficient for its recruitment. Either the SH3 or C-terminal SH2 domain of PLCγ1, with the participation of Vav1, c-Cbl and Slp76, are required to stabilize PLCγ1 recruitment. All three PLCγ1 SH domains are required for phosphorylation of PLCγ1 Y783, which is critical for enzyme activation. These novel findings entailed revision of the currently accepted model of PLCγ1 recruitment and activation in T lymphocytes. Introduction Ligation of the T-cell antigen receptor (TCR) initiates a cascade of molecular events leading to T-cell activation, cellular proliferation and cytokine production. Proximal events that immediately follow TCR engagement include activation of protein kinases and phosphorylation of multiple enzymes and adaptor molecules (Burack et al, 2002; Samelson, 2002). The phosphorylation of LAT, a lipid raft-associated adaptor protein, creates docking sites for Src-homology 2 (SH2) domain-containing proteins (Zhang et al, 1998). Slp76, another adaptor protein phosphorylated following TCR engagement (Wu and Koretzky, 2004), is recruited to LAT through its constitutive interaction with a small adaptor protein Gads (Liu et al, 2001). The LAT–Gads–Slp76 complex creates a platform for the recruitment of multiple signaling molecules, including phospholipase Cγ1 (PLCγ1), the adaptors Grb2 and Nck, the Rho-family GTPase exchange factor Vav, the adaptor with ubiquitin-ligase activity c-Cbl, and the Tec-family kinase Itk (Zhang et al, 1998, 2000; Bunnell et al, 2000; Lewis et al, 2001; Tybulewicz et al, 2003; Wu and Koretzky, 2004). Recruitment and activation of PLCγ1 is a key step in the T-cell activation process triggered by the TCR (Desai et al, 1990; Bonvini et al, 2003). The activated enzyme hydrolyzes phosphatidylinositol-4,5-bisphosphate to inositol-1,4,5-trisphosphate (IP3), which stimulates the release of Ca2+ from intracellular stores, and diacylglycerol, which activates protein kinase C and RasGRP-dependent signaling pathways (Katan, 1998; Ebinu et al, 2000; Rhee, 2001). The increase in intracellular free Ca2+ concentration triggered by IP3 plays a crucial role in the induction of numerous T-cell activation-associated responses (Desai et al, 1990). Two forms of PLCγ have been identified. PLCγ1 is ubiquitously expressed, whereas PLCγ2 is limited to certain cell types primarily of hematopoietic lineage. T cells express predominantly the PLCγ1 form (Katan, 1998). A common structural feature of all PLCs is a split catalytic domain comprised of two conserved subdomains. In PLCγ, these subdomains are separated by a regulatory region, which includes two SH2 domains followed by a single Src-homology 3 (SH3) domain (Katan, 1998; Rhee, 2001). PLCγ1 contains at least five potential sites of tyrosine phosphorylation. Among them only Y775 and Y783, located between the C-terminal SH2 (SH2C) domain and the SH3 domain, are essential for enzyme activation in vivo (Irvin et al, 2000; Rhee, 2001; Serrano et al, 2005). Despite the vital function of PLCγ1 in T cells and the ubiquitously pivotal role of PLCs in signal transduction in general, the mechanism of PLCγ1 activation following TCR engagement is incompletely understood. Several proteins participating in formation of the TCR-proximal signaling complex have been implicated in the regulation of PLCγ1 activity. Impaired Ca2+ mobilization has been reported in Jurkat cell lines deficient in LAT (Finco et al, 1998; Zhang et al, 2000), Slp76 (Yablonski et al, 1998), Vav1 (Cao et al, 2002), as well as in c-Cbl-deficient (Naramura et al, 1998) or Vav-deficient thymocytes (Reynolds et al, 2002) and Vav-deficient (Costello et al, 1999) or Itk-deficient T cells (Liu et al, 1998; Schaeffer et al, 1999). LAT has been identified as a primary docking site for PLCγ1 following TCR engagement (Zhang et al, 2000; Zhu et al, 2003). The recruitment of PLCγ1 to LAT occurs through specific binding of the N-terminal SH2 (SH2N) domain of PLCγ1 to the phosphorylated Y132 (pY132) of LAT (Stoica et al, 1998; Zhang et al, 2000). Mutations of either SH2N of PLCγ1 or Y132 of LAT abrogated PLCγ1–LAT association and inhibited PLCγ1 phosphorylation and activation (Stoica et al, 1998; Irvin et al, 2000; Zhang et al, 2000; Lin and Weiss, 2001; Paz et al, 2001; Zhu et al, 2003). No other in vivo binding partners for either LAT-pY132 or the SH2N domain of PLCγ1 have been identified in T cells to date. On the other hand, the SH2C domain of PLCγ1 is substantially less selective than the SH2N domain. A chimeric protein consisting of GST fused to the SH2C domain is able to precipitate a range of phosphorylated proteins from activated Jurkat T cells (Stoica et al, 1998; Irvin et al, 2000; Bonvini et al, 2003). However, it is still unclear which of these proteins interact with PLCγ1 via its SH2C domain in vivo. The loss-of-function mutation of the SH2C domain (R694K) did not affect PLCγ1 interaction with LAT and was reported to have a mild to negligible effect on the PLCγ1 phosphorylation in Jurkat T cells (Stoica et al, 1998; Irvin et al, 2000). Yet, expression of SH2C-mutated PLCγ1 failed to reverse the impaired IL-2 transcriptional activation in Jurkats expressing low levels of PLCγ1 (Irvin et al, 2000) or rescue IP3 production in PLCγ-deficient B cells (DeBell et al, 1999). The active conformation of PLCγ1 involves intramolecular association of the SH2C domain with phosphorylated Y783 (pY783) (Poulin et al, 2005). Without this association the enzyme is only moderately active. Thus, it has been suggested that, although required for the PLCγ1 activity, the SH2C domain is dispensable for PLCγ1 recruitment and phosphorylation (Irvin et al, 2000; Bonvini et al, 2003). The SH3 domain of PLCγ1 has been shown to interact constitutively with c-Cbl in T cells (Rellahan et al, 2003). Several studies have demonstrated that the SH3 domain of PLCγ1 can also bind other proline-rich domain-containing proteins, including Slp76 (Yablonski et al, 2001), Sos (Kim et al, 2000), Itk (Perez-Villar and Kanner, 1999) and PIKE (Ye et al, 2002). The significance of these interactions and their occurrence in vivo following TCR engagement remains unclear. Mutation of the SH3 domain has been reported to have no effect on PLCγ1 phosphorylation (DeBell et al, 1999). Moreover, in Jurkat cells expressing low levels of endogenous PLCγ1, the SH3-mutated PLCγ1 was able to recover impaired IL-2 transcriptional activation more efficiently than wild-type (WT) PLCγ1 (Irvin et al, 2000). It has been hypothesized that the SH3 domain is dispensable for PLCγ1 phosphorylation and may negatively regulate PLCγ1 activity in T cells (Irvin et al, 2000; Bonvini et al, 2003; Rellahan et al, 2003). To summarize, the currently accepted model of PLCγ1 regulation in T cells postulates that the SH2N domain of PLCγ1 is both necessary and sufficient for its recruitment and phosphorylation following TCR engagement, whereas the SH2C and SH3 domains of PLCγ1 are dispensable for this purpose. Our current study contradicts both these propositions. Using a combination of biochemical methods and real-time fluorescent imaging, we show here that the SH2N domain of PLCγ1 is necessary but not sufficient for its recruitment to the LAT-nucleated complex. Furthermore, all three SH domains of PLCγ1 are required for the efficient phosphorylation and activation of PLCγ1 in T cells. In addition, the results of this study contribute new information on the role of other signaling proteins in the process of PLCγ1 activation. Thus, we propose a different model of PLCγ1 regulation in T cells that can account for both our new findings and previously reported data. Results To visualize the recruitment of PLCγ1 following TCR stimulation, we have expressed full-length PLCγ1 fused to the monomeric version of yellow fluorescent protein (Zacharias et al, 2002) (PLCwt-YFP) in WT Jurkat E6 T cells. Our imaging analysis was based on a previously published technique (Bunnell et al, 2002). In this protocol, T cells were dropped onto a surface coated with a stimulatory monoclonal antibody binding the TCR. This resulted in TCR clustering and recruitment of multiple signaling molecules to the points of contact with the stimulatory surface (Bunnell et al, 2002; Barda-Saad et al, 2005). Following the initial engagement, the cells spread out on the planar surface over a period of 2–3 min for the Jurkat cells and up to 30 min for human peripheral blood lymphocytes (Bunnell et al, 2002; Barda-Saad et al, 2005). Similar clusters of signaling molecules have recently been observed upon T-cell contact with lipid bilayers containing antigen–MHC complexes (Campi et al, 2005). Stimulation of E6 cells stably expressing PLCwt-YFP resulted in formation of PLCγ1 clusters within seconds of the initial contact. The cluster formation continued during the cell spreading, then gradually diminished with eventual dissipation within 4–6 min after the beginning of the process (Figure 1A; Supplementary video 1). To evaluate whether the clustering of PLCwt-YFP represented its recruitment to signaling complexes, we have created a Jurkat E6 cell line expressing both PLCwt-YFP with either LAT or Slp76 fused with the Cerulean variant of cyan fluorescent protein (Rizzo et al, 2004) (LAT-CFP and Slp76-CFP, respectively). It has been shown previously that upon cell stimulation, LAT and Slp76 form clusters that colocalize with the sites of tyrosine phosphorylation, with the TCR and with other molecules recruited to the TCR-proximal signaling complex (Bunnell et al, 2002; Barda-Saad et al, 2005; Campi et al, 2005). As is evident from Figure 1B and C, the PLCwt-YFP clusters colocalize completely with LAT-CFP and Slp76-CFP, indicating that the PLCwt-YFP clustering represents recruitment of PLCγ1 to the LAT-nucleated signaling complex. Figure 1.Visualization of PLCγ1 recruitment to signaling clusters in Jurkat E6 cells. (A) Jurkat E6 cells expressing PLCwt-YFP were seeded on stimulatory coverslips. Images obtained 0.5, 2 and 5 min into the spreading process are shown. The size bar corresponds to 5 μm. (B) Jurkat E6 cells expressing LAT-CFP and PLCwt-YFP were seeded on stimulatory coverslips. Images obtained 1.5 min into the spreading process are shown. The size bar corresponds to 5 μm. (C) Same as (B) for cells expressing Slp76-CFP and PLCwt-YFP. (D) Jurkat E6 cells expressing mutant PLCγ1-YFP conjugates as indicated were seeded on stimulatory coverslips. Images obtained 1–1.5 min into the spreading process are shown. The size bar corresponds to 5 μm. (E) Murine primary CD4+ T cells expressing PLCγ1-YFP conjugates as indicated were seeded on stimulatory coverslips and fixed 5 min into the spreading process. The size bar corresponds to 5 μm. Download figure Download PowerPoint To assess the role of its SH domains in the process of PLCγ1 recruitment, we have created Jurkat E6 cell lines expressing PLCγ1-YFP conjugates bearing the previously described loss-of-function point mutations in the SH3 domain (SH3*-YFP), the SH2C domain (SH2C*-YFP) or the SH2N domain (SH2N*-YFP) (Stoica et al, 1998; Irvin et al, 2000). The mutation of the SH2N domain abrogated the TCR-induced clustering of PLCγ1 (Figure 1D), consistent with the notion that the SH2N domain serves as a binding site for LAT and, as such, is necessary for PLCγ1 recruitment to the LAT-nucleated complex. The mutations of either the SH3 or the SH2C domain did not affect the clustering of PLCγ1 (Figure 1D). It has been suggested that these domains may negatively regulate PLCγ1 activity, possibly through binding of c-Cbl, and as such may be responsible for the dissipation of the clusters (Stoica et al, 1998; Bonvini et al, 2003; Rellahan et al, 2003). Therefore, disruption of both these domains might enhance or prolong the clustering of PLCγ1. We have created an additional PLCγ1-YFP variant bearing mutations in both the SH3 and the SH2C domains (SH3*SH2C*-YFP). Surprisingly, however, SH3*SH2C*-YFP completely failed to cluster, similar to SH2N*-YFP (Figure 1D). The clusters were undetectable at all stages of cell spreading, suggesting that the inhibition of clustering was absolute and not relative to the duration of TCR stimulation. Similar results were obtained using primary murine CD4+ T cells transiently expressing the PLCγ1-YFP forms mentioned above (Figure 1E). To confirm the results obtained using the imaging technique, we evaluated the interaction of the various PLCγ1-YFP forms with LAT by precipitating the YFP conjugates with an anti-GFP serum and blotting with an antibody recognizing the phosphorylated Y132 residue (pY132) of LAT. Because maximal PLCγ1 phosphorylation is observed by 1 min after stimulation under similar conditions (Houtman et al, 2005), 1-min stimulation was chosen for this and other biochemical experiments in this study. In agreement with the results described above, phospho-LAT co-precipitated with the clustering conjugates—PLCwt-YFP, SH3*-YFP and SH2C*-YFP (Figure 2A and B). However, no co-precipitation of LAT was observed with the nonclustering SH2N*-YFP and SH3*SH2C*-YFP (Figure 2A and B). This result further supports the conclusion that the observed clusters of PLCγ1-YFP (Figure 1) represent recruitment of PLCγ1 to LAT and that the concomitant mutation of both the SH3 and SH2C domains block this recruitment. These results indicate that contrary to the currently accepted model, the SH2N domain alone is not sufficient for the PLCγ1 recruitment to the LAT-nucleated cluster. Although the SH3 and the SH2C domains, taken separately, are dispensable for the PLCγ1 recruitment, at least one of them is required in combination with the SH2N domain to maintain the association of PLCγ1 with activated LAT. Figure 2.Effect of SH domain mutations on PLCγ1 recruitment and phosphorylation. (A) Jurkat E6 cells (1 × 107) expressing the indicated PLCγ1-YFP variants were either stimulated with C305 for 1 min or left unstimulated. The cells were lysed and PLCγ1-YFP conjugates were precipitated with anti-GFP. The precipitates were resolved on SDS–PAGE and probed with anti-phospho-LAT-pY132 (upper blot). The same membrane was stripped and probed with anti-GFP (lower blot). The data are representative of three independent experiments. (B) Quantitative analysis of blots obtained in experiments represented by (A). The data represent densitometry of LAT-pY132 bands as a percentage of band intensity obtained in cells expressing PLCwt-YFP. Each bar represents an average over four independent experiments. Error bars indicate s.e. (C) Jurkat E6 cells expressing the indicated PLCγ1-YFP variants were either stimulated with OKT3 for 1 min or left unstimulated and lysed. The lysates were resolved on SDS–PAGE and probed with either anti-phospho-PLCγ1-pY783 (upper blot) or anti-PLCγ1 (lower blot). The data are representative of five independent experiments. (D) Quantitative analysis of blots obtained in experiments in (C). The data represent densitometry of PLCγ1-YFP-pY783 bands as a percentage of endogenous PLCγ1-pY783 band intensity in corresponding lanes. The results were normalized to account for the relative amounts of PLCγ1-YFP and endogenous PLCγ1 in each cell line. Each bar represents an average over five independent experiments. Error bars indicate s.e. Download figure Download PowerPoint It has been reported previously that mutations of either the SH3 or SH2C domains had little or no effect on the general tyrosine phosphorylation of PLCγ1 (Stoica et al, 1998; DeBell et al, 1999; Irvin et al, 2000). Yet, these findings did not correlate well with the functional impact of the mutations on downstream signaling. PLCγ1 contains several tyrosine residues that can be phosphorylated upon stimulation. However, only two of them, Y783 and the recently discovered Y775, are necessary for enzyme activation (Irvin et al, 2000; Rhee, 2001; Serrano et al, 2005). Therefore, we have assessed the effect of the SH domain mutations on phosphorylation of these critical tyrosine residues using specific antibodies binding these phosphorylated sites—anti-pY783 (Figure 2C and D) and anti-pY775 (not shown). The ability to distinguish the endogenous PLCγ1 and the PLCγ1-YFP conjugates as separate bands on Western blots provided a clear advantage for analyzing these results. Comparison between the level of phosphorylation of the YFP conjugates with that of the endogenous PLCγ1 in the same cells allowed more accurate assessment of the effect produced by the mutants and prevented skewed results because of a possible general effect of the mutants on the stimulation. The results clearly demonstrate (Figure 2C and D) that upon stimulation PLCwt-YFP is phosphorylated to the same extent as endogenous PLCγ1, ruling out the possibility of YFP interfering with phosphorylation. However, the loss-of-function mutation in either of the three SH domains strongly inhibits phosphorylation of Y783. Moreover, despite the fact that SH3*-YFP and SH2C*-YFP are recruited to the LAT-nucleated signaling complex, Y783 phosphorylation in these mutants is inhibited more severely than that in SH2N*-YFP, which is not recruited (Figure 2C and D). Virtually identical phosphorylation pattern was obtained using anti-pY775 (not shown). Note that the mutations did not interfere with the ability of the proteins to act as a substrate for tyrosine kinases, as pervanadate stimulation induced Y783 phosphorylation in all PLC-YFP conjugates at levels comparable with those obtained in endogenous PLCγ1 (Supplementary Figure S1). These results suggest that both the SH3 and SH2C domains are indispensable for the efficient phosphorylation and activation of PLCγ1 and that PLCγ1 recruitment to the signaling complex alone is not sufficient for this purpose. The deleterious effect of the SH domain mutations on recruitment and activating phosphorylation of PLCγ1 might be explained by disruption of PLCγ1 interactions with other signaling proteins normally mediated by these domains. Precipitation of PLCwt-YFP from the stimulated E6 cells reveals four clear bands of tyrosine-phosphorylated proteins co-precipitating with PLCγ1 (Figure 3A, second lane). The 36–38 kDa band has already been identified as LAT (Figure 2A). The other three bands correspond in sizes to Slp76, Vav1 and c-Cbl, all of which have been implicated in the PLCγ1 regulation in T cells (Naramura et al, 1998; Yablonski et al, 1998; Costello et al, 1999; Cao et al, 2002; Reynolds et al, 2002; Rellahan et al, 2003). Application of antibodies specific to Slp76, phosphorylated Vav1 (pVav1) or phosphorylated c-Cbl (pc-Cbl) confirmed the identity of these bands (Figure 3B). Mutation of any of the SH domains abrogated co-precipitation of PLCγ1 with Slp-76 (Figure 3B). Consistent with the previously reported constitutive interaction between PLCγ1 and c-Cbl mediated via the SH3 domain of PLCγ1 (Rellahan et al, 2003), co-precipitation of these two proteins was completely blocked in the PLCγ1 mutants lacking a functional SH3 domain (Figure 3B and C). Precipitation of Vav1 was substantially inhibited with either SH3*-YFP or SH2C*-YFP and was undetectable with either SH2N*-YFP or SH3*SH2C*-YFP (Figure 3B and D). These results suggest that the SH domains of PLCγ1 mediate its interactions with Slp76, Vav1 and c-Cbl. The loss of these interactions might account for the impaired recruitment and phosphorylation of the SH domain mutants. Figure 3.Co-precipitation of PLCγ1-YFP variants with various signaling proteins. (A) Jurkat E6 cells (1 × 107) expressing PLCwt-YFP were either stimulated for 1 min with C305 or left unstimulated and lysed. PLCwt-YFP was precipitated with anti-GFP. The precipitates (three left lanes) and a sample from stimulated whole-cell lysate WCL (right lane) were resolved on SDS–PAGE and probed with anti-phosphotyrosine 4G10 antibody. NC lane represents a control experiment performed without cell lysate. All lanes were from the same gel. The three left lanes were obtained at equal exposure times. WCL lane was obtained at a shorter exposure time. HC, heavy chain of Ig; LC, light chain of Ig. The data are representative of three independent experiments. (B) Jurkat E6 cells (1 × 107) expressing the indicated PLCγ1-YFP variants were either stimulated with C305 for 1 min or left unstimulated. The cells were lysed and PLCγ1-YFP conjugates were precipitated with anti-GFP. The precipitates were resolved on SDS–PAGE and probed with anti-phospho-c-Cbl-pY774 (top blot). The same membrane was stripped and probed with anti-GFP (second from top blot). In a different experiment, the precipitates were resolved on SDS–PAGE and probed consecutively with anti-phospho-Vav1-pY160, anti-Slp76 and anti-GFP (lower blots from top to bottom). NS, nonspecific band. The data are representative of at least four independent experiments. (C) The data represent densitometry of phospho-c-Cbl bands from experiments in (B) as a percentage of band intensity obtained in cells expressing PLCwt-YFP. Each bar represents an average of five independent experiments. Error bars indicate s.e. (D) Same as (C) for phospho-Vav1 bands. Each bar represents an average of four independent experiments. Download figure Download PowerPoint Co-precipitation does not necessarily indicate direct interaction between the protein molecules. Therefore, we have applied the fluorescence resonance energy transfer (FRET) technique to address this issue. As FRET can occur between fluorescent species separated no further than ∼10 nm, detection of FRET between two protein molecules strongly suggests their direct interaction. Unfortunately, we have not been able to detect FRET between full-length PLCγ1-YFP and either LAT-CFP or Slp76-CFP, although direct interaction between PLCγ1 and LAT is well documented (Stoica et al, 1998; Zhang et al, 2000). This may be due to the size and structure of PLCγ1, which places YFP at a greater distance from the interacting proteins than is allowed for FRET. Therefore, we have created a truncated version of PLCγ1-YFP (PLCs-YFP) by removing the C-terminal part of the PLCγ1 catalytic domain, thereby placing YFP closer to the SH domains of PLCγ1. Figure 4 demonstrates that PLCs-YFP is recruited to the signaling clusters similarly to PLCwt-YFP. Furthermore, a FRET signal has been detected between PLCs-YFP and either LAT-CFP, c-Cbl-CFP, Vav1-CFP or Slp76-CFP (Figure 4A–D) suggesting direct interaction between these proteins. Note that FRET between PLCs-YFP and c-Cbl-CFP, in contrast to the other proteins, is not limited to the clusters, which is likely due to the constitutive nature of the PLCγ1 and c-Cbl interaction. Interestingly, the C-terminus labeled Slp76 (Slp76-CFP) exhibited substantially lower FRET efficiency with PLCs-YFP than its N-terminus labeled counterpart (CFP-Slp76) (Figure 4D and E). This sensitivity of the observed FRET efficiency to the structural variations within the protein complex suggests that the obtained FRET signal reflects actual protein interactions and is not merely due to high protein density within the cluster. Predictably, no significant FRET was observed between LAT-CFP and PLCs-YFP mutated at the SH2N domain of PLCγ1 (Figure 4F). Figure 4.FRET analysis between PLCγ1-YFP and various CFP-conjugated proteins. (A) Jurkat E6 cells expressing PLCs-YFP and LAT-CFP were seeded on a stimulatory coverslip and fixed 2 min into the spreading process. FRET efficiency values are presented in a pseudo-colored scale, with black color representing saturated pixels. The average value of FRET efficiency±s.e. obtained for a given pair is shown. The size bar corresponds to 5 μm. (B) Same as (A) for cells expressing PLCs-YFP and c-Cbl-CFP. (C) Same as (A) for cells expressing PLCs-YFP and Vav1-CFP. (D) Same as (A) for cells expressing PLCs-YFP and CFP-Slp76. (E) Same as (A) for cells expressing PLCs-YFP and Slp76-CFP. (F) Same as (A) for cells expressing PLCs-SH2N*-YFP and LAT-CFP. Download figure Download PowerPoint To further evaluate the role of Slp76 and Vav1 in PLCγ1 recruitment, we expressed PLCγ1-YFP conjugates in cells deficient in either of these proteins. Expression of PLCwt-YFP in the Slp76-deficient Jurkat J14 cells revealed strong inhibition of PLCγ1 recruitment to the signaling clusters (Figure 5A). This result was unexpected, as normal co-precipitation of PLCγ1 with LAT in the J14 cells has been previously shown (Yablonski et al, 1998). In the course of an independent study, we have discovered that the expression of c-Cbl in the J14 cells used in our experiment is substantially reduced compared to those observed in E6 cells. To assess whether the reduced level of c-Cbl can account for the impaired PLCγ1 recruitment, we have created a J14 cell line expressing c-Cbl-CFP (J14-Cbl) at a level similar to the level of c-Cbl in E6 cells (Supplementary Figure S2). Reconstitution of J14 cells with c-Cbl restored normal clustering of PLC-YFP and SH2C*-YFP (Figure 5B). Colocalization of PLCγ1 and c-Cbl clusters is worth noting. However, c-Cbl-CFP was unable to rescue the clustering of SH3*-YFP (Figure 5B) observed in the WT Jurkats (Figure 1D). These results suggest that the SH3-mediated interaction of PLCγ1 with c-Cbl (Rellahan et al, 2003) stabilizes PLCγ1 recruitment to the signaling complex. Although reconstitution of the J14 cells with c-Cbl restored recruitment of PLCγ1, it was unable to rescue PLCγ1 activity (Figure 5C). Yet, reconstitution of J14 cells with Slp76 (J14-Slp) restored calcium mobilization (Figure 5C) and PLCwt-YFP clustering (not shown) in these cells. Therefore, in the presence of Slp76, c-Cbl is either redundant for PLCγ1 activation or the low levels of c-Cbl present in J14 cells are sufficient to maintain its functionality under these conditions. Figure 5.Effect of Slp76 and c-Cbl deficiencies on PLCγ1 recruitment and activation. (A) Jurkat J14 cells expressing PLCwt-YFP were seeded on a stimulatory coverslip. An image obtained 1 min into the spreading process is shown. The size bar corresponds to 5 μm. (B) Jurkat J14 cells expressing c-Cbl-CFP and an indicated PLCγ1-YFP conjugate were seeded on stimulatory coverslips. Images obtained 1–1.5 min into the spreading process are shown. The size bar corresponds to 5 μm. (C) Time course of the response in cytosolic calcium concentration obtained from the indicated cell lines after stimulation with OKT3. AU, arbitrary units. (D) CD4+ T cells from c-Cbl KO mice expressing PLCwt-YFP were seeded on a stimulatory coverslip, fixed 5 min into the spreading process and stained with anti-pY antibody. The size bar corresponds to 5 μm. Download figure Download PowerPoint To differentiate between these two possibilities, we have transiently expressed PLCwt-YFP in CD4+ T cells derived from c-Cbl knockout (KO) mice. Contrary to the results obtained in WT CD4+ T cells (Figure 1E), PLCwt-YFP clustering was abolished in c-Cbl-deficient T cells, despite normal TCR stimulation as indicated by multiple phospho-tyrosine clusters (Figure 5D). In addition, it has been shown previously that calcium elevation in T cells from c-Cbl KO mice is strongly inhibited (Chiang et al, 2004). These results together indicate that c-Cbl at some level is required for PLCγ1 recruitment and activation. Impaired calcium mobilization has also been demonstrated in Vav1-deficient T cells (Costello et al, 1999; Cao et al, 2002; Reynolds et al, 2002). However, the general tyrosine phosphorylation of PLCγ1 was not affected by Vav1 deficiency in Jurkat T cells (Reynolds et
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