Regulation of Protein Kinase C u Function during T Cell Activation by Lck-mediated Tyrosine Phosphorylation*

Protein kinase C u (PKC u ) is a novel Ca 2 1 -independent PKC isoform, which is selectively expressed in skeletal muscle and hematopoietic cells, especially T cells. In T cells, it colocalizes with the T cell antigen receptor (TCR) z CD3 complex in antigen-stimulated T cells and is involved in the transcriptional activation of the inter-leukin-2 gene. In the present study, we report that PKC u is tyrosine phosphorylated in Jurkat T cells upon TCR z CD3 activation. The Src family protein-tyrosine kinase, Lck, was critical in TCR-induced tyrosine phosphorylation of PKC u . Lck phosphorylated and was associated with the regulatory domain of PKC u both in vitro and in intact cells. This association was constitutive, but it was enhanced by T cell activation, with both Src-homology 2 and Src-homology 3 domains of Lck contrib-uting to it. Tyrosine 90 (Tyr-90) in the regulatory domain of PKC u was identified as the major phosphorylation site by Lck. A constitutively active mutant of PKC u (A148E) could enhance proliferation of Jurkat T cells and synergized with ionomycin to induce nuclear factor of T cells activity. However, mutation of Tyr-90 into phenylalanine markedly reduced (or abolished) these activities. These results suggest that Lck plays an important role in tyrosine phosphorylation of PKC u , which may in turn modulate the physiological functions of PKC

the cytosolic to the particulate (membrane) fraction. Studies indicate that PKC is also important during T cell activation. This is indicated by the ability of physiological T cell receptor (TCR) ligands to activate PKC and induce its translocation from the cytosol to the particulate fraction; by the ability of PKC inhibitors, or PKC depletion by prolonged phorbol ester treatment, to block lymphocyte signaling and activation; by the requirement for persistent PKC activation during mitogenic T cell activation; and, finally, by the diminished TCR⅐CD3-mediated proliferation in PKC-deficient T cells (reviewed in Ref. 3).
PKC is a novel Ca 2ϩ -independent PKC isoform. It is characterized by a unique tissue distribution, i.e. in skeletal muscle, lymphoid organs, and hematopoietic cell lines, in particular T cells (4 -6); by isoenzyme-specific activation requirements and substrate preferences in vitro (7,8); and by its presence in the particulate and detergent-insoluble (i.e. cytoskeletal) fraction in resting T cells (unlike, e.g. PKC␣ and -␤) (9). Previous reports have shown that among several PKC isoforms tested, only PKC was capable of significantly stimulating Ras-dependent transcription from an AP-1 element in EL4 leukemic T cells (10). PKC also specifically cooperates with calcineurin and plays a critical role in c-Jun NH 2 -terminal kinase activation and induction of the interleukin-2 gene (11,12). Recent reports indicated that among different T cell-expressed PKC isoforms, PKC was the only one to colocalize precisely with the TCR⅐CD3 complex in the contact region between antigen-specific T cells and antigen-presenting cells. Importantly, this colocalization occurred at a high stoichiometry and correlated with positive activation signals leading to proliferation, as opposed to activation conditions, which result in anergy or apoptosis (13,14). These findings strongly suggest that PKC plays specialized role(s) in T cells as a specific constituent of signaling cascades that are involved in TCR⅐CD3-mediated T cell activation.
One of the earliest signaling events in T cell activation via the TCR⅐CD3 complex is the activation of the Src and Syk families of protein-tyrosine kinases (PTKs), which in turn leads to the phosphorylation of numerous cellular proteins (15). There is evidence that cross-talk among PTKs and Ser/Thr kinases occurs commonly in different cell types and serves as an important regulatory mechanism. In this regard, recent studies have shown that a novel PKC, PKC␦, can be phosphorylated on tyrosine residues upon activation (16). Because of the close structural relationship between PKC␦ and PKC (5) and the finding that PKC colocalizes to the activated TCR complex (which includes activated PTKs) in antigen-specific T cells (13), we have decided to examine whether PKC can become phosphorylated on tyrosine residues.

MATERIALS AND METHODS
Antibodies and Reagents-Anti-Lck and -glutathione S-transferase (GST) monoclonal antibodies (mAbs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-phosphotyrosine Tyr(P) mAb (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY), * This work was supported by by National Institutes of Health Grants CA35299 (to A. A.) and AI09881 (to Y. L.). This is publication No. 322 from the La Jolla Institute for Allergy and Immunology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 1 The abbreviations used are: PKC, protein kinase C; TCR, T cell antigen receptor; PTK, protein-tyrosine kinase; mAb, monoclonal antibodies; Tyr(P), phosphotyrosine; GST, glutathione S-transferase; SH, Src-homology; RD, regulatory domain; NFAT, nuclear factor of T cells; CD, catalytic domain. and the anti-PKC mAb was from Transduction Laboratories (Lexington, KY). The anti-human CD3 mAb, OKT3, was purified from culture supernatants of the corresponding hybridoma by protein A-Sepharose chromatography. The anti-hemagglutinin (clone 12CA5) and -Xpress tag mAbs were obtained from Roche Molecular Biochemicals and Invitrogen (Carlsbad, CA), respectively. A goat anti-mouse Ig antibody was obtained from Pierce.
Plasmids and Transfections-Full-length wild-type, constitutively active (A148E) or dominant-negative (K409R) human PKC (10) and Lck (20) cDNAs were generated as described previously. The PKC plasmids were subcloned into the BamHI and XbaI sites of the pEF4/ His-C mammalian expression vector (Invitrogen) by standard techniques. This vector encodes in-frame 6xHis and Xpress TM tags upstream of the insert. A constitutively active PKC-A148E plasmid in which Tyr-90 has been mutated to phenylalanine (Y90F) was made by site-directed mutagenesis. The cDNAs encoding the regulatory domain (PKC-RD, amino acid residues 1-378) or catalytic domain (PKC-CD, residues 379 -706) of PKC were subcloned into a mammalian expression vector, pEFneo (21), which has been tagged with an hemagglutinin epitope. A GST-PKC-RD fusion protein was prepared by cloning the corresponding cDNA into the pGEX-5X-1 Escherichia coli expression vector and purifying the expressed, isopropyl-1-thio-␤-D-galactopyranoside-induced protein on glutathione-Sepharose beads (22). The NFAT/ AP-1 and AP-1-luciferase reporter constructs were provided by G. Crabtree and M. Karin, respectively. A chloramphenicol acetyltransferase reporter gene driven by five repeats of the NFAT site (3 element) derived from the tumor necrosis factor ␣ promoter was obtained from A. Rao. Jurkat-TAg and COS-1 cells were transiently transfected with 5-10 g of cDNA by electroporation (260 V, 950 microfarads). Cells were cultured for 48 -60 h before they were used in various assays.
Immunoprecipitation, Binding Reactions, and Immunoblotting-Cells were lysed in lysis buffer containing 1% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM NaF, 5 mM NaPP, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml aprotinin and leupeptin. Cell lysates were mixed with antibody for 1 h at 4°C and then incubated with 30 l of protein G-Sepharose beads (Amersham Phar-macia Biotech) for an additional hour. Binding reactions containing 10 g of GST fusion proteins and cell lysates were incubated for 2 h at 4°C, followed by the addition of 20 l of glutathione-Sepharose 4B beads and incubation for 1 h at 4°C. Precipitates were washed five times with lysis buffer and boiled in 30 l of sample buffer for 5 min. Samples were subjected to SDS-polyacrylamide gel electrophoresis analysis and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were immunoblotted with primary antibodies overnight at 4°C or for 2 h at room temperature. After a brief wash, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were washed and visualized by the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech).
In Vitro Lck Kinase Assay-COS-1 cells transiently transfected with Lck were lysed, and Lck was immunoprecipitated from 1 ϫ 10 6 cells as described above. Immunoprecipitates were washed four times in lysis buffer and one time in 50 mM HEPES (pH 7.0). 100 l of reaction mixture containing 50 mM HEPES (pH 7.0), 10 mM MgCl 2 , 10 mM MnCl 2 , 1 M ATP, 10 Ci of [␥-32 P]ATP, 0.1% Nonidet P-40, and 10 g of peptide substrate were added to immunoprecipitates and incubated at 30°C for 30 min. The reactions were terminated by placing the samples on ice. After a brief spin, 25 l of the reaction supernatant were transferred to SpinZyme phosphocellulose units (Pierce) and washed as per the manufacturer's instructions. 32 PO 4 incorporation was determined in a Beckman LS 6500 scintillation counter.
Reporter Assay-Jurkat-TAg cells were transfected with 5 g of the appropriate reporter plasmid together with 5 g of the indicated expression plasmids. Identical amounts of the corresponding empty vectors were used as controls. Transfection efficiencies were monitored by cotransfection of a thymidine kinase promoter luciferase reporter and using the Dual Luciferase kit (Promega, Madison, WI) according to the manufacturer's instructions. Cells were cultured for 24 h and either left unstimulated or activated for the final 6 h of culture with the indicated stimuli. The cells were lysed, and luciferase activity was determined as described previously (23). The results are expressed as normalized luciferase activity of triplicate samples.
Proliferation Assay-Jurkat-Tag cells were transfected with the appropriate plasmid and plated on 96-well plates (1 ϫ 10 4 cells/well). Cells were cultured for 48 h, pulsed with 0.5 Ci of [ 3 H]thymidine for the last 6 h, and then harvested. [ 3 H]thymidine incorporation was determined in a Beckman LS 6500 scintillation counter.

PKC Is Tyrosine Phosphorylated upon T Cell Activation-
Recent studies have shown that activation of different receptors leads to tyrosine phosphorylation of PKC␦ in various cell types (16, 24 -27). Because of the similarity between PKC␦ and PKC we have decided to examine whether PKC can also become phosphorylated on tyrosine. As shown in Fig. 1A, PKC from resting Jurkat T cells contained a low level of Tyr(P). Anti-CD3 or pervanadate stimulation caused a marked increase in this phosphorylation. Unlike PKC␦ in a murine myeloid progenitor cell line 32D (16), PKC was not phosphoryl-

FIG. 1. Tyrosine phosphorylation of PKC in T cells and the role of Lck. A,
Jurkat T cells were left unstimulated or stimulated with cross-linked OKT3 (2 g/ ml) for 5 min, phorbol ester (PMA, 50 ng/ml) for 10 min, or pervanadate (100 M) for 10 min. PKC was immunoprecipitated from 2 ϫ 10 7 cells. B, Jurkat T cells were stimulated with OKT3 for the indicated period of time, and PKC was immunoprecipitated from 2 ϫ 10 7 cells. C, COS-1 cells were cotransfected with wildtype PKC and empty pEF vector or Lck. PKC was immunoprecipitated from 1 ϫ 10 7 cells after 60 h. D, endogenous PKC was immunoprecipitated from 2 ϫ 10 7 Jurkat T cells, and an in vitro kinase assay was performed by adding purified Lck kinase to the immunoprecipitates (IP). ated on tyrosine in Jurkat T cells when stimulated with phorbol ester.
To determine the kinetics of OKT3-induced tyrosine phosphorylation of PKC, Jurkat T cells were either left unstimulated or treated with OKT3 for different times. As shown in Fig.  1B, OKT3-induced tyrosine phosphorylation of PKC peaked at 1 min, decreased thereafter, and returned to the baseline level within 30 min.
PKC Is Phosphorylated by Lck in Vivo and in Vitro-To determine which T cell-expressed PTKs mediate the phosphorylation of PKC, COS-1 cells were cotransfected with PKC plus Lck, Fyn, ZAP-70, Syk, Itk (Emt), or combinations of Lck plus ZAP-70 or Lck plus Itk. PKC was immunoprecipitated, and its tyrosine phosphorylation was analyzed by anti-Tyr(P) immunoblotting. All these tyrosine kinases, with the exception of Lck, did not induce detectable tyrosine phosphorylation of PKC (data not shown). As shown in Fig. 1C, PKC was phosphorylated on tyrosine residue(s) in cells coexpressing Lck. This phosphorylation was not present in PKC plus empty vector-cotransfected cells. Moreover, immunoprecipitated endogenous PKC was phosphorylated in vitro on tyrosine by purified Lck (Fig. 1D). PKC expression in the immunoprecipitates was monitored by immunoblotting and was found to be equivalent among different groups in each experiment (Fig. 1, bottom panels).
To address the question whether Lck and PKC associate with each other in vivo, Jurkat T cells were cotransfected with Lck and PKC expression plasmids. Probing of Lck immunoprecipitates from these cells with an anti-PKC mAb revealed that PKC coimmunoprecipitated with Lck; however, the amount of PKC associated with Lck did not change following anti-CD3 stimulation (Fig. 2C). When the reverse experiments were conducted using the anti-PKC mAb for immunoprecipitation, no Lck could be detected in association with PKC. It is possible that our immunoprecipitating antibody, which binds to the amino-terminal region of PKC, disrupts the association between Lck and PKC, because we found that the regulatory domain of PKC is involved in the interaction with Lck (Fig.  2B).
Overlay binding experiments were next carried out to examine whether the association between PKC and Lck is direct. Endogenous PKC from Jurkat cells was immunoprecipitated and transferred onto nitrocellulose membranes. The membranes were incubated with GST-Lck fusion proteins, and membrane-bound fusion proteins were detected with an anti-GST mAb. As shown in Fig. 3, the SH3, SH2, and the full regulatory domain of Lck (Lck/Nϩ3ϩ2) bound directly to PKC. This binding was not significantly affected by stimulation, with the exception of pervanadate stimulation, which enhanced the binding of the Lck SH2 domain to the membranebound PKC.
To address the role of Lck versus ZAP-70 in the phosphorylation of PKC, we compared the tyrosine phosphorylation of PKC in wild-type Jurkat T to that occurring in two mutant Jurkat cell lines, i.e. Lck-deficient (J.CaM1.6 (18)) or ZAP-70deficient (P116 (19)) Jurkat T cells. As shown in Fig. 4, the basal tyrosine phosphorylation of PKC was abrogated in the Lck-deficient cells, whereas the OKT3-or pervanadate-mediated tyrosine phosphorylation was greatly reduced in the same cells. In contrast, the basal as well as the prominent pervanadate-induced tyrosine phosphorylation of PKC was maintained in P116 cells, but its anti-CD3-induced phosphorylation was reduced to a level approaching the basal one. These data suggest that Lck plays a critical role in the tyrosine phosphorylation of PKC in T cells and that ZAP-70 may contribute to this event under basal conditions or in anti-CD3-stimulated cells, although it is largely dispensable for the pervanadateinduced phosphorylation of PKC.
Tyr of PKC was prominently phosphorylated on tyrosine in COS-1 cells coexpressing Lck (Fig. 5A, left panels), whereas tyrosine phosphorylation of the catalytic domain was not detectable under the same conditions (Fig. 5A, right panels). Next, a series of PKC-derived 15-mer peptides containing the various tyrosine residues present in the regulatory domain were used as substrates in in vitro Lck kinase assays. All of these peptides contained tyrosine in the center position, with the exception of the peptide representing Tyr-237 and Tyr-239 (Fig. 5B). Only the peptide containing Tyr-90 was a good substrate for Lck isolated by immunoprecipitation from Lck-overexpressing COS-1 cells (Fig. 5C).
To determine whether Tyr-90, which is located in the V1 region within the regulatory domain of PKC, represents a substrate for Lck in intact cells, we replaced this tyrosine residue with phenylalanine by site-directed mutagenesis and then compared the tyrosine phosphorylation of this mutant versus wild-type PKC in transfected COS-1 or Jurkat cells. Both full-length PKC and its regulatory domain were assayed in these experiments. When cotransfected with Lck into COS-1 cells, the level of Tyr(P) in both the regulatory domain (Fig. 6A, top left panel) and the full-length (Fig. 6A, top right panel) Y90F-mutated proteins was markedly reduced (Ն90%) in comparison with the corresponding wild-type PKC proteins, despite the similar expression levels of both PKC (middle panels) and Lck (bottom panels) in the two transfected groups. Tyrosine phosphorylation of PKC was not observed in cells transfected with empty vector instead of Lck (data not shown; Fig. 1C). Similarly, the anti-CD3-or pervanadate-induced tyrosine phsophorylation of the transfected PKC-RD in Jurkat T cells was greatly reduced when Tyr-90 was mutated to phenylalanine (Fig. 6B, top panel). All groups expressed similar levels of the transfected PKC-RD (bottom panel). Together, these experiments reveal that Tyr-90 in the regulatory domain of PKC is most likely the major phosphorylation site for Lck.
Mutation of Tyr-90 Leads to Deficient PKC Function-To determine whether phosphorylation of Tyr-90 is important for the proper function of PKC in T cells, we compared two forms of constitutively active PKC (A148E), i.e. one containing the wild-type Tyr-90 and another in which the Y90F mutation has been introduced, in several functional assays. First, we evaluated the effect of mutating Tyr-90 on the in vitro catalytic activity of transfected PKC immunoprecipitated from transfected COS-1 cells (which do not express endogenous PKC), using myelin basic protein as a substrate. No significant dif- ferences were detected between wild-type and Y90F-mutated PKC either in the presence or absence of lipid cofactors (data not shown). Next, we evaluated the effect of the mutation on two downstream events that we recently found to be selectively induced by PKC, i.e. enhanced proliferation of Jurkat T cells; and second, we activated the NFAT-luciferase reporter gene in conjunction with a second signal provided by Ca 2ϩ ionophore. As shown in Fig. 7A, the constitutively active PKC mutant containing Tyr-90 enhanced the proliferation of Jurkat cells by ϳ50%. The actual level of enhancement is most likely considerably higher given the fact that only a relatively small fraction of the cells actually expresses the A148E mutant under these transient transfection conditions. Under the same conditions, the double PKC mutant (A148E/Y90F) was devoid of this activity. Similarly, the A148E/Y90F double mutant was deficient in inducing the activity of a reporter gene driven by an NFAT/AP-1 element derived from the interleukin-2 gene promoter (Fig. 7B). These deficiencies did not reflect lower expression of the Y90F mutant, because immunoblotting with a tagspecific antibody indicated that the Y90F mutant was expressed as well as, or even better than, the single A148E mutant (Fig. 7, A and B, bottom panels).
To further evaluate the specificity of this effect, as well as rule out the possibility that mutation of Tyr-90 nonselectively inactivates the enzyme or confers upon it a dominant-negative phenotype, we compared the ability of the same PKC mutants to activate reporter genes driven by isolated AP-1 or NFAT response elements. The results demonstrate that the double mutant was fully active in inducing AP-1 activity (Fig. 7C) but was deficient relative to the single A/E mutant in stimulating NFAT activity (Fig. 7D). These results indicate that Tyr-90 in PKC plays a role in NFAT, but not AP-1, activation and, furthermore, that the Tyr-90-mutated PKC does not behave in a nonselective inhibitory manner. DISCUSSION Protein kinase C isozymes consist of an amino-terminal regulatory domain and a highly conserved carboxyl-terminal catalytic domain. The activation and localization of PKC enzymes are regulated by the binding of lipid cofactors and, in the case of conventional PKCs, also Ca 2ϩ , to the regulatory domain of PKC (2). PKC is also regulated by trans-and autophosphorylation on serine and threonine residues in the activation loop and the carboxyl-terminal region of the catalytic domain, modifications that are necessary for processing catalytically competent enzymes and for the correct subcellular localization of PKC (30 -33). In a recent report, threonine 250 of PKC␣ has been identified as an autophosphorylation site upon 12-O-tetradecanoylphorbol-13-acetate stimulation (34).
Previous studies have shown that Src family PTKs are involved in the phosphorylation of PKC␦ both in intact cells and in vitro (16, 25, 27-29, 35, 38). We tested a series of T cellexpressed PTKs, which are important in TCR-mediated signaling, for their ability to phosphorylate PKC on tyrosine in cotransfected COS cells. Only Lck, a Src family PTK, could phosphorylate PKC significantly, and furthermore, Lck also phosphorylated PKC directly in vitro. An important role for Lck in phosphorylating PKC is supported by the finding that PKC tyrosine phosphorylation induced by both OKT3 and pervanadate was greatly reduced in an Lck-deficient Jurkat cell line, JCaM1.6. However, our findings do not exclude some contribution by other PTKs, e.g. ZAP-70, to the tyrosine phosphorylation of PKC.
Several recent studies reported that Src family PTKs, i.e. Lyn and Src, associate with PKC␦ (27)(28)(29). Whereas phosphorylated Tyr-52 in PKC␦ was shown to associate with the SH2 domain of Lyn and this interaction in intact mast cells was dependent on cross-linking of the high affinity receptor for IgE (27), the interaction between PKC␦ and Src did not require the SH2 domain of the latter (28) and occurred constitutively (27). In another recent study, the c-Abl PTK was shown to interact with PKC␦ through its SH3 domain (41). Our results indicate that PKC is associated with Lck in Jurkat T cells and that the regulatory domain of PKC mediates this interaction. Analysis of this association in vitro demonstrated that it was direct and involved the SH2 and SH3 domains of Lck in both unstimulated and activated T cells. However, the kinase activity of PKC was not required for this interaction (data not shown). Although the association between Lck and PKC was readily demonstrable in transfected T cells, we could not consistently detect it in intact, nontransfected cells (data not shown). However, whether or not this association occurs in intact T cells, it is clear that Lck can phosphorylate PKC on tyrosine in vitro and in vivo and, moreover, that Lck is required for the inducible tyrosine phosphorylation of PKC in T cells.
Tyrosine residues 52 and 187 in the regulatory domain of PKC␦ have been shown to be phosphorylated upon cellular stimulation (42,43). PKC␦ was also found to be phosphorylated on multiple tyrosine residues in the catalytic domain, and phosphorylation of Tyr-512 and -523 was demonstrated to be critical for the activation of PKC␦ by H 2 O 2 (40). In the present study, we showed that only the regulatory domain of PKC was phosphorylated by Lck in vitro. Among the nine tyrosine residues in the regulatory domain, Tyr-90 was the only good substrate of Lck in vitro, although weak phosphorylation of peptides containing Tyr-28 and Tyr-237/239 above background levels was also observed. Replacement of Tyr-90 with phenylalanine revealed that the tyrosine phosphorylation of PKC-RD in transfected COS cells was nearly abolished. Similarly, tyrosine phosphorylation of the same mutant was greatly reduced in anti-CD3-or pervanadate-stimulated Jurkat T cells in the context of the PKC-RD. However, we noticed that Y90F-mutated full-length PKC could still be phosphorylated on tyrosine in stimulated T cells (data not shown). Because several tyrosine residues in the catalytic domain are highly conserved among all members of the PKC family, the most likely explanation for this observation is that some PTK(s) can phosphorylate tyrosine residues in the catalytic domain of PKC, in agreement with a recent report (40). However, the inability of Lck to phosphorylate the PKC-CD in transfected COS cells indicates a kinase other than Lck may phosphorylate the catalytic domain of PKC.
The consequences of tyrosine phosphorylation of PKC on its enzymatic activity are controversial. Several reports showed that tyrosine-phosphorylated PKC␦ had decreased activity (25,28,35), whereas others indicated the opposite (16,40) or reported a modulation in its substrate specificity (24). Li et al. (43) had identified Tyr-187 as a phosphorylation site in PKC␦ upon 12-O-tetradecanoylphorbol-13-acetate or platelet-derived growth factor stimulation. However, mutation of this residue to phenylalanine did not alter its activity or known functions (43). Immune complex kinase assays failed to reveal differences in catalytic activity between wild-type PKC and the Y90F mutant; furthermore, Y90F-mutated PKC did not display altered subcellular distribution and could still associate well with Lck in Jurkat T cells (data not shown). These findings suggest that Tyr-90 may not be involved in regulating the activation, enzyme activity, or subcellular localization of PKC. In addition, although Tyr-90 is the major phosphorylation site by Lck, it most likely is not the major binding site for Lck, consistent with the fact that the relevant motif (Tyr-Ser-Leu-Ala) is very different from the optimal Tyr(P)-containing motif for binding the SH2 domain of Src family PTKs (44). Rather, PKC binding to the Lck SH3 domain and/or the binding of other Tyr(P) residues in PKC to the Lck SH2 domain may be more important factors in their interaction. Nevertheless, we did observe that mutation of Tyr-90 to phenylalanine had functional consequences. Thus, a constitutively active PKC (A148E) mutant could enhance proliferation of Jurkat T cells, whereas a Y90F/A148E double mutant was incapable of inducing this effect. Similarly, the Y90F/A148E double mutant also was deficient in its ability to cooperate with a Ca 2ϩ signal in the induction of interleukin-2 promoter NFAT/ AP-1 activity. Furthermore, this deficiency occurred at the level of NFAT, but not AP-1, activation. The indirect mechanism by which the Y90F mutation reduces the activity of PKC in these functional assays remains to be determined. One possibility, currently under investigation, is that Tyr-90 might FIG. 7. The Y90F PKC mutant is functionally deficient. A, Jurkat-TAg cells were transfected with empty vector (pEF), PKC-A148E, or PKC-Y90F/ A148E. The cells were cultured for 48 h and pulsed with 0.5 Ci of [ 3 H]thymidine for the last 6 h. Background proliferation in pEF-transfected cells was ϳ6000 cpm. B, Jurkat-TAg cells were transfected with empty vector, PKC-A148E, or PKC-Y90F/A148E, in the presence of an NFAT/ AP-1 reporter plasmid derived from the interleukin-2 promoter. The cells were left unstimulated or stimulated for the final 6 h with ionomycin (500 ng/ml). Luciferase activity in cell lysates was determined in triplicate. The bottom panels show the expression levels of the transfected PKC enzymes, which was determined by immunoblotting with an anti-Xpress tag antibody. C and D, Jurkat-TAg cells were cotransfected with the indicated PKC plasmids or empty vector plus AP-1 (C) or NFAT (D) reporter constructs. Reporter activity was assessed as in B. WCL, whole cell lysate. represent a binding site for another signaling intermediate that contains Tyr(P)-binding domains (e.g. an SH2 domain) and can indirectly regulate the function of PKC.
In summary, our results show that TCR⅐CD3 ligation induces tyrosine phosphorylation of PKC. We found that the Src family protein-tyrosine kinase, Lck, was critical in TCR-induced tyrosine phosphorylation of PKC. Furthermore, Lck associated with PKC in vitro and in intact T cells. We also identified Tyr-90 in the regulatory domain of PKC as the major tyrosine phosphorylation site by Lck and demonstrated that tyrosine-to-phenylalanine mutation at this position reduced the ability of PKC to induce NFAT/AP-1 activity and enhance proliferation in Jurkat T cells. These results suggest that Lck plays an important role in tyrosine phosphorylation of PKC, which may in turn modulate the physiological functions of PKC.