38 by Syk in Lck-Negative T Cells

Cell. Signal. Vol. 10, No. 6, pp. 407–413, 1998 Copyright  1998 Elsevier Science Inc.

ISSN 0898-6568/98 $19.00 PII S0898-6568(97)00139-3

Phosphorylation of the Grb2and Phosphatidylinositol 3-Kinase p85–binding p36/38 by Syk in Lck-Negative T Cells Maria von Willebrand, Scott Williams, Pankaj Tailor and Tomas Mustelin* Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA

ABSTRACT. Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activiation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85DiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass of 36–38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells. cell signal 10;6:407–413, 1998.  1998 Elsevier Science Inc. KEY WORDS. Syk, Lck, Phosphatidylinositol 3-kinase, Extracellular signal regulated kinase, Grb2, T-cell antigen receptor, Tyrosine phosphorylation

INTRODUCTION Activation of T lymphocytes by engagement of their T-cell antigen receptors (TCRs) is associated with the activation of the heterodimeric isoforms of phosphatidylinositol 3-kinase (PI3K), resulting in the intracellular accumulation of inositol phospholipids phosphorylated at the D3-position of the inositol ring [1]. These lipids act as second messengers and allosterically activate the Ser/Thr-specific protein kinase c-Akt, also known as protein kinase B [2, 3], and some Ca21-independent isoforms of protein kinase C [4–6]. The role of these protein kinases in T-cell activation has not yet been clearly defined. The heterodimeric PI3Ks consist of an 85- or 55-kDa reg*Author to whom all correspondence should be addressed. Tel: (619) 5583547; Fax: (619) 558-3526; E-mail: [email protected] Abbreviations: HA–haemagglutinin; iSH2–inter-SH2 region; MAPK–mitogen-activated protein kinase; MBP–myelin basic protein; NFAT–nuclear factor of activated T cells; p85DiSH2–p85 lacking the inter-SH2 region; PI3K–phosphatidylinositol 3-kinase; PTyr–phosphotyrosine; TCR–T-cell antigen receptor. Received 7 July 1997; and accepted 20 August 1997.

ulatory subunit, p85a, p85b, or p55PIK, respectively [7–10], and a 110-kDa catalytic subunit (p110a or p110b) [11, 12]. The adaptor subunit contains a number of domains that mediate protein–protein interactions and are commonly found in signalling proteins: one Src homology 3 (SH3) domain, two proline-rich regions, two SH2 domains, and a region with similarity to the breakpoint cluster region gene. p55PIK lacks the N-terminal SH3 and breakpoint cluster region– homology domains [10]. The catalytic p110 subunit binds through interaction of the region between the SH2 domains of p85 (inter-SH2 domain, or iSH2) and the extreme N-terminus of the catalytic subunit [12, 13]. Regulation of PI3K activity occurs through interaction of the two subunits [13, 14], autophosphorylation at Ser-608 of p85 [15], and translocation to the plasma membrane [16]. In addition, a number of reports have demonstrated that binding of cellular proteins to PI3K domains can increase its enzymatic activity: the SH3 domain of Src family kinases binding to one or both of the proline-rich regions of p85 [17], phosphotyrosine (PTyr)-containing peptides binding to the SH2 domains of p85 [18, 19], and GTP-Ras binding to the cata-

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lytic p110 subunit [20]. The relative importance of these mechanisms in T-cell activation is not well understood. We have previously shown that PI3K activity is required for optimal activation of the Erk2 mitogen-activated protein kinase (MAPK) in TCR-triggered T cells [21]. Both the relatively specific PI3K inhibitor wortmannin and, more importantly, a mutated “dominant negative” form of p85 that lacked the p110-binding iSH2 region (p85DiSH2) reduced Erk2 activation in response to anti-CD3 antibodies [21]. In contrast, phorbol ester–induced MAPK activation was unaffected, and an inactive analogue of wortmannin, WM12, had no effects. We have also recently [22] found that expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enables the TCR to activate Erk2. This system enables us to investigate the function of Syk in lymphocyte signalling and to dissect the molecules that relay the signals from Syk to the activation of Erk2. In this paper, we provide evidence that PI3K plays a polsitive role in TCR-induced Syk-mediated Erk2 activation upstream of Mek phosphorylation and activation. We also found that Syk phosphorylates a 36–38-kDa protein that binds to the SH2 domains of PI3K and Grb2. This protein may provide a mechanism for the recruitment of PI3K and may link Syk to other downstream events. MATERIALS AND METHODS Antibodies and Reagents The anti-CD3ε mAb OKT3 and the anti-Myc tag epitope mAb 9E10 were purified from ascites fluid by protein A Sepharose chromatography. The anti-PTyr mAb 4G10 and anti-p85 rabbit serum were from Upstate Biotechnology Inc. (Lake Placid, NY), the anti-influenza haemagglutinin (HA) tag epitope mAb 12CA5 was from Boehringer Mannheim (Indianapolis, IN), anti-Erk2 polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-phospho-Erk and anti-phospho-Mek antibodies were from New England Biolabs Inc. (Beverly, MA). The Myctagged Erk2 cDNA was from Dr. C. Marshall (Ludwig Institute for Cancer Research). The p85DiSH2 construct has been described previously [21]; it is in the pEF/HA vector and encodes amino acids 1–439 plus 563–724 of bovine p85a with an added HA epitope, YPYDVPDYA, at its N-terminus. The expression plasmid encoding Syk with the same N-terminal tag and in the same vector has been published before [22]. The glutathione S-transferase (GST) fusion proteins containing the two SH2 domains of p85, GST-p85-NC-SH2, has been described before [23]. GST-Grb2-SH2 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cells and Transient Transfections JCaM1 cells were kept at logarithmic growth in RPMI supplemented with 10% heat-inactivated foetal calf serum, l-glutamine, and antibiotics. Transient transfections were carried out by electroporation as described before [22–24]. Electroporation conditions typically contained 20 3 106 cells

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and a total of 15 mg of plasmid DNA (5 mg of each plasmid, in some 10 mg of p85DiSH2), and in each transfection the DNA amount was kept constant by the addition of empty vector. Immunoprecipitations, Immunoblots, and in Vitro Erk2 Kinase Assays These were performed exactly as described before [22]. Use of GST Fusion Proteins GST fusion proteins were used at 5 mg/mL in lysates and were bound to glutathione-Sepharose as described before [23–25]. RESULTS Anti-CD3-Induced Activation of Erk2 Is Inhibited by p85DiSH2 and Wortmannin MAPK is a key integration point in signalling by growth factor and cytokine receptors, as well as lymphocyte antigen receptors [26–28]. We have previously shown that expression of Syk in the Lck-negative JCaM1 cell line reconstitutes the TCR-induced activation of the Erk2 MAPK [22]. We have also shown that PI3K activity is required for an optimal activation of Erk2 upon TCR stimulation in wildtype (Lck-expressing) Jurkat T cells [21]. To test if PI3K is also required for Syk-mediated TCR-induced Erk2 activation, we transiently transfected JCaM1 cells with Myc-tagged Erk2 alone or together with Syk, p85DiSH2, or Syk plus p85DiSH2. Two days after transfection, the cells were treated with OKT3 for 5 min and lysed, the tagged Erk2 was immunoprecipiated with the anti-Myc mAb 9E10, and its kinase activity was measured with myelin basic proteins (MBP) as a substrate. As shown in Figure 1a, the transfected Erk2 was not activated in JCaM1 cells (lanes 1 and 2) unless Syk was co-expressed (lanes 3 and 4). Expression of p85DiSH2 alone did not support Erk2 activation (lanes 5 and 6), whereas Erk2 was activated in the presence of both Syk and p85DiSH2 (lanes 7 and 8), although its activity remained considerably lower than in the presence of Syk alone. This reduction is very similar to that seen in parental Jurkat transfected with Myc-Erk2 and p85DiSH2 [21]. A control blot showed that both the 72-kDa Syk and the 71-kDa p85DiSH2 were expressed in the transfected cells. To further support the notion that PI3K activity is important for TCRinduced Syk-mediated Erk2 activation, we transfected JCaM1 cells with Myc-Erk2 and Syk and treated one-half of the cells with 300 nM of wortmannin. As shown in Figure 1b, the activation of Erk2 was reduced in the wortmannintreated cells to a similar extent as in the cells expressing p85DiSH2, although they still contained as much Erk2 protein as the untreated cells. We have previously shown that addition of wortmannin directly to the Erk2 assay has no effect on this kinase [21]. We conclude from these experiments that PI3K activity plays a positive role in TCR-induced Syk-mediated activiation of Erk2 but that PI3K is not absolutely required for this pathway, because some activation can occur even when PI3K is greatly inhibited.

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of the same filter with anti-Erk2 antibodies showed that all samples contained similar amounts of the Erk2 protein (Fig. 2a, lower panel). This result indicates that PI3K is required for the efficient phosphorylation of Erk2, a reaction catalysed by the protein kinase Mek [29]. Therefore, we next blotted the same lysates with anti-phospho-Mek antibodies, which recognise only the serine-phosphorylated activated form of Mek. As shown in Figure 2b, phospho-Mek was detected in Syk-transfected JCaM1 cells (lanes 3 and 4) but not in untransfected (lanes 1 and 2) or p85DiSH2-transfected cells (lanes 5 and 6). In the presence of both Syk and p85DiSH2, the appeareance of phospho-Mek was much reduced (lanes 7 and 8), as was the case in wortmannintreated Syk-transfected cells (lanes 9 and 10). Control blots showed that Syk and p85DiSH2 were expressed at comparable levels in the samples (Fig. 2c and d). Thus, PI3K seems to be required for TCR-induced Syk-mediated phosphorylation of both Erk2 and Mek. This explains why Erk2 activation was reduced and suggests that the PI3K-sensitive step is upstream of Mek phosphorylation and activation. Anti-CD3-Induced Syk-Mediated Phosphorylation of PI3K-Binding Proteins

FIGURE 1. PI3K activity is required for TCR-induced Erk2 ac-

tivation in Syk-expressing JCaM1 cells. (a) Kinase assay (upper panel) of immunoprecipitates obtained with the anti-Myc tag mAb 9E10 from JCaM1 cells transiently co-transfected with Myc-tagged Erk2 (all lanes) together with empty pEF/HA (lanes 1 and 2), HA-tagged Syk (lanes 3 and 4), HA-tagged p85DiSH2 (lanes 5 and 6), or Syk and p85DiSH2 (lanes 7 and 8); (middle panel), anti-Erk2 blot of the same filter, and (lower panel), antiHA tag blot of total cell lysates of the same transfectants. (b) Similar kinase assay (upper panel) of anti-Myc tag immnoprecipitates from JCaM1 cells co-transfected with Myc-tagged Erk2 and Syk. The cells were treated with medium (lanes 1 and 2) or 300 nM wortmannin (lanes 3 and 4) for the last 18 h prior to stimulation of the cells with anti-CD3 as in (a); (lower panel), anti-Erk2 blot of the same filter.

p85DiSH2 and Wortmannin Also Block the Phosphorylation of Erk and Mek To further study this positive role of PI3K for the MAPK pathway, we transfected JCaM1 cells with Syk or p85DiSH2 or both and treated part of the Syk-transfected cells with wortmannin. After anti-CD3 stimulation, total cell lysates were immunoblotted with anti-phospho-Erk antibodies, which detect only the tyrosine and threonine phosphorylated activated form of Erk. As shown in Figure 2a, expression of Syk enabled the TCR complex to induce the appearance of phospho-Erk (lanes 3 and 4). This response was severely reduced in cells expressing both Syk and p85DiSH2 (lanes 7 and 8) and in cells expressing Syk and treated with wortmannin (lanes 9 and 10). A control blot

The inhibitory effect of the p85DiSH2 construct suggests that Syk induces a signalling event that causes the activation or translocation of PI3K to the plasma membrane through its p85 subunit and that the “dominant negative” p85DiSH2 competes with endogenous p85 for this recruitment but obviously fails to bring a catalytic p110 subunit along (owing to its lack of iSH2 region). Because Syk requires its kinase activty to induce the MAPK response [22] and p85 contains two SH2 domains, the most likely model predicts that Syk phosphorylates one or several cellular proteins that subsequently bind the SH2 domains of p85 and thereby cause the recruitment of PI3K to the plasma membrane, where its substrate phospholipids are located. To test this possibility experimentally, we transfected JCaM1 cells with empty vector or Syk and assayed the lysates for cellular phosphoproteins binding to a GST-fusion protein containing both SH2 domains of p85, GST-p85-NC. To reduce the occurrence of multimeric protein complexes, we made lysates in either regular lysis buffer or in the presence of SDS to at least partly denature all cellular proteins. As shown in Figure 3a, GST-p85-NC-SH2 precipitated a few phosphoproteins from untransfected JCaM1 cells (lanes 1–4) and a similar set from Syk-transfected resting cells (lanes 9 and 11). Control GST (lanes 5–8) did not bind any proteins. Upon stimulation of the cells with anti-CD3, only the Syk-expressing cells showed a clear increase in three phosphoproteins that bound to GST-p85-NC-SH2 (lanes 10 and 12). These proteins had a mass of 21, 36–38, and 72–74 kDa. The 21-kDa protein was recognized by anti-TCRz antisera (now shown), and a barely detectable increase in its phophorylation was also seen in vector-transfected cells (lanes 2 and 4). The 72–74kDa protein co-migrated with Syk; but, although the band was not recognized by anti-HA tag or anti-Syk antibodies,

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FIGURE 2. PI3K activity is required for TCR-induced phosphorylation of both Erk and Mek in Syk-expressing JCaM1 cells. (a) Anti-

phospho-Erk immunoblot (upper panel) and anti-Erk2 blot (lower panel) of total lysates of JCaM1 cells transiently transfected with the indicated expression plasmids and left untreated or treated with 300 nM wortmannin overnight and then stimulated with the antiCD3 mAb OKT3 for 5 min, as indicated. (b) Anti-phospho-Mek immunoblot (upper panel) and anti-Mek blot (lower panel) of the same samples as in (a). (c) Anti-HA tag immunoblot of the same filter. (d) Anti-p85 immunoblot of the same filter.

we cannot exclude the possibility that it represents the presence of Syk at quantities below the detection limit of our reagents. Our earlier attempts to co-immunoprecipitate Syk with PI3K consistently failed to show any significant association. Thus, p36/38 and perhaps TCRz are likely candidates for the PI3K-recruiting Syk target. The PI3K-Binding p36/38 Also Binds the SH2 Domain of Grb2 A prominent 36–38-kDa phosphoprotein has been reported by several laboratories to bind PI3K, PLCg1, and Grb2 in T cells [30–33]. Efforts to clone the cDNA for this molecule led to the identification of the Lnk protein [34] as one component of the band, which may also contain the c-Crk and CrkL proteins [35] and perhaps others. We utilized the high affinity of the Grb2-SH2 domain for this protein [32] to evaluate whether the 36–38 kDa protein phosphorylated by Syk and bound by p85-SH2 domains in JCaM1 is indeed the same protein or group of proteins. JCaM1 cells transfected with Syk (or vector control) were stimulated with anti-CD3, and the lysates were pre-cleared with GSTGrb2-SH2 or control GST and then incubated with GSTp85-NC-SH2. Analysis of the bound phosphoproteins by anti-phosphotyrosine (PTyr) immunoblotting (Fig. 4a) showed that the 36–38-kDa band was completely removed by preadsorption of the lysates with GST-Grb2-SH2 (lanes 5 and 6). In contrast, the 21- and 72–74-kDa proteins were not re-

moved, and pre-adsorption with control GST had no effect (lanes 3 and 4). Analysis of the phosphoproteins removed by the pre-adsorption step (Fig. 4b) showed that p36/38 was present in the sample for the stimulated Syk-expressing cells but not in other samples. Thus, p36/38 is heavily phosphorylated upon TCR-triggering of Syk-expressing cells, and it has a good affinity for the Grb2-SH2 domain. This confirms its identity as the previously described p36/38 that binds both PI3K and Grb2.

DISCUSSION TCR-trggering leads to the activation of a cascade of serine/ threonine-specific kinases, including c-Raf [26, 36–38], Mek [29, 39–41), and Erk2 [42]. Activation of c-Raf occurs through complex formation with the Ras proto-oncogene protein in its active GTP-bound state [43], which induces translocation to the membrane, followed by both tyrosine [44] and serine phosphorylation [45]. Activated c-Raf subsequently activates Mek by phosphorylating it at two serine residues [39, 40, 46]. In intact cells, however, this reaction can also be catalysed by other Raf family members or Mek kinase (Mekk). Thus activation of Mek can be achieved by more than one pathway. Activation of Erk2 is achieved by Mek-mediated phosphorylation at both tyrosine 185 and threonine 183 [47]. Once activated, Erk2 phosphorylates and activates other protein kinases and a number of tran-

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FIGURE 3. Expression of Syk induces the TCR-dependent tyrosine phosphorylation of PI3K-binding proteins in JCaM1 cells. (a) Anti-

phosphotyrosine (PTyr) immunoblot of cellular phosphoproteins binding the GST-p85-NC protein (lanes 1–4 and 9–12) or control GST (lanes 5–8) from lysates of JCaM1 cells transfected with the indicated expression plasmids. Prior to lysis, the cells were left untreated or were treated with OKT3 for 5 min as indicated, and lysates were prepared in regular lysis buffer or a denaturing SDS-containing buffer as indicated. (b) Anti-HA tag immunoblot of total lysates of the vector (lane 1) of Syk-transfected (lane 2) cells from the same experiment.

scription factors completing the receptor-induced signalling to the nucleus. The signalling events that couple the TCR to the activation of Ras and Raf are incompletely understood. The TCR initially activates a complex series of tyrosine phosphorylation events [48] catalysed by phosphotyrosine kinases (PTKs) of the Src and Syk families [28, 49, 50]. Because these enzymes regulate the activity, subcellular localisation, and substrate interactions of each other, it becomes difficult

to dissect the role of each PTK. In addition, so many cellular proteins are phosphorylated on tyrosine that it is a major undertaking to evaluate the contribution of each substrate to the downstream activation events. We have therefore utilised our observation that expression of Syk in the Lcknegative JCaM1 cell line (which contains very low levels of endogenous Syk) reconstitutes the capacity of the TCR to induce a full activation of the MAPK cascade and transcriptional activation of a reporter gene driven by two nuclear

FIGURE 4. Depletion of the PI3K-binding 36–38 kDa phosphoprotein by GST-Grb2-SH2. (a) Anti-PTyr immunoblot of cellular

phosphoproteins binding the GST-p85-NC protein (all lanes) from lysates or JCaM1 cells transfected with empty pEF/HA (lanes 1 and 2) or HA-tagged Syk in the same vector (lanes 3–6). Prior to the addition of the GST-p85-NC, the lysates were pre-cleared with GST (lanes 1–4) or GST-Grb2-SH2 (lanes 5 and 6). Prior to lysis, the cells were left untreated or were treated with OKT3 for 5 min as indicated. (b) Anti-PTyr immunoblot of the material removed in the pre-clearing step of the same experiment. (c) Anti-HA tag immunoblot of total lysates of the vector (lane 1) of Syk-transfected (lane 2) cells from the same experiment.

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factor of activated T cells (NFAT)/AP-1 elements taken from the interleukin 2 gene 59 promoter [22]. In these cells, only a few proteins are phosphorylated on tyrosine upon TCR stimulation, thus limiting the potential signalling intermediates to a manageable few. Importantly, these cells express normal levels of the Zap-70 kinase, which, however, remains unphosphorylated and non-functional in the absence of Lck [22]. In contrast, Syk does not need Lck for activation [22, 50–52] and is catalytically much more active than Zap even in its activated form [53]. The Fyn kinase is also present in JCaM1 at low levels, but its participation in TCR signalling seems to be minimal [54]. Thus, in Syk-transfected JCaM1 cells TCR-induced tyrosine phosphorylation is catalysed by Syk or PTKs downstream of Syk, with little or no participation by Src family PTKs. In normal T cells, the same phosphorylation events may be catalyzed by Zap. In Syk-expressing JCaM1 cells TCR-induced Erk2 activation proceeds with kinetics similar to that seen in parental Jurkat, peaking at 3–5 min, and it is inhibited by the “dominant negative” N17-Ras [22]. In the present study, we show that PI3K activity is required for TCR-induced Sykdependent Erk2 activation in these cells, in a manner that is inhibited by the “dominant negative” p85DiSH2, suggesting that signals from Syk cause the p85-dependent recruitment and functional activation of PI3K. Of the few cellular proteins phosphorylated on tyrosine residues [22], the SH2 domains of p85 bound three proteins at 21, 36–38, and 72–74 kDa. Of these proteins, the smallest corresponded to TCRz, and its binding to the SH2 domains of p85 depended on the presence of both SH2 domains [23]. The binding of PI3K to phosphorylated TCR subunits has been reported before [55, 56] but seems to be of low stoichiometry and questionable significance. The 36–38-kDa phosphoprotein was identified as the Grb2-SH2 binding phosphoprotein, observed by several investigators as a major ligand also for PI3K [30–33]. The 72–74-kDa phosphoprotein may be the transfected Syk, but it was present in low quantities, perhaps owing to its association with TCRz. Taken together, p36/38 seems the most likely candidate for the Syk substrate that recruits PI3K upon receptor-induced tyrosine phosphorylation. This issue can be addressed more critically when the cDNA(s) for the molecule(s) becomes available. The function of PI3K, at least in part, is to produce 3phosphorylated inositol phospholipids, mainly phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5triphosphate, which act as second messengers for c-Akt and some isoforms of protein kinase C [2–6]. In addition, these lipids may be important for the membrane translocation of proteins having pleckstrin homology domains, which can bind these lipds directly [57]. The step in the TCR-induced Erk2 activation in which PI3K activity is important seems to be an event between Syk and the phosporylation of Mek. A similar inhibition of Mek was also observed by Ferby and co-workers [58], who studied the activation of Erk2 in neutrophils by platelet-activating factor, and by Karnitz and coworkers [59], in interleukin 2–stimulated T cells. We speculate that PI3K products are required for the activation of

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FIGURE 5. Model for the involvement of Erk2 activation in

Syk-expressing JCaM1 cells. The p36/38 protein is proposed as the link between Syk and PI3K, which is feeding into the MAPK pathway at the level of Ras (through physical interaction of the two molecules), Raf (through protein kinase B or C–mediated phosphorylation of Raf), or through Mekk–mediated activation of Mek. These possibilities are not mutually exclusive. PMA, phorbol ester.

Ras or Raf, suggesting that protein kinase B or C or a pleckstrin homology domain–containing protein participates in the regulation of these enzymes. The capacity of phorbol esters to activate Raf [60] or another Mek activator, Mekk [61] and the insensitivity of phorbol ester–induced activation of MAPK to PI3K inhibition [21] are compatible with a positive role for protein kinase B or C in Mek activation through Raf or Mekk. This model (Fig. 5), which has been suggested before for the insulin receptor system [62], will require more experiments but suggests an important role for PI3K in T cell activation. This work was supported by the Finska La¨karesa¨llskapet, the Finnish Cancer Organizations, and by National Institutes of Health Grants AI35603 and GM48960. This is publication no. 186 from the La Jolla Institute for Allergy and Immunology.

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