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Signaling via the T Cell Antigen Receptor Induces Phosphorylation of Stat1 on

Serine 727

Ana M. GameroΨ and Andrew C. Larner †

From the Department of Immunology, The Lerner Research Institute, The Cleveland

Clinic Foundation, Cleveland, Ohio 44195

Running Title: TCR activation induces serine phosphorylation of Stat1

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

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SUMMARY

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 tyrosine 701 (Y701). However, to effectively stimulate gene

transcription, it must also be phosphorylated on serine 727 (S727). We show that

engagement of T cell antigen receptor (TCR)/CD3 complex in either Jurkat cells or

peripheral blood lymphocytes stimulates phosphorylation of S727 but not tyrosine 701

(Y701) of Stat1. This process does not require the expression of tyrosine kinases

Lck and Zap70. Interestingly, pretreatment of T cells with the Src kinase inhibitor PP1

completely abrogated CD3-mediated serine phosphorylation of Stat1 whereas

inhibitors to MEK1 and PI3-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 S727. Surprisingly, Stat1 mediated transcriptional

activity in response to IFN-α 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.

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INTRODUCTION

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 (PTK)

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 protein tyrosine kinases (PTK) 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 (SH2)-phosphotyrosine and

translocate to the nucleus where they are able to bind DNA and regulate gene

transcription (10). Serine 727 (S727) is conserved in the transactivation domains of

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Stat1, Stat3 and Stat4. This site contains a mitogen- activated protein kinase (MAPK)

recognition motif, PXnS/TP (P=proline; S/T,serine or threonine; X, any amino acid: 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 S727 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-α. These results suggest that Stat1 is another

component in T cell signal transduction pathways.

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EXPERIMENTAL PROCEDURES

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, NC) were cultured in

RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (Gibco-

BRL), 2mM L-glutamine, penicillin (10 U/ml) and streptomycin (10 µg/ml) at 37oC and

5% CO2. Peripheral blood lymphocytes (PBL) were isolated from whole blood by

density gradient centrifugation using Ficoll-Hypaque (Amersham–Pharmacia Biotech,

NJ) followed by removal of monocytes by plastic adherence at 37oC.

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, Harvard, 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).

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Stimulation of Cells

For T cell receptor (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 37oC followed by

addition of 10 µg/ml of anti-mouse IgG (Sigma, St Louis, MO) at 37oC for the indicated

times. In some experiments, cells were treated for 30 min at 37oC prior to CD3

stimulation with MEK1 specific inhibitor PD98059 (50 µM), p38 MAPK inhibitor

SB203580 (10 µM), MEK1 inhibitor U01206 (10 µM), PI3 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 PBS and lysed

in a buffer containing 1% Triton X-100, 50 mM Tris, pH 7.5, 150 mM NaCl, 2mM EDTA,

1 mM sodium orthovanadate, 1mM PMSF, 10 mM β-glycerophosphate. Lysates were

vortexed, incubated on ice for 10 min and insoluble material cleared by centrifugation

at 12,000 rpm for 10 min at 4oC.

Immunoblot Analysis

Proteins (30 µg of whole cell extract) were separated on 8% SDS-PAGE gels and

transferred to PVDF 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, San Francisco, CA) and

enhanced chemiluminescence, ECL, (Amersham-Pharmacia, Piscataway, NJ).

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Luciferase Assay

Jurkat cells were transiently transfected using Superfect Reagent (Qiagen Inc.,

Valencia, CA) with 2 µg of 3 x 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 37oC, cells were left

untreated or stimulated with anti-CD3 OKT3 antibody as described above, IFN-α or

the combination of anti-CD3 plus IFN-α 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

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RESULTS

Phosphorylation of Serine 727 of Stat1 is Induced by TCR/CD3 Stimulation

The participation of Stat transcription factors in T cell antigen receptor (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 since 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 crosslinking with an anti-CD3

monoclonal antibody (OKT3) which mimicks 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 serine 727 (S727). As shown in

Figure 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 1hour.

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 peripheral

blood lymphocytes (PBLs) from normal volunteers to determine if 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

S727 of Stat1 (Fig. 1B). Detection of phosphorylated Stat1 on S727 with similar

kinetics as seen with Jurkat cells was observed as early as 15 min, and the signal

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was sustained up to 1 hour post-stimulation. Interferons (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-α for 30 min. Serine phosphorylation of Stat1 in whole cell extracts prepared from

these cells was analyzed by immunoblot analysis. As shown in Figure 1C, CD3

stimulation induced phosphorylation of S727 of Stat1 with similar levels to those

detected with IFN-α treatment.

CD3 Stimulation of T Cells Does Not Induce Tyrosine Phosphorylation of Stat1

Previous studies have shown that Stat3 becomes phosphorylated on S727 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

Figure 1D, CD3-mediated T cell activation failed to induce tyrosine phosphorylation of

Stat1 over a course of one hour. 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-α 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.

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CD28 Costimulation Triggers Stat1 S727 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). Since 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 S727 Stat1 (Fig. 2A). The levels of

phosphorylated Stat1 S727 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 vs. 4). The kinetics of activation

of Stat1 S727 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, CD28 stimulation or by the combination of both activators. Figure

2B demonstrates that like Jurkat cells, these activators also stimulate

phosphorylation of Stat1 S727 in normal peripheral T cells. Again, signaling via both

CD3 and CD28 did not enhance phosphorylation of S727 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. 2A and 2B).

Serine phosphorylation of Stat3 was observed in response to CD28 costimulation

with similar kinetics as seen with Stat1 (data not shown).

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Lck and Zap-70 Tyrosine Kinases Are Not Required in CD3-Mediated

Phosphorylation of Stat1 S727

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 S727 with similar kinetics to those observed in parental cells (Fig. 3A and 3B).

These findings indicate that phosphorylation of Stat1 S727 in response to CD3

stimulation occurs independently of Lck and Zap-70 kinases.

CD3-Mediated Phosphorylation of Stat1 S727 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 PBL

with MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126 did not block the

induction of serine phosphorylation of Stat1 but effectively inhibited the activation of

ERK1/2 MAPK (Figure 4A and 4B). 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 S727. The p38 MAPK

specific inhibitor SB203580 had no effect in blocking activation of Stat1 S727 in Jurkat

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cells and PBLs. Because PI3-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 p70S6K

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.

4A and 4B). This was also accompanied by inhibition of ERK1/2 MAPK activation (Fig.

4A and 4B, lower panels).

CD3 Stimulation of T cells Augments Stat1 Dependent Transcriptional Activity to

IFN-

To understand the possible significance of CD3-mediated phosphorylation of S727 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-α. 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-α or the combination of anti-CD3 plus IFN-α (Fig. 5). Incubation of

cells with anti-CD3 induced a 2 ± 0.5 fold increase in reporter activity compared to

untreated cells. IFN-α treatment of cells stimulated the IRF-GAS luciferase reporter

11.8 ± 0.3 fold using 100 U/ml of cytokine or 20.9 ± 2 fold in cells incubated with 1000

U/ml. Interestingly, CD3 stimulation of these cells in the presence of IFN-α

significantly enhanced the level of transcriptional activity. For instance, compare 20.9

± 2, with IFN-α (1000 U/ml) alone versus 50.7± 2.3 with IFN-α plus anti-CD3. These

findings indicate that IFN-α and CD3 signals synergyze to augment Stat dependent

gene transcription.

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DISCUSSION

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 S727 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, TNFα

and LPS, can trigger Stat1 phosphorylation on S727 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 TNFα-mediated induction of apoptosis (32).

Our data indicate that TCR signals can synergize with IFN-α 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-γ (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-α

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.

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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 S727 in response to CD3

stimulation, which can be blocked by the MEK1 inhibitor, PD98059 (19). The inability

of PI3-kinase and p70S6K kinase inhibitors (data not shown) to affect Stat1

phosphorylation of S727 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 S727 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 suggest 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-γ-

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

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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 kinases regulated signaling events.

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34. Takaoka, A., Tanaka, N., Mitani, Y., Miyazaki, T., Fujii, H., Sato, M., Kovarik, P.,

Decker, T., Schlessinger, J., and Taniguchi, T. (1999) EMBO J. 18, 2480-2488

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FOOTNOTES

*This work was supported by the National Institutes of Health Grants CA77741 and

CA77736 (to A. C. L.)

ΨRecipient of a National Research Service Award from the National Institutes of

Health

1The 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

PI3, phosphatidylinositol 3

IFN, interferon

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FIGURE LEGENDS

Figure 1. CD3/TCR stimulation induces phosphorylation of serine 727 in Stat1. (A)

Jurkat and (B) Peripheral blood lymphocytes (PBLs) 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 U/ml of IFN-α. (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).

Figure 2. CD28 costimulation induces serine phosphorylation of Stat1. (A) Jurkat

cells and (B) peripheral blood lymphocytes were left untreated or stimulated with anti-

CD3, anti-CD28 or anti-CD3 + anti-CD28 antibodies followed by crosslinking with

anti-mouse IgG for 30 min and cell lysates were prepared. Proteins were separated

by SDS-PAGE and immunoblotted with anti-phosphoserine S727 or anti-

phosphotyrosine Y701 Stat1 antibodies. Membranes were reprobed with anti-Stat1

antibody to verify for equal loading of protein (lower panels).

Figure 3. Lck and Zap-70 tyrosine kinases are not required for TCR mediated

serine phosphorylation of Stat1. (A) Lck deficient Jurkat and (C) Zap-70 deficient

Jurkat cells 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

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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).

Figure 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.

Figure 5. CD3 stimulation synergyzes with IFN to augment Stat1-mediated gene

transcription. Jurkat cells were transiently transfected with the Stat dependent 3x

IRF-GAS luciferase reporter. Cells were left untreated or stimulated with anti-CD3,

different doses of IFN-α or by the combination of anti-CD3 and IFN-α 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 mean ± standard deviation of duplicate samples.

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IFNα

Stat1

Stat1

pS727Stat1

Stat1

pS727Stat1

Stat1

pS727Stat1

pTyr701Stat1αpTyr701Stat1β

A

B

C

D

C D 3: 0 5 ’ 1 5’ 3 0’ 1 h

C D 3: 0 1 5’ 3 0’ 1 h

- CD 3 I F Nα

- 30’ 1h 15’CD3

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Figure 1

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B

A

Stat1

Stat1

pS727Stat1

pS727Stat1

Stat1

pTyr701Stat1α

Stat1

pTyr701Stat1β

pTyr701Stat1βpTyr701Stat1α

Figure 2

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C D 3: 0 15 ’ 3 0’ 1 h

pS727Stat1

Stat1

Lck -A

pS727Stat1

Zap-70 -

Stat1

C D 3: 0 1 5’ 3 0’ 1 h

B

Figure 3

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Figure 4

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0

10

20

30

40

50

60

0 100 500 1000

IFNα (Units/ml)

- CD3

+ CD3

Figure 5

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Ana M. Gamero and Andrew C. Larner727

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

published online March 23, 2000J. Biol. Chem. 

  10.1074/jbc.M910149199Access the most updated version of this article at doi:

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