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J. Biol. Chem., Vol. 275, Issue 22, 16574-16578, June 2, 2000

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Signaling via the T Cell Antigen Receptor Induces Phosphorylation of Stat1 on Serine 727^*

Ana M. Gamero and Andrew C. Larner^§

From the Department of Immunology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, December 21, 1999, and in revised form, February 16, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Stat1 transcription factor plays a pivotal role in both, the antiviral and antigrowth actions of interferons. Stat1 acquires the ability to bind DNA by becoming phosphorylated on Tyr⁷⁰¹. However, to effectively stimulate gene transcription, it must also be phosphorylated on Ser⁷²⁷. We show that engagement of T cell antigen receptor (TCR)/CD3 complex in either Jurkat cells or peripheral blood lymphocytes stimulates phosphorylation of Ser⁷²⁷ but not Tyr⁷⁰¹ of Stat1. This process does not require the expression of tyrosine kinases Lck and Zap-70. Interestingly, pretreatment of T cells with the Src kinase inhibitor PP1 completely abrogated CD3-mediated serine phosphorylation of Stat1, whereas inhibitors to MEK1 and phosphatidylinositol 3-kinase had no effect. Phosphorylation of Ser⁷²⁷ of Stat1 in T cells is not restricted to TCR/CD3 but also results when cells are stimulated via the costimulatory molecule CD28. The combination of CD3 and CD28 did not augment phosphorylation of Stat1 Ser⁷²⁷. Surprisingly, Stat1-mediated transcriptional activity in response to IFN- was enhanced with CD3 stimulation, whereas CD3 alone had little effect. These findings suggest that Stat1 is a signaling molecule in TCR signaling and may play a role in T cell function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Engagement of the T cell receptor (TCR)/CD3¹ activates signaling pathways critical for T cell development and function (1, 2). One of the earliest detectable events in this process is the rapid activation of several protein-tyrosine kinases (PTKs) including Fyn, Lck, and Zap-70 and protein-tyrosine phosphatase, CD45. Ultimately, transcription factors become activated and initiate gene expression critical for T cell activation and survival (1, 2).

Lck and Fyn are two critical cytoplasmic PTKs of the Src family involved in T cell maturation. Both PTKs can partially substitute for the activity of each other. Mice deficient in Lck show a profound block in T cell development at the early double positive CD4⁺/CD8⁺ stage (3), whereas mice deficient in Fyn display minor aberration in T cell differentiation, yet their single positive T cells show reduced proliferation in response to mitogens (4). Interestingly, mice lacking both Lck and Fyn display a complete block in T cell maturation at two stages: CD4/CD8 to CD4⁺/CD8⁺ and CD4⁺/CD8⁺ to CD4⁺ and CD8⁺ (5).

Another key player in T cell maturation is Zap-70, a member of the Syk/Zap-70 family of cytoplasmic PTKs (6). Disruption of the Zap-70 gene in mice leads to a block in T cell differentiation similar to those detected in Lck-deficient mice (7). A subgroup of patients with severe combined immunodeficiency lack expression of Zap-70, and their T cells are refractory to T cell stimulation (8, 9).

Signal transducers and activators of transcription (Stat) become activated in response to cytokines, growth factors, and osmotic stress (10, 11). Upon stimulation, Janus family PTKs (Jaks) become activated and tyrosine phosphorylate Stats. This allows Stats to dimerize through Src homology 2 phosphotyrosine and translocate to the nucleus where they are able to bind DNA and regulate gene transcription (10). Ser⁷²⁷ is conserved in the transactivation domains of Stat1, Stat3, and Stat4. This site contains a mitogen-activated protein kinase (MAPK) recognition motif, PX_n(S/T)P (n = 1 or 2) that must be phosphorylated for Stats to gain maximal transcriptional activity (12). Cytokine-induced tyrosine phosphorylation of Stats can occur independently of serine phosphorylation (13). The identity of the Stat serine kinase has remained elusive. Both ERK2 and p38 MAPK have been shown to be activated in response to IFNs, and dominant negative forms of these proteins can inhibit transcriptional reporter activity of Stat1 (14, 15).

Stats also play a pivotal role in T cell function (16). T cells in mice deficient in Stat4 and Stat6 are impaired in the development of Th1 and Th2 responses, respectively (17, 18). Stimulation of T lymphoblasts via their TCR receptor has been shown to induce serine but not tyrosine phosphorylation of Stat3 (19). This process is mediated by MEK/ERK1/2 signaling pathways (19). In contrast, a different study showed that stimulation of an allogen-specific CD4⁺ human T cell line with anti-CD3 antibody or bacterial superantigen triggered both serine and tyrosine phosphorylation of Stat3 with similar kinetics (20). Furthermore, CD3 stimulation of murine T cells triggers tyrosine phosphorylation of Stat5, a process mediated by the Src kinase Lck, and this stimulation induces transient association of Stat5 with the TCR (21). Here we show that signaling through the TCR/CD3 and CD28 costimulation induces serine but not tyrosine phosphorylation of Stat1 in Jurkat cells, a human T cell leukemia line. This observation can be seen in nontransformed naive, primary human T cells. Phosphorylation of Ser⁷²⁷ of Stat1 can be effectively blocked by the Src kinase inhibitor PP1 in response to TCR/CD3 stimulation and occurs independently of Lck and Zap-70 tyrosine kinases. Moreover, CD3 stimulation augments Stat1-dependent gene transcription in response to IFN-. These results suggest that Stat1 is another component in T cell signal transduction pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells and Cell Culture-- The human leukemic T cell line Jurkat, subclone E6, mutant lines derived from this clone, Lck-deficient (J.Cam.1.6), (a gift from A. Weiss, UCSF, San Francisco, CA) and Zap-70-deficient (P116) (a gift from R. Abraham, Duke University, Durham, NC), were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 2 mM L-glutamine, penicillin (10 units/ml) and streptomycin (10 µg/ml) at 37 °C and 5% CO₂. Peripheral blood lymphocytes (PBLs) were isolated from whole blood by density gradient centrifugation using Ficoll-Hypaque (Amersham Pharmacia Biotech) followed by removal of monocytes by plastic adherence at 37 °C.

Antibodies and Chemical Reagents-- Antiserum that specifically recognizes the phosphorylated form of serine 727 of Stat1 was used at 1:10000 dilution (a generous gift of D. Frank, Dana-Farber Cancer Institute, Boston, MA). Phosphospecific antibodies against tyrosine 701 of Stat1 and active ERK1/2 were purchased from New England Biolabs Inc. (Beverly, MA) and used at 1:2000 dilution. Monoclonal antibodies to Stat1 and pan-ERK were obtained from Transduction Laboratories (Lexington, KY) and used at 1:1000 dilution. Anti-CD3 monoclonal antibody OKT3 was from Ortho Biotech, Inc. (Raritan, NJ). Anti-CD28 was purchased from Pharmingen (Palo Alto, CA). The inhibitors H7, PD98059, U0126, PP1, SB203580, wortmannin, and LY294002 were purchased from Calbiochem (San Diego, CA).

Stimulation of Cells-- For TCR/CD3 stimulation, cells were left untreated or incubated with 5 µg/ml anti-CD3 OKT3 antibody. For CD28 stimulation, a final dilution of 1:200 dilution of CD28 antibody was used. Cells were incubated for 10 min at 37 °C followed by addition of 10 µg/ml of anti-mouse IgG (Sigma) at 37 °C for the indicated times. In some experiments, cells were treated for 30 min at 37 °C prior to CD3 stimulation with MEK1-specific inhibitor PD98059 (50 µM), p38 MAPK inhibitor SB203580 (10 µM), MEK1 inhibitor U0126 (10 µM), phosphatidylinositol 3-kinase inhibitors LY294002 (10 µM), wortmannin (100 nM), serine/threonine kinase inhibitor H7 (50 µM), or Src kinase inhibitor PPI (10 µM). Cells were then washed once with cold phosphate-buffered saline and lysed in a buffer containing 1% Triton X-100, 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mM -glycerophosphate. Lysates were vortexed and incubated on ice for 10 min, and insoluble material was cleared by centrifugation at 12,000 rpm for 10 min at 4 °C.

Immunoblot Analysis-- Proteins (30 µg of whole cell extract) were separated on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). Membranes were immunoblotted with the indicated antibodies using concentrations and conditions recommended by manufacturers. Immunoblots were developed using horseradish peroxidase-conjugated secondary antibodies (Zymed Laboratories Inc., San Francisco, CA) and ECL (Amersham Pharmacia Biotech).

Luciferase Assay-- Jurkat cells were transiently transfected using Superfect Reagent (Qiagen Inc., Valencia, CA) with 2 µg of 3 × IRF-GAS luciferase reporter plasmid. To normalize for luciferase activity and control for transfection efficiency, 0.5 µg of pRL-TK (Promega, Madison, WI) was also included. After overnight incubation at 37 °C, cells were left untreated or stimulated with anti-CD3 OKT3 antibody as described above, IFN- or the combination of anti-CD3 plus IFN- for 6 h. Cell lysates were prepared, and luciferase activity was measured with a Luminometer (Dynatech Laboratories, Chantilly, VA) using the dual luciferase reporter system according to the manufacturer (Promega).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosphorylation of Serine 727 of Stat1 Is Induced by TCR/CD3 Stimulation-- The participation of Stat transcription factors in TCR signaling was unknown until it was discovered that Stat3 and Stat5 become activated in response to TCR/CD3 stimulation (19, 21). We set out to examine whether TCR/CD3 stimulation may also lead to activation of Stat1 in T cells because this transcription factor is critical in the generation of antiviral and antibacterial responses as demonstrated in mice deficient in Stat1 gene, suggesting a defect in T cell-mediated immunity (22, 23). To address this, Jurkat cells were stimulated by cross-linking with an anti-CD3 monoclonal antibody (OKT3), which mimics TCR activation. Total cell extracts were prepared and proteins were separated by SDS-PAGE. The serine phosphorylation status of Stat1 was monitored by Western blot analysis using an antibody that specifically recognizes Stat1 phosphorylated on Ser⁷²⁷. As shown in Fig. 1A, serine phosphorylated Stat1 was detected as early as 15 min after CD3 stimulation of Jurkat cells, and this form of Stat1 continued to be activated up to 1 h. Reprobing the blots for Stat1 demonstrates that equal amounts of the protein are present in all the samples (Fig. 1A, lower panel). Despite the extensive use of Jurkat cells as a model to study TCR signaling pathways, these cells are transformed and grow in the absence of exogenous growth factors. We wanted to extend our observations to a more physiological relevant T cell; therefore we assayed PBLs from normal volunteers to determine whether Stat1 was serine phosphorylated in response to CD3 stimulation. PBLs were incubated for various times with anti-CD3, and whole cell extracts were analyzed for phosphorylation of Ser⁷²⁷ of Stat1 (Fig. 1B). Detection of phosphorylated Stat1 on Ser⁷²⁷ with similar kinetics as seen with Jurkat cells was observed as early as 15 min, and the signal was sustained up to 1 h post-stimulation. IFNs activate serine and tyrosine phosphorylation of Stat1. We decided to examine whether the level of activation of serine phosphorylation of Stat1 induced after CD3 stimulation was similar to that of IFN stimulation. PBLs were stimulated with either CD3 antibody or IFN- for 30 min. Serine phosphorylation of Stat1 in whole cell extracts prepared from these cells was analyzed by immunoblot analysis. As shown in Fig. 1C, CD3 stimulation induced phosphorylation of Ser⁷²⁷ of Stat1 with similar levels to those detected with IFN- treatment.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   CD3/TCR stimulation induces phosphorylation of serine 727 in Stat1. Jurkat cells (A) and PBLs (B) were left untreated or stimulated with 5 µg/ml of anti-CD3 (OKT3) antibody and then incubated with 10 µg/ml of rabbit anti-mouse IgG for the indicated times. Proteins were separated by SDS-PAGE and probed with anti-phosphoserine Stat1 antibody (upper panels). C, same as in B except PBLs were stimulated with 1000 units/ml of IFN-. D, same as in A except the membrane was probed with anti-phosphotyrosine Stat1 antibody. Immunoblots were reprobed with anti-Stat1 antibody to verify for equal loading of protein (lower panels).

CD3 Stimulation of T Cells Does Not Induce Tyrosine Phosphorylation of Stat1-- Previous studies have shown that Stat3 becomes phosphorylated on Ser⁷²⁷ after TCR stimulation. However, there are conflicting data about whether tyrosine phosphorylation of Stat3 occurs in T cells (19, 20). In light of this observation, we decided to examine whether CD3 stimulation leads to tyrosine phosphorylation of Stat1 in Jurkat cells by immunoblot analysis using phosphospecific antibodies that recognize only the tyrosine phosphorylated form of these proteins. As shown in Fig. 1D, CD3-mediated T cell activation failed to induce tyrosine phosphorylation of Stat1 over a course of 1 h. The lack of Stat1 tyrosine phosphorylation was not due to a defect in Stat activation in these cells because treatment of Jurkat cells with IFN- induced tyrosine phosphorylation of Stat1 (Fig. 1D). Similar results were obtained when normal PBLs were analyzed (Fig. 2B). This suggests that activation of the TCR complex only stimulates serine phosphorylation of Stat1.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   CD28 costimulation induces serine phosphorylation of Stat1. Jurkat cells (A) and PBLs (B) were left untreated or stimulated with anti-CD3, anti-CD28, or anti-CD3 + anti-CD28 antibodies followed by cross-linking with anti-mouse IgG for 30 min, and cell lysates were prepared. Proteins were separated by SDS-PAGE and immunoblotted with anti-phosphoserine Ser727 or anti-phosphotyrosine Tyr701 Stat1 antibodies. Membranes were reprobed with anti-Stat1 antibody to verify for equal loading of protein (lower panels).

CD28 Costimulation Triggers Stat1 Ser⁷²⁷ Phosphorylation-- CD28 is a molecule expressed on T cells that provides a second signal to T cell activation when bound to its ligand, B7/CD80 (24). B7/CD28 interactions lead to cytokine gene expression and rescues T cells from entering an unresponsive state or anergy (25). Because CD28 plays a pivotal role in T cell activation and is intimately linked with activation through the TCR, we set out to explore the possibility that stimulation through CD28 may also result in the activation of Stat1. Stimulation of Jurkat cells with anti-CD28 antibody induced the phosphorylation of Ser⁷²⁷ Stat1 (Fig. 2A). The levels of phosphorylated Stat1 Ser⁷²⁷ were similar to those observed with CD3 stimulation. No increase in the level of serine phosphorylation of Stat1 was observed by the combination of CD3+CD28 stimulation (lanes 2 and 3 versus lane 4). The kinetics of activation of Stat1 Ser⁷²⁷ phosphorylation were the same as with CD3 stimulation (data not shown). This suggested that the signaling machinery that leads to Stat1 activation in T cells through TCR/CD3 or CD28 receptor stimulation may be shared. To show that CD28-mediated Stat1 activation could also occur in primary T cells, PBLs were activated by CD3, by CD28 stimulation, or by the combination of both activators. Fig. 2B demonstrates that like Jurkat cells, these activators also stimulate phosphorylation of Stat1 Ser⁷²⁷ in normal peripheral T cells. Again, signaling via both CD3 and CD28 did not enhance phosphorylation of Ser⁷²⁷ of Stat1. Similar to the CD3 response, CD28 or the combination of CD3 and CD28 stimulation does not trigger tyrosine phosphorylation of Stat1 in Jurkat or in primary T cells (Fig. 2). Serine phosphorylation of Stat3 was observed in response to CD28 costimulation with similar kinetics as those seen with Stat1 (data not shown).

Lck and Zap-70 Tyrosine Kinases Are Not Required in CD3-mediated Phosphorylation of Stat1 Ser⁷²⁷-- Signaling cascades regulated via the TCR require the expression and activation of Lck and Zap-70 tyrosine kinases, which are critical in T cell differentiation and function (3, 9). Recently, Lck has been shown to be required for TCR-mediated activation of Stat5 (21). To examine further whether these signaling molecules were required for TCR induced serine phosphorylation of Stat1, we made use of variant Jurkat cell lines, which lack expression of either Lck or Zap-70 (26, 27). Treatment of either Lck- or Zap-70-deficient Jurkat cells with anti-CD3 antibody stimulated phosphorylation of Stat1 Ser⁷²⁷ with similar kinetics to those observed in parental cells (Fig. 3). These findings indicate that phosphorylation of Stat1 Ser⁷²⁷ in response to CD3 stimulation occurs independently of Lck and Zap-70 kinases.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Lck and Zap-70 tyrosine kinases are not required for TCR-mediated serine phosphorylation of Stat1. Lck-deficient Jurkat (A) and Zap-70-deficient Jurkat cells (B) were left untreated or stimulated with anti-CD3 (OKT3) antibody followed by incubation with rabbit anti-mouse IgG for the indicated times, and cell lysates were prepared. Proteins were separated by SDS-PAGE and probed with anti-phosphoserine Stat1 antibody (upper panels). Immunoblots were reprobed with anti-Stat1 antibody to verify for equal loading of protein (lower panels).

CD3-mediated Phosphorylation of Stat1 Ser⁷²⁷ Is inhibited by Src Kinase Inhibitor PP1 and Is Independent from the MEK/MAPK Pathway-- The fact that neither Lck nor Zap-70 are required for TCR stimulated serine phosphorylation of Stat1 was surprising. We therefore decided to explore what other kinases might be implicated for this event. Pretreatment of either Jurkat cells or PBLs with MEK inhibitors PD98059 and U0126 did not block the induction of serine phosphorylation of Stat1 but effectively inhibited the activation of ERK1/2 MAPK (Fig. 4). The H7 serine/threonine kinase inhibitor used at 50 µM, a concentration reported to effectively block serine phosphorylation of Stats (28), did not affect TCR/CD3-mediated phosphorylation of Stat1 Ser⁷²⁷. The p38 MAPK-specific inhibitor SB203580 had no effect in blocking activation of Stat1 Ser⁷²⁷ in Jurkat cells and PBLs. Because phosphatidylinositol 3 (PI-3)-kinase is important in T cell activation, we also evaluated the role of this kinase in Stat1 serine phosphorylation. Again, both LY294002 and wortmannin, potent inhibitors of PI-3 kinase, did not alter Stat1 serine phosphorylation in Jurkat cells (Fig. 4A). Similar responses were detected when primary T cells were evaluated or when rapamycin, an inhibitor of p70^S6K kinase was used (data not shown). Surprisingly, the Src kinase inhibitor PP1, completely blocked CD3-induced serine phosphorylation of Stat1 in both Jurkat and primary T cells (Fig. 4). This was also accompanied by inhibition of ERK1/2 MAPK activation (Fig. 4, lower panels in both A and B).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   The Src kinase inhibitor PP1 abrogates TCR-mediated serine phosphorylation of Stat1. A, Jurkat cells were pretreated for 30 min with PD98059 (50 µM), U0126 (10 µM), H7 (50 µM), PP1 (10 µM), SB203580 (10 µM), Ly294002 (10 µM), or wortmannin (100 nM) followed by CD3 stimulation for 30 min. Proteins were resolved by SDS-PAGE, and immunoblot analysis was performed with anti-phosphoserine Stat1 antibody. Membranes were then reprobed with anti-Stat1 antibody to verify for equal loading of proteins (middle panel). Activation of MAPK was also analyzed in the same samples by using a phospho-specific antibody that recognizes ERK1/ERK2 when dually phosphorylated (lower panels). B, the same as A except primary PBLs were used for the analysis. DMSO, dimethyl sulfoxide.

CD3 Stimulation of T cells Augments Stat1-dependent Transcriptional Activity to IFN--- To understand the possible significance of CD3-mediated phosphorylation of Ser⁷²⁷ of Stat1 in T cells, we wanted to determine whether CD3 stimulation of T cells would have an effect on Stat1-dependent transcriptional responses induced by IFN-. To address this possibility, Jurkat cells were transiently transfected with an IRF-GAS luciferase reporter, and cell lysates were prepared after 6 h of stimulation with anti-CD3 antibody, IFN-, or the combination of anti-CD3 plus IFN- (Fig. 5). Incubation of cells with anti-CD3 induced a 2 ± 0.5-fold increase in reporter activity compared with untreated cells. IFN- treatment of cells stimulated the IRF-GAS luciferase reporter 11.8 ± 0.3-fold using 100 units/ml of cytokine or 20.9 ± 2-fold in cells incubated with 1000 units/ml. Interestingly, CD3 stimulation of these cells in the presence of IFN- significantly enhanced the level of transcriptional activity. For instance, compare 20.9 ± 2-fold induction, with IFN- (1000 units/ml) alone versus 50.7 ± 2.3 with IFN- plus anti-CD3. These findings indicate that IFN- and CD3 signals synergize to augment Stat-dependent gene transcription.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   CD3 stimulation synergizes with IFN- to augment Stat1-mediated gene transcription. Jurkat cells were transiently transfected with the Stat-dependent 3× IRF-GAS luciferase reporter. Cells were left untreated or stimulated with anti-CD3, different doses of IFN-, or a combination of anti-CD3 and IFN- for 6 h and harvested for determination of firefly luciferase activity in cell extracts. Values were normalized against Renilla luciferase activity and shown as fold induction with respect to untreated samples. This is a representative experiment of two that were performed. Results are shown as means ± S.D. of duplicate samples.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we report that triggering of the antigen receptor complex in T cells results in the phosphorylation of serine 727 but not tyrosine 701 of Stat1. This activation differs in B cells, where engagement of the B cell receptor induces both serine and tyrosine phosphorylation of Stat1 (29). This finding indicates that the transcription factor Stat1 is a component in TCR signal transduction. The role that phosphorylated Ser⁷²⁷ Stat1 and Stat3 play in T cell biology remains to be elucidated. It is widely accepted that tyrosine phosphorylation of Stats allows these proteins to dimerize, translocate to the nucleus, and bind DNA sequences found in promoters of Stat target genes. Serine phosphorylation is required to augment the transcriptional activity of Stats (12). A variety of activators such as ultraviolet light irradiation, TNF, and LPS can trigger Stat1 phosphorylation on Ser⁷²⁷ in the absence of tyrosine phosphorylation (30, 31). Serine 727 of Stat1 also appears to play a critical role in the constitutive expression of Stat1 target genes such as members of the caspase family of proteins as well as in the protection of TNF-mediated induction of apoptosis (32). Our data indicate that TCR signals can synergize with IFN- to increase Stat-dependent transcriptional response. A similar observation has been made in monocytes where pretreatment with LPS resulted in an enhanced transcriptional activity in response to IFN- (30). In such a study, it was postulated that tyrosine phosphorylated Stat1 may be a better substrate for a Stat1 serine kinase. We can speculate that a similar mechanism may also occur with signals derived from IFN- and CD3 stimulation in T cells. It is thus likely that CD3-mediated serine phosphorylation of Stat1 might modulate one or several of the biological outcomes in T cells.

The identity of the Stat1 serine kinase that mediates CD3/CD28 triggered phosphorylation of Stat1 is not clear. Pharmacological inhibitors PD98059 and SB203580, which block the activation of MEK1 and p38 MAPK, respectively, fail to inhibit CD3-stimulated serine phosphorylation of Stat1. In addition, the broad spectrum serine/threonine kinase inhibitor, H7, that has been reported to inhibit serine phosphorylation of Stats is also without effect on CD3-mediated phosphorylation of this protein. This is in contrast to Stat3 phosphorylation on Ser⁷²⁷ in response to CD3 stimulation, which can be blocked by the MEK1 inhibitor, PD98059 (19). The inability of phosphatidylinositol 3-kinase and p70^S6K kinase inhibitors (data not shown) to affect Stat1 phosphorylation of Ser⁷²⁷ indicated that these proteins also were not involved in Stat1 activation. All TCR-mediated signaling cascades require the activity of one or several tyrosine kinases, including Lck and Zap-70. To address whether these PTKs were needed for the serine phosphorylation of Stat1, we examined Jurkat cell variants that lack expression of Lck or Zap-70. Triggering the TCR complex in these cells did not alter the induction of Stat1 serine phosphorylation. By contrast, the Src kinase inhibitor PP1 could effectively block phosphorylation of Stat1 Ser⁷²⁷ and ERK1/2 activation. This result demonstrates that neither Lck nor Zap-70 may be involved in Stat1 activation. However, the fact that the Src kinase inhibitor PP1 did prevent serine phosphorylation of Stat1 suggests that another tyrosine kinase in this family is involved in mediating this process. One kinase that is well known to be required for TCR function is Fyn. It is interesting that TCR activation of the tyrosine kinase Pyk2 requires Fyn but neither Lck nor Zap-70 (33). Pyk2 has also been reported to be required for IFN--stimulated serine phosphorylation of Stat1 (34). One might speculate that TCR-stimulated Fyn activity allows for the activation of Pyk2 followed by activation of a MAPK member responsible for serine phosphorylation of Stat1. Experiments are in progress to test this hypothesis. The fact that Stat1 can be selectively serine phosphorylated through engagement of the TCR provides another potential physiological role for this transcription factor. Relating this event to T cell-mediated responses and/or clonal selection will allow for further understanding of the pleotropic actions of Stat1 through a variety of tyrosine kinase-regulated signaling events.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants CA77741 and CA77736 (to A. C. L.).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.

Recipient of a National Research Service Award from the National Institutes of Health.

^§ To whom correspondence should be addressed: Dept. of Immunology, Lerner Research Inst., NB-30, Cleveland Clinic Foundation, Cleveland, OH 44195. Tel.: 216-445-9045; Fax: 216-444-8372; E-mail: larnera@ccf.org.

Published, JBC Papers in Press, March 23, 2000, DOI 10.1074/jbc.M910149199

	ABBREVIATIONS

The abbreviations used are: TCR, T cell receptor; Stat, signal transducers and activators of transcription; PBL, peripheral blood lymphocyte; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/Erk kinase; PAGE, polyacrylamide gel electrophoresis; IFN, interferon; PTK, protein-tyrosine kinase.

	REFERENCES

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