Address correspondence to V.L.J. Tybulewicz, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Phone: 4420-8913-8699; Fax: 4420-8906-4477; E-mail: vtybule{at}nimr.mrc.ac.uk
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Abstract |
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Key Words: thymus Itk Tec Rac1 SLP-76
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Introduction |
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Vav1 is a 95-kD protein first isolated as an oncogene in a 3T3 fibroblast transformation assay (2). Subsequently, two related proteins, Vav2 and Vav3, have been identified (35). Vav1 was shown to be rapidly tyrosine phosphorylated after stimulation of both the TCR and BCR (68). Analysis of its sequence showed that it contained a number of domains characteristic of signal transducing proteins (9, 10). It has a Dbl homology domain common to GDP/GTP exchange factors (GEFs)* for Rho-family GTPases. Vav1 itself was shown to be a specific GEF for Rac1, Rac2, and RhoG, and to be activated by tyrosine phosphorylation (1113). In addition Vav1 contains an array of SH3-SH2-SH3 domains, suggestive of an adaptor-like function. Its SH2 domain has been proposed to bind to a phosphotyrosine on the adaptor Src homology 2 domaincontaining leukocyte phosphoprotein (SLP)-76 which itself is part of a complex including phospholipase C (PLC)1 and the adapters linker for activation of T cell (LAT) and Gads which assembles after TCR stimulation (1417).
The first indications of the importance of Vav1 in TCR signaling came from studies in which we and others showed that TCR-induced proliferation of T cells from Vav1-deficient mice was greatly reduced, in part as a result of decreased IL-2 production (1820). Subsequently, it was found that overexpression of Vav1 in Jurkat T cells led to hyperactivation of TCR-induced activation of the transcription factor nuclear factor (NF)-AT (21). Further analysis showed that both positive and negative selection was compromised in Vav1-/- mice, consistent with Vav1 transducing TCR signals required for both of these selective events (22, 23). Biochemical studies demonstrated that in Vav1-/- CD4+ T cells, TCR-induced calcium flux, ERK activation, and induction of the NF-B transcription factor were defective (24). In addition it has been shown that Vav1-/- T cells show reduced TCR capping, as well as TCR clustering at the immunological synapse between a T cell and an antigen-presenting cell (2527).
In this study we address the mechanism by which Vav1 transduces TCR signals to the activation of an intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4+CD8+ double positive (DP) thymocytes, we show that Vav1 regulates PLC1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLC
1 and the adaptor molecule SLP-76. We show that this latter function is independent of PI3K.
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Materials and Methods |
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Stimulation of Thymocytes for Biochemical Analysis.
For all biochemical analysis, thymi were disaggregated in air-buffered (AB) IMDM. Cells were preincubated with the hamster antimouse CD3 mAb (10 µg/ml 2C11; BD PharMingen) on ice for 30 min, washed, and then incubated in AB IMDM for 5 min at 37°C before cross-linking of the antibodies with goat anti-Armenian hamster IgG antiserum (75 µg/ml; Jackson Immunoresearch Laboratories). Where PI3K inhibitors were used, thymocytes were preincubated with 100 nM wortmannin (Sigma-Aldrich) or 10 µM Ly294002 (Sigma-Aldrich) at 37°C for 30 min before standing on ice for 5 min. Preincubation with anti-CD3
on ice then proceeded as above, still in the presence of inhibitor. Subsequent cross-linking of 2C11 also occurred in the presence of inhibitor. For control samples where no inhibitor was used, an equivalent volume of DMSO carrier was added.
Immunoblotting and Immunoprecipitation.
All chemicals were obtained from Sigma-Aldrich unless otherwise indicated. Cells were typically stimulated at 2 x 108 cells per milliliter in AB IMDM for the specified times and stimulation stopped by the addition of an equal volume of 2x lysis buffer (2% n-octyl ß-D-glucopyranoside, 50 mM Tris-Cl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 20 mM NaF, 10 mM disodium pyrophosphate, 2 mM sodium orthovanadate, 1:50 [vol/vol] protease inhibitor cocktail [P8340]; Sigma-Aldrich]). Cell lysates were cleared by centrifugation at 15,340 g for 15 min at 4°C and used for immunoprecipitations or if total cytoplasmic lysates were to be analyzed directly, an equal volume of 2x Laemmli sample buffer was added before boiling for 3 min. Immunoprecipitations, immunoblotting, and SDS-PAGE were performed by standard procedures. The following antibodies were used for immunoprecipitation and immunoblotting: anti-PLC1 rabbit polyclonal Ab (sc-81; Santa Cruz Biotechnology, Inc.); anti-LAT rabbit polyclonal Ab M41 (gift from M. Turner, Babraham Institute, Babraham, UK); antiSLP-76 sheep polyclonal Ab (gift from G. Koretzky, University of Massachusetts, Worcester, MA); anti-Tec rabbit polyclonal Ab (Upstate Biotechnology). Anti-Itk rabbit polyclonal Ab (USB) was used for immunoprecipitation. The following antibodies were used for immunoblotting: anti-phosphotyrosine mAb RC20 conjugated directly to HRP (BD Transduction Labs); anti-Itk mAb 2F12 (gift from L. Berg, University of Pennsylvania, Philadelphia, PA); anti-Gads rabbit polyclonal Ab (gift from J. McGlade, Hospital for Sick Children, Toronto, Canada); anti-phosphotyrosine783-PLC
1 (Biosource International); antiphosphothreonine308-Akt, antiphosphoserine473-Akt, anti-Akt, anti-phosphotyrosine319-ZAP-70, and anti-ZAP-70 rabbit polyclonal Ab (Cell Signaling Technology); and anti-Rac1 mAb (USB). For immunoblots, antibody binding was revealed with goat antimouse IgG-HRP (Santa Cruz Biotechnology, Inc.) for mouse mAb, goat antirabbit IgG-HRP (Cell Signaling Technology) or Protein A-HRP (Amersham Pharmacia Biotech) for rabbit polyclonal Ab, and donkey antisheep IgG-HRP (Serotec) for sheep polyclonal Ab. For immunoprecipitations, immunocomplexes were isolated using protein A or protein G Plus agarose (Santa Cruz Biotechnology, Inc.) for rabbit or sheep polyclonal Ab, respectively.
For densitometric analysis the blots were scanned, bands of interest were quantitated and in-lane background was subtracted. To determine specific phosphorylation, the signal from phosphorylated bands was divided by the signal from the appropriate loading control and all values were normalized to the maximum response (set to 100%). Signals below detection were set to 0%.
Rac1 Activation Assay.
For analysis of Rac1 activation, cells were stimulated at 3 x 107 cells per milliliter, lysed by the addition of an equal volume of 2x pulldown buffer (2% Triton X-100, 300 mM NaCl, 20 mM MgCl2, 2 mM sodium orthovanadate, 100 mM NaF, 2 mM PMSF, and 20 µg/ml leupeptin), and cleared by centrifugation at 15,340 g at 4°C for 2 min followed by the addition of 0.03 vol of a slurry containing Glutathione-Sepharose beads (Amersham Pharmacia Biotech) bound to bacterially expressed GST-Pak1-RBD (fusion protein of GST with amino acids 1125 of rat Pak1, the Rac binding domain of Pak1). Samples were rotated at 4°C for 5 min, washed twice in 1x wash buffer (0.1% Triton X-100, 50 mM Tris, pH 7.5, 500 mM NaCl, 10 mM MgCl2) before elution of Gst-Pak1-RBD-bound protein using Laemmli sample buffer at 95°C.
Intracellular Calcium Analysis.
Intracellular calcium concentrations were analyzed by flow cytometry using Indo-1 loaded thymocytes as described previously, except that the cells were only stained with anti-CD3 (22). CD3
was cross-linked by the addition of goat antihamster IgG (75 µg/ml). Where required, cells were preincubated with wortmannin or Ly294002 (concentrations as above) at 37°C for 30 min before the addition of Indo-1AM. Inhibitors were present throughout the anti-CD3
prebinding and cross-linking stages.
Inositol 1,4,5-Trisphosphate Measurement.
Thymocytes were stimulated in AB IMDM (100 µl) by cross-linking of CD3 as described above. The stimulations were terminated by the addition of 15 µl ice-cold 6.1 M TCA followed by 15-min incubation on ice. The samples were centrifuged at 1,400 g, 4°C for 15 min, and the supernatant extracted with 10 vol water-saturated diethyl ether, neutralized with 10 µl 1 M NaHCO3, and the final volume of the aqueous phase was adjusted to 200 µl with water. Inositol 1,4,5-trisphosphate (IP3) was quantitated in duplicate or triplicate 100 µl samples using a competitive [3H]IP3 binding assay (NEN Life Sciences) according to the manufacturer's instructions.
Determination of Phosphatidylinositol 4,5-Bisphosphate Levels.
Pellets obtained after TCA-mediated cell lysis as above were washed with 1 M TCA, 1 mM EDTA, and then water. The pellets were extracted in methanol/chloroform/HCl (0.9 ml; 80:40:1), for 15 min at room temperature with intermittent vortexing. To this, chloroform (0.3 ml) and 0.1 M HCl (0.6 ml) was added and 0.4 ml of the lower phase was removed. After evaporation of solvent under vacuum, 1 M KOH (0.25 ml) was added, and lipids were hydrolyzed at 95°C for 15 min, which converts phosphatidylinositol 4,5-bisphosphate (PIP2) primarily to IP3. Hydrolyzed extracts were passed through Dowex columns (0.4 ml) preequilibrated with 5 ml 0.1 M HCl followed by 25 ml water. IP3 was eluted from the column in 1.25 ml of water. Ether extraction followed by neutralization with NaHCO3 and IP3 estimation was performed as above. PIP2 levels were calculated from the IP3 values as described in the Biotrak IP3 assay kit (Amersham Pharmacia Biotech).
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Results |
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A key second messenger driving intracellular calcium fluxes in lymphocytes is IP3, which is generated by the action of PLC on PIP2. As shown in Fig. 1 C, the TCR-driven production of IP3 is greatly diminished in Vav1-/- DP thymocytes relative to Vav1+/+ cells, which likely accounts for the large decrease in intracellular calcium flux. This result is similar to the reduced TCR-induced IP3 generation that we previously demonstrated in Vav1-/- CD4+ T cells (24).
The defect in IP3 production could in principle be due to a failure of PLC activation or localization, or to a shortage of its substrate PIP2. It has been proposed that the GTPase Rac1 may activate phosphatidylinositol 4-phosphate 5-kinase (PIP5K), the enzyme that generates PIP2 from its precursor phosphatidylinositol 4-phosphate (31), and thus Vav1, as a GEF for Rac1 could potentially regulate PIP5K and thus the supply of PIP2. Indeed it has been shown that in Vav1-deficient B cells, CD19-induced activation of PIP5K is defective (32). To examine if a shortage of PIP2 might account for the defective IP3 production in Vav1-/- thymocytes, we directly determined PIP2 levels in cells stimulated through the TCR. Our results show that Vav1-/- cells had levels of PIP2, at least as high if not higher than those seen in Vav1+/+ cells (Fig. 1 C). Thus it seems unlikely that the defective TCR-induced IP3 generation is caused by a lack of substrate for PLC. However since our analysis measures total cellular PIP2, we cannot exclude that there is a small pool of PIP2 used by PLC in which there is a selective shortage of the phospholipid.
Vav1 Is Required for Normal Tyrosine Phosphorylation of PLC1.
Generation of IP3 by signaling from antigen receptors is mediated by the PLC1 and 2 isoforms of the enzyme whose activation is in part mediated by tyrosine phosphorylation (33). Of these isoforms, PLC
1 is likely to be more important for TCR signaling, since, while PLC
2 is required for normal antigen receptor signaling in B cells, it appears to be dispensable for TCR signaling (34, 35). Thus we analyzed TCR-induced tyrosine phosphorylation of PLC
1 in Vav1+/+ and Vav1-/- DP thymocytes. Using anti-phosphotyrosine Ab, we saw a clear reduction in TCR-induced PLC
1 phosphorylation in Vav1-/- cells (Fig. 2
A). Three distinct sites of phosphorylation have been identified on PLC
1: tyrosines 771, 783, and 1254 (36). Mutation of tyrosine 783 (Y783) to phenylalanine in PLC
1 blocked the phosphorylation-induced activation of the enzyme, suggesting that this residue is critical for regulation of PLC
1 (36). Using antisera specific for phospho-Y783 PLC
1, we found that in Vav1-/- DP thymocytes, the TCR-induced phosphorylation of this residue is reduced, consistent with the defective activation of the lipase (Fig. 2 A).
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Vav1 Regulates the Association of PLC1 with SLP-76.
While tyrosine phosphorylation is essential for the activation of PLC1, it is likely that the enzyme is regulated in other ways as well. In particular it needs to be recruited to the plasma membrane and to associate with a protein complex containing the adaptor proteins linker for activation of T cells (LAT), Gads, and SLP-76 (17). The transmembrane adaptor protein LAT is phosphorylated on multiple tyrosine residues creating docking sites to which several other proteins are recruited via SH2 domainphosphotyrosine interactions (38). These include the Gads adaptor molecule which binds inducibly to LAT through its SH2 domain and constitutively to the cytoplasmic adaptor SLP-76 via its COOH-terminal SH3 domain (39, 40). PLC
1 appears to be recruited to this complex through at least two interactions. It binds to phosphorylated LAT in a TCR stimulation-dependent manner through its NH2-terminal SH2 domain and constitutively to SLP-76 via its SH3 domain and both interactions are required for normal TCR-induced phosphorylation (41, 42).
Since Vav1 is also thought to be recruited to this complex (via SLP-76), we investigated whether its formation might be defective in Vav1-/- DP thymocytes. We immunoprecipitated LAT from Vav1+/+ and Vav1-/- cells stimulated through the TCR and immunoblotted the precipitates with anti-PLC1, anti-Gads, and antiSLP-76 antibodies. Our results show that in DP thymocytes, TCR stimulation induces the recruitment of all three of these molecules to LAT, and that the extent of complex formation is similar in Vav1+/+ and Vav1-/- cells (Fig. 3
A). We examined the same associations in reverse by immunoprecipitating PLC
1 or SLP-76 and analyzing the precipitates for the presence of LAT. In both cases we were unable to detect LAT itself, probably due to limitations of antibody sensitivity, however using anti-phosphotyrosine antibodies we could readily see the inducible association of a 36-kD phosphoprotein (pp36) with both PLC
1 and SLP-76, which comigrated with LAT (data not shown). Our results showed that the extent of TCR-induced association of pp36/LAT with both PLC
1 and SLP-76 was similar in both Vav1+/+ and Vav1-/- cells (data not shown).
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Vav1 Is Required for TCR-induced PI3K Activation.
The phosphorylation and subsequent activation of Itk and Tec is controlled at several levels. First, the kinases are recruited to the plasma membrane. This is achieved by the binding of phosphatidylinositol 3,4,5-trisphosphate (PIP3), produced by the action of PI3K on PIP2, to the PH domain of Itk or Tec (43). Mutations in the PH domain that block binding of PIP3, or treatment of cells with inhibitors of PI3K inhibit the antigen receptorinduced activation of both kinases (44, 45). Second, it has been proposed for Itk that the inactive kinase exists in a self-inhibited state with intramolecular interactions repressing activity (46). Binding of ligands to various domains of Itk is proposed to promote unfolding of these intramolecular interactions, eventually leading to activation of the kinase after phosphorylation by a Src-family kinase.
To investigate whether the defective phosphorylation of Itk and Tec in Vav1-deficient cells might be due to a failure to activate PI3K, we analyzed the phosphorylation of the serine/threonine kinase Akt as a surrogate for PI3K activity. Akt is activated by at least three steps: membrane recruitment driven by binding of phosphatidylinositol 3,4-bisphosphate to the PH domain, phosphorylation on threonine 308 (T308) by PDK1, a PIP3-activated kinase, and finally phosphorylation on serine 473 (S473) by a kinase whose identity is unclear (47). Since phosphatidylinositol 3,4-bisphosphate is derived from PIP3 by the action of the inositol phosphatase SHIP, the first two steps in the activation of Akt are dependent on PI3K. Analysis of TCR-induced phosphorylation of Akt on T308 showed that it was readily induced in Vav1+/+ DP thymocytes, but was undetectable in Vav1-/- cells (Fig. 4 A). Furthermore, analysis of S473 phosphorylation showed that once again Vav1+/+ cells showed clear TCR-induced phospho-S473-Akt, while Vav1-/- cells were greatly diminished in this response (Fig. 4 A). Taken together these results strongly suggest that the TCR stimulationinduced activation of PI3K was defective.
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Vav1 Regulates PLC1 Activation via PI3K-dependent and -independent Pathways.
To investigate whether the role of Vav1 in transducing TCR signals to the activation of PLC1 was limited to the activation of PI3K, we compared the effects of the Vav1 mutation with those of inhibitors of PI3K (Ly294002 and wortmannin). Treatment of Vav1+/+ DP thymocytes with either inhibitor caused a small decrease in the TCR-induced calcium flux relative to untreated cells (Fig. 5
A, and data not shown). However inhibitor-treated Vav1+/+ cells still showed a much larger TCR-induced calcium flux than Vav1-/- cells (Fig. 5 A). To control for efficacy of the inhibitors we examined TCR-induced Akt phosphorylation in Vav1+/+ or Vav1-/- cells treated with Ly294002 or wortmannin. As expected, both inhibitors completely abolished TCR-induced Akt phosphorylation on S473 (data not shown). Taken together we conclude that in addition to regulating the activation of PI3K, Vav1 must also signal through a PI3K-independent pathway leading to the activation of an intracellular calcium flux.
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Finally, we examined whether the defect in complex formation between PLC1 and SLP-76 is dependent on PI3K. Treatment of Vav1+/+ DP thymocytes with Ly294002 caused no reproducible change in the TCR-inducible association of PLC
1 and SLP-76 (Fig. 5 B). In contrast Vav1-/- cells once again showed a decrease in both the constitutive and inducible associations of these molecules (Fig. 5 B). Reblotting of the same PLC
1 immunoprecipitates for the Gads adaptor protein, showed that its association with PLC
1 mirrored that of SLP-76, as would be expected given the constitutive SLP-76-Gads association. Vav1+/+ cells showed TCR-inducible association of PLC
1 and Gads which was not affected by Ly294002, whereas in Vav1-/- cells, the basal and inducible association was greatly decreased (Fig. 5 B). These observations suggest that the PI3K-independent pathway by which Vav1 transduces TCR signals to the induction of PLC
1 phosphorylation and hence intracellular calcium flux may involve regulation of the association of PLC
1 with the SLP-76-Gads adaptor molecules (Fig. 6)
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Discussion |
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We show here that after TCR stimulation, levels of PIP2 in Vav1-/- DP cells remain as high if not higher than in Vav1+/+ cells (Fig. 1 C), strongly suggesting that the defect in IP3 production is due to a failure to activate PLC1 rather than a shortage of its substrate. In contrast, it has been proposed that in Vav1-/- B cells, the defective CD19-induced calcium flux is due to a shortage of PIP2, caused by defective activation of PIP5K (32). An earlier report showed an interaction between Rac1 and PIP5K (31), thus Vav1 could regulate this lipid kinase via its GEF activity toward Rac1. However, a more recent report questions this, showing that PIP5K is regulated by the GTPase ARF6, not Rac1 (50). How can we can reconcile our data with the observed reduction in PIP5K activity in CD19-stimulated Vav1-/- B cells? One possibility is the difference in cell type and stimulus. Alternatively, we note that rather than measuring PIP2 levels, this study measured the synthesis of PIP2 after CD19 engagement and showed that it is reduced in Vav1-/- cells. If the levels of PIP2 are controlled by negative feedback, as is common in many biosynthetic pathways, then a failure of PLC
and PI3K activation would mean that PIP2 is not consumed and would not need to be replenished, thus giving rise to the observed decreased rates of PIP2 synthesis in Vav1-/- cells.
PLC1 activity is regulated at several levels. It is recruited to the plasma membrane through interactions with membrane-associated protein complexes or via binding of PIP3 to its PH domain and is phosphorylated on at least three tyrosine residues (33, 51). Our data shows Vav1 regulates TCR-induced PLC
1 phosphorylation, and this is likely to account for at least some of the defective PLC
1 activity in Vav1-/- DP cells. In contrast, we previously reported that TCR-induced PLC
1 phosphorylation was normal in Vav1-/- CD4+ splenic T cells (24). This could be due to a difference in signaling pathways used by the TCR in T cells versus DP thymocytes. However, we note that in our original studies we were hampered by the low number of T cells we could purify and the insensitivity of the antibodies being used, which resulted in a very low signal to background ratio, making it difficult to see partial decreases in PLC
1 phosphorylation. In contrast, in the current work, we have used a more sensitive anti-PLC
1 Ab for immunoprecipitation and have had access to a much larger number of cells from the DP thymocyte system, and this may account for why we see a defect in PLC
1 phosphorylation. Furthermore, the defect was also evident using antiserum specific for phospho-Y783 of PLC
1 (Fig. 2 A). Mutational analysis has shown that phosphorylation of this residue is critical for PLC
1 activation (36), so its reduction in Vav1-/- DP thymocytes is likely to at least partially account for the failure to activate the enzyme. We note, however, that the reduction in PLC
1 phosphorylation is less pronounced than that in TCR-induced IP3 production. This may be because Vav1 does something other than just regulate PLC
1 phosphorylation; as we have shown, Vav1 is required for normal PLC
1/SLP-76 association. Alternatively, the relationship between PLC
1 phosphorylation and activity may be nonlinear, if for example several tyrosines need to be phosphorylated to activate the lipase.
To understand the defect in TCR-induced PLC1 phosphorylation, we systematically studied most molecules known from genetic studies to be required for this event. While phosphorylation of ZAP-70 on Y319 and total tyrosine phosphorylation of LAT and SLP-76 was normal, the phosphorylation and hence probably activation of Itk and Tec kinases was defective. While mice defective in a single Tec-family kinase show mild defects in thymocyte development and TCR signaling, compound mutations in Itk and Rlk cause a phenotype remarkably similar to that in Vav1-/- mice (37). As in Vav1 mutants, Itk-/-, Rlk-/- mice show defective positive selection, decreased TCR-driven T cell proliferation and IL-2 production. Furthermore, biochemical analysis showed reduced TCR-induced calcium flux, IP3 production, PLC
1 phosphorylation, and ERK activation. Unfortunately, we have been unable to determine if Rlk phosphorylation is affected in Vav1-/- cells, for lack of a sufficiently sensitive antibody and the phenotype of the Itk/Tec double mutant has not yet been reported, so we cannot compare it directly to the Vav1-/- mice. However, extrapolating from the phenotype of the Itk-/-, Rlk-/- mice, it seems likely that the reduced Itk and Tec activation will contribute significantly to the observed PLC
1 phosphorylation defect. While the importance of Tec-family kinases for phosphorylation and activation of PLC
isoforms has been demonstrated in a number of studies, the precise sites of phosphorylation have not been determined. Our data suggests that tyrosine 783 of PLC
1 may be an important direct target of Tec-family kinases.
The defective activation of PI3K in Vav1-/- DP thymocytes is the most likely cause for the failure to phosphorylate Itk and Tec. Both kinases contain a PH domain through which they are recruited to the plasma membrane after binding of PIP3, and this binding is a prerequisite for phosphorylation and activation. However, we cannot exclude that Vav1 may regulate these kinases by other mechanisms. Itk has been proposed to exist in an inhibited state maintained by intramolecular interactions (46). Binding of other proteins to domains of Itk involved in these internal associations would open up the kinase allowing its activation by phosphorylation. Vav1 has been reported to bind directly to Itk and Tec, and thus may contribute to their activation by displacing these intramolecular associations (52, 53).
The requirement for Vav1 in TCR-induced activation of PI3K was unexpected, since it has been proposed that PI3K is upstream of Vav1 (54). In these studies it was shown that PIP3 binds to the PH domain of Vav1 and activates its GEF activity. Indeed a recent study has shown that inhibition of PI3K leads to a reduction in TCR-induced Vav phosphorylation (55). Taken together with our results it seems that PI3K is both upstream and downstream of Vav1, potentially creating a positive feedback loop (Fig. 6). In agreement with our conclusions, recent work from other groups has shown that Vav-family proteins may also regulate antigen receptor-induced PI3K activation in mast cells and B cells (56, 57).
It is unclear how Vav1 regulates PI3K. The effect may be direct, in view of a report showing association of Vav1 with p85, a regulatory subunit of class 1 PI3K (58, 59). Alternatively PI3K may be activated by the Rac1 GTPase, since some reports have shown association between these proteins (31, 48, 49). Interestingly, a genetic analysis of PI3K and Rac1 activation in the Jurkat T cell line concluded that in a pathway leading from TCR stimulation to Akt activation, Rac1 was upstream of PI3K whereas in the same cells, cytoskeletal rearrangements were induced by a TCR signaling pathway in which PI3K was upstream of Rac1 (60). Our results are consistent with the idea that in thymocytes, the TCR signaling pathway leading to Akt phosphorylation is mediated by Vav1 via Rac1 and then PI3K activation.
It is most likely that PI3K contributes to PLC1 activation and TCR-induced calcium fluxes by regulating Tec-family kinases as described above, however there are other possibilities. PLC
1 has been shown to bind PIP3 through its PH domain and thus PI3K may regulate PLC
1 activation directly (51). In addition, we have shown that Vav1 also contributes to PLC
1 phosphorylation and activation through a PI3K-independent pathway, by demonstrating that the effects of the Vav1 mutation were more severe than PI3K inhibitors (Fig. 5 A and B). Furthermore, we identified a potential route for such a pathway by showing that Vav1 regulates the association of PLC
1 with the adapters SLP-76 and Gads and that this association was PI3K-independent (Fig. 5 B). This is likely to be significant for the activation of the lipase as mutations in SLP-76 which block its ability to bind PLC
1 lead to reduced TCR-induced PLC
1 phosphorylation (42).
The observation that in Vav1-/- cells, the association of LAT with PLC1, Gads, and SLP-76 was normal, whereas that of PLC
1 with Gads and SLP-76 was affected suggests that there may be two distinct signaling complexes containing PLC
1. Only one of these includes LAT, whereas the other, Vav1-dependent complex, apparently does not. An interesting possibility is that this complex is nucleated around a different adaptor protein which substitutes for LAT. How Vav1 may regulate the assembly of such a complex is unclear. Since Vav1 binds to SLP-76 after TCR stimulation, it may stabilize the complex by directly binding to PLC
1, or it might affect the ability of SLP-76 to bind to PLC
1 by an allosteric mechanism.
In conclusion, we have shown that in primary DP thymocytes Vav1 transduces TCR signals to the phosphorylation and activation of PLC1 by at least two independent pathways (Fig. 6). First, Vav1 transduces a signal which leads to the activation of PI3K and phosphorylation of Tec-family kinases, and second, independently of PI3K, Vav1 regulates the formation of a signaling complex between PLC
1 and the adaptor proteins Gads and SLP-76.
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Acknowledgments |
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This work was supported by the Medical Research Council (United Kingdom).
Submitted: October 2, 2001
Revised: March 11, 2002
Accepted: March 19, 2002
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Footnotes |
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References |
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