J Biol Chem, Vol. 275, Issue 5, 3603-3609, February 4, 2000

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

Yuhong Liu, Stephan Witte, Yun-Cai Liu, Melissa Doyle, Chris Elly, and Amnon Altman^§

From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Protein kinase C  (PKC) is a novel Ca²⁺-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)·CD3 complex in antigen-stimulated T cells and is involved in the transcriptional activation of the interleukin-2 gene. In the present study, we report that PKC is tyrosine phosphorylated in Jurkat T cells upon TCR·CD3 activation. The Src family protein-tyrosine kinase, Lck, was critical in TCR-induced tyrosine phosphorylation of PKC. Lck phosphorylated and was associated with the regulatory domain of PKC 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 contributing to it. Tyrosine 90 (Tyr-90) in the regulatory domain of PKC was identified as the major phosphorylation site by Lck. A constitutively active mutant of PKC (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, which may in turn modulate the physiological functions of PKC during TCR-induced T cell activation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Protein kinase C (PKC)¹ is a family of serine/threonine kinases that play critical roles in the regulation of differentiation and proliferation in many cell types and in the response to diverse stimuli (reviewed in Refs. 1 and 2). Products of the 10 known mammalian PKC genes are classified into four subfamilies of Ca²⁺-dependent (or conventional, PKC, -, and -), Ca²⁺-independent (or novel, PKC, -, -, and -), atypical (PKC and /), and PKCµ/D enzymes. Activity of PKC enzymes is regulated by phosphorylation and binding of defined cofactors. Enzyme activation is associated with its redistribution among different cellular compartments, commonly from 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 TCR·CD3-mediated proliferation in PKC-deficient T cells (reviewed in Ref. 3).

PKC is a novel Ca²⁺-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 NH₂-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 TCR·CD3 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 TCR·CD3-mediated T cell activation.

One of the earliest signaling events in T cell activation via the TCR·CD3 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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), 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^TM tag mAbs were obtained from Roche Molecular Biochemicals and Invitrogen (Carlsbad, CA), respectively. A goat anti-mouse Ig antibody was obtained from Pierce.

GST fusion proteins containing the amino-terminal SH2 and SH3 or the combined amino-terminal, SH2, and SH3 domains of Lck (GST-Lck/N, GST-Lck/SH2, GST-Lck/SH3, and GST-Lck/N+3+2, respectively) were generated as described previously (17). Synthetic 15-mer peptides containing the various tyrosine residues present in the regulatory domain of PKC (Fig. 5B) were from QCB, Hopkinton, MA.

Cells and Stimulation-- Simian virus 40 large T antigen-transfected human leukemic Jurkat T cells (Jurkat-TAg), wild-type Jurkat cells, and Lck-deficient (J.CaM1.6 (18)) or ZAP-70-deficient (P116 (19)) variant Jurkat cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM minimal Eagle's medium nonessential amino acids, 10 mM HEPES, and 50 µM -mercaptoethanol and antibiotics. In some experiments, the cells were stimulated for the indicated time periods with OKT3 (2 µg/ml) followed by cross-linking with a secondary goat anti-mouse Ig antibody, or with sodium orthovanadate (100 µM). COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, and antibiotics.

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 µg 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 Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin and leupeptin. Cell lysates were mixed with antibody for 1 h at 4 °C and then incubated with 30 µl of protein G-Sepharose beads (Amersham Pharmacia Biotech) for an additional hour. Binding reactions containing 10 µg of GST fusion proteins and cell lysates were incubated for 2 h at 4 °C, followed by the addition of 20 µl 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 µl 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 × 10⁶ cells as described above. Immunoprecipitates were washed four times in lysis buffer and one time in 50 mM HEPES (pH 7.0). 100 µl of reaction mixture containing 50 mM HEPES (pH 7.0), 10 mM MgCl₂, 10 mM MnCl₂, 1 µM ATP, 10 µCi of [-³²P]ATP, 0.1% Nonidet P-40, and 10 µg 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 µl of the reaction supernatant were transferred to SpinZyme phosphocellulose units (Pierce) and washed as per the manufacturer's instructions. ³²PO₄ incorporation was determined in a Beckman LS 6500 scintillation counter.

Reporter Assay-- Jurkat-TAg cells were transfected with 5 µg of the appropriate reporter plasmid together with 5 µg 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 × 10⁴ cells/well). Cells were cultured for 48 h, pulsed with 0.5 µCi of [³H]thymidine for the last 6 h, and then harvested. [³H]thymidine incorporation was determined in a Beckman LS 6500 scintillation counter.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 phosphorylated on tyrosine in Jurkat T cells when stimulated with phorbol ester.

View larger version (28K): [in this window] [in a new window]   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 µg/ml) for 5 min, phorbol ester (PMA, 50 ng/ml) for 10 min, or pervanadate (100 µM) for 10 min. PKC was immunoprecipitated from 2 × 107 cells. B, Jurkat T cells were stimulated with OKT3 for the indicated period of time, and PKC was immunoprecipitated from 2 × 107 cells. C, COS-1 cells were cotransfected with wild-type PKC and empty pEF vector or Lck. PKC was immunoprecipitated from 1 × 107 cells after 60 h. D, endogenous PKC was immunoprecipitated from 2 × 107 Jurkat T cells, and an in vitro kinase assay was performed by adding purified Lck kinase to the immunoprecipitates (IP).

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).

PKC Is Associated with Lck-- Several recent studies reported that PKC associates with Src family PTKs, and is phosphorylated on tyrosine, in transformed cells (28, 29) and in activated mast cells (27). To investigate whether PKC associates with Lck, we first performed in vitro binding assays using GST-Lck fusion proteins. Whole cell lysates from unstimulated, OKT3-, or pervanadate-stimulated Jurkat cells were incubated with GST alone, GST-Lck/N+3+2 (amino acid residues 1-244), GST-Lck/SH2 (residues 120-226), GST-Lck/SH3 (residues 54-120), or GST-Lck/N (residues 1-95). The precipitates were then analyzed with an anti-PKC antibody. As shown in Fig. 2A, endogenous PKC from unstimulated cells was precipitated by GST-Lck/N+2+3, GST-Lck/SH2, and GST-Lck/SH3 proteins, but not by GST or GST-Lck/N. The association between PKC and GST-Lck/N+2+3 was enhanced when the cells were stimulated with pervanadate, whereas the association between PKC and GST-Lck/SH3 or GST-Lck/SH2 was enhanced by either OKT3 or pervanadate stimulation. Conversely, a GST-PKC-RD fusion protein (amino acids 1-378) was capable of binding Lck present in T cell lysates (Fig. 2B).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   PKC is associated with Lck. Whole cell lysates (WCL) from from 4 × 107 Jurkat T cells were incubated with 10 µg of the indicated GST-Lck (A) or PKC-RD (B) fusion proteins, which were precipitated with glutathione-Sepharose 4B beads. Bound proteins were detected by immunoblotting with anti-PKC (A) or -Lck (B) antibodies. C, Jurkat-TAg cells were transfected with Lck and PKC and left unstimulated or stimulated with OKT3 for 1 min. Lck was immunoprecipitated from 2 × 107 cells after 60 h. IP, immunoprecipitate.

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/N+3+2) 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 membrane-bound PKC.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   PKC is directly associated with Lck. PKC was immunoprecipitated from 2 × 107 Jurkat T cells, resolved on SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membrane. The membranes were overlaid with the indicated GST-fusion proteins and blotted with an anti-GST mAb. IP, immunoprecipitate.

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-70-deficient (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 pervanadate-induced phosphorylation of PKC.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Lck is critical in tyrosine phosphorylation of PKC in vivo. Cells were left unstimulated or stimulated with OKT3 for 1 min or with pervanadate for 5 min. PKC was immunoprecipitated from 2 × 107 cells, and its Tyr(P) content (top panel) or expression level (bottom panel) was determined by immunoblotting with the indicated antibodies. IP, immunoprecipitate.

Tyr-90 of PKC Is the Major Phosphorylation Site by Lck-- To map the site(s) in PKC phosphorylated by Lck, we first determined whether the RD, the CD, or both can be phosphorylated by Lck in transiently transfected cells. The regulatory domain 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).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Mapping of the major Lck phosphorylation site in PKC. A, COS-1 cells were transfected with PKC-RD (left panels) or PKC-CD (right panels) together with empty vector or an Lck expression plasmid. The regulatory and catalytic domains were immunoprecipitated from 1 × 107 cells with anti-PKC or anti-HA mAbs, respectively, and their tyrosine phosphorylation was monitored by anti-Tyr(P) immunoblotting (upper panels). The membranes were reprobed with anti-PKC or anti-HA mAbs to assess the expression levels of the respective PKC domains (bottom panels). IP, immunoprecipitate. B, the sequence of 15-mer synthetic peptides containing tyrosine residues in the regulatory domain of PKC. The positions of the highlighted tyrosine residues in the sequence of PKC are indicated by numbers above the sequence. C, Lck was immunoprecipitated from transfected COS-1 cells and assayed in an in vitro kinase assay using the synthetic PKC peptides as substrates. neg ctrl indicates a negative control group that lacked peptide substrate.

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 (90%) 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.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   The effect of mutating Tyr-90 in PKC on its tyrosine phosphorylation. A, COS-1 cells were cotransfected with the regulatory domain of PKC (left panels) or with full-length PKC (right panels) expression plasmids, which were mutated at Tyr-90 (Y90F) or left unmutated (WT), plus Lck. PKC was immunoprecipitated from 1 × 107 cells after 60 h, and its tyrosine phosphorylation was assessed by anti-Tyr(P) immunoblotting (upper panels). The same membranes were reprobed with an anti-PKC antibody (middle panels), and aliquots of cell extracts (1 × 106 cells) were immunoblotted with an anti-Lck antibody (bottom panels). B, Jurkat-TAg cells were transfected with the regulatory domain of wild-type (WT) or Y90F-mutated PKC, and the cells were left unstimulated or stimulated for the final 6 h with a cross-linked anti-CD3 mAb (OKT3; 2 µg/ml) or with pervanadate (100 µM). PKC was immunoprecipitated from 1 × 107 cells after 60 h, and its tyrosine phosphorylation (upper panel) or expression level (bottom panel) was monitored as in A. IP, immunoprecipitate.

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 differences 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 Ca²⁺ ionophore. As shown in Fig. 7A, the constitutively active PKC mutant containing Tyr-90 enhanced the proliferation of Jurkat cells by ~50%. 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 tag-specific 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).

View larger version (45K): [in this window] [in a new window]   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 µCi of [3H]thymidine for the last 6 h. Background proliferation in pEF-transfected cells was ~6000 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.

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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 Ca²⁺, 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).

In addition to serine and threonine phosphorylation, several studies have also shown that one PKC isoform, PKC, can be phosphorylated on tyrosine residues. PKC is phosphorylated on tyrosine in Ras (35), v-Src (28), or insulin-like growth factor-1 (36) -transformed cells; in 12-O-tetradecanoylphorbol-13-acetate (16), platelet-derived growth factor (16), epidermal growth factor (25), thrombin (26), or carbachol (37) -stimulated cells; and upon cross-linking of the high-affinity receptor for IgE in rat basophilic leukemia cells (24). Similarly, Src family protein kinases were found to phosphorylate PKC in vitro (27, 38). In addition, PKC was found to become tyrosine phosphorylated in insulin-stimulated cells (39), and tyrosine phosphorylation of several PKC isoforms (PKC, -I, -, -, -, and -) was observed in COS cells in response to H₂O₂ stimulation (40). In the present study, we demonstrate that PKC undergoes tyrosine phosphorylation in T cells upon TCR·CD3 ligation or pervanadate stimulation.

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 cell-expressed 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 (27-29). 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 H₂O₂ (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 Ca²⁺ 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 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 TCR·CD3 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.

	ACKNOWLEDGEMENTS

We thank Drs. G. Baier, G. Crabtree, M. Karin, and A. Rao for providing various plasmids, and Dr. R. T. Abraham for the P116 cells.

	FOOTNOTES

^* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: ICON Clinical Research, 63225 Langen, Germany.

^§ To whom correspondence should be addressed: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Tel.: 858-558-3527; Fax: 858-558-3526; E-mail: amnon@liai.org.

	ABBREVIATIONS

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.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES