STAT3 Is a Serine Kinase Target in T Lymphocytes

Interleukin 2 (IL-2) induces tyrosine phosphorylation of STATs 3 and 5 (signal transducer and activator of transcription). We now show that IL-2 regulation of STAT3 proteins in T cells is a complex response involving activation of two forms of STAT3: 90-kDa STAT3α and an 83-kDa carboxyl-terminal truncated STAT3β. The phosphorylation of STAT proteins on serine residues is also required for competent STAT transcription. A critical serine phosphorylation site in STAT3α is at position 727. In this study we have produced an antisera specific for STAT3α proteins phosphorylated on serine 727 and used this to monitor the phosphorylation of this residue during T lymphocyte activation. Our results show that phosphorylation of STAT3α on serine 727 is not constitutive in quiescent T cells but can be induced by the cytokine IL-2. Interestingly, triggering of the T cell antigen receptor complex or activation of protein kinase C with phorbol esters also induces phosphorylation of serine 727 but without simultaneously inducing STAT3 tyrosine phosphorylation or DNA binding. Hence, the present results show that STAT3 serine phosphorylation can be regulated independently of the tyrosine phosphorylation of this molecule. IL-2 and T cell antigen receptor complex induction of STAT3α serine 727 phosphorylation is dependent on the activity of the MEK/ERK pathway. Previous studies have identified H-7-sensitive kinase pathways that regulate STAT3 DNA binding. We show that H-7-sensitive pathways regulate STAT3 DNA binding in T cells. Nevertheless, we show that H-7-sensitive kinases do not regulate STAT3 tyrosine phosphorylation or phosphorylation of serine 727. These results thus show that STAT3 proteins are targets for multiple kinase pathways in T cells and can integrate signals from both cytokine receptors and antigen receptors. Interleukin 2 (IL-2) induces tyrosine phosphorylation of STATs 3 and 5 (signal transducer and activator of transcription). We now show that IL-2 regulation of STAT3 proteins in T cells is a complex response involving activation of two forms of STAT3: 90-kDa STAT3α and an 83-kDa carboxyl-terminal truncated STAT3β. The phosphorylation of STAT proteins on serine residues is also required for competent STAT transcription. A critical serine phosphorylation site in STAT3α is at position 727. In this study we have produced an antisera specific for STAT3α proteins phosphorylated on serine 727 and used this to monitor the phosphorylation of this residue during T lymphocyte activation. Our results show that phosphorylation of STAT3α on serine 727 is not constitutive in quiescent T cells but can be induced by the cytokine IL-2. Interestingly, triggering of the T cell antigen receptor complex or activation of protein kinase C with phorbol esters also induces phosphorylation of serine 727 but without simultaneously inducing STAT3 tyrosine phosphorylation or DNA binding. Hence, the present results show that STAT3 serine phosphorylation can be regulated independently of the tyrosine phosphorylation of this molecule. IL-2 and T cell antigen receptor complex induction of STAT3α serine 727 phosphorylation is dependent on the activity of the MEK/ERK pathway. Previous studies have identified H-7-sensitive kinase pathways that regulate STAT3 DNA binding. We show that H-7-sensitive pathways regulate STAT3 DNA binding in T cells. Nevertheless, we show that H-7-sensitive kinases do not regulate STAT3 tyrosine phosphorylation or phosphorylation of serine 727. These results thus show that STAT3 proteins are targets for multiple kinase pathways in T cells and can integrate signals from both cytokine receptors and antigen receptors. The cytokine interleukin-2 (IL-2) 1The abbreviations used are: IL-2, interleukin 2; IFN, interferon; MAP, mitogen-activated protein; MEK, MAP kinase kinase; PDBu, phorbol 12,13-dibutyrate; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; SIEM, sis-inducible element mutant; TCR, T cell antigen receptor complex. controls G1-S phase progression, T cell clonal expansion, and functional differentiation (1Smith K.A. Science. 1988; 240: 1169-1176Crossref PubMed Scopus (1906) Google Scholar). High affinity IL-2 receptors trigger activation of tyrosine kinases notably the Janus kinases (JAKs) 1 and 3 (2Johnston J. Kawamura M. Kirken R. Chen Y. Blake T. Shibuya K. Ortaldo J. McVicar D. O'Shea J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (510) Google Scholar, 3Witthuhn B. 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IL-2-activated transcription factors include members of the STAT (signal transducer and activator of transcription) family: STAT3 and STAT5 (15Lin J.-X. Migone T.-S. Tsang M. Friedmann M. Weatherbee J.A. Zhou L. Yamauchi A. Bloom E.T. Mietz J. John S. Leonard W.J. Immunity. 1995; 2: 331-339Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 16Wakao H. Harada N. Kitamura T. Mui A.L.-F. Miyajima A. EMBO J. 1995; 14: 2527-2535Crossref PubMed Scopus (214) Google Scholar, 17Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (731) Google Scholar, 18Brunn G.J. Falls E.L. Nilson E.A. Abraham R.T. J. Biol. Chem. 1995; 270: 11628-11635Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 19Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A.D. Nelms K. Paul W.E. Ihle J.N. Mol. Cel. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (304) Google Scholar). STAT activation involves tyrosine phosphorylation which allows for Src homology (SH) 2 domain-mediated homodimerization or heterodimerization. Activated and tyrosine-phosphorylated STATs then translocate to the nucleus to form sequence-specific DNA binding complexes and enable cytokine-mediated gene transcription (reviewed in Ref. 20Darnell J.E.J. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar). Serine kinases are critical for STAT activation and can regulate STAT DNA binding or transcriptional activity (21Schmitt Ney M. Happ B. Ball R.K. Groner B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3130-3134Crossref PubMed Scopus (100) Google Scholar, 22Lutticken C. Coffer P. Yuan J. Schwartz C. Caldenhoven E. Schindler C. Kruijer W. Heinrich P.C. Horn F. FEBS Lett. 1995; 360: 137-143Crossref PubMed Scopus (93) Google Scholar, 23Boulton T.G. Zhong Z. Wen Z. Darnell J.E.J. Stahl N. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6915-6919Crossref PubMed Scopus (192) Google Scholar, 24Zhang X. Blenis J. Li H.-C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (525) Google Scholar, 25Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1755) Google Scholar, 26Cho S.S. Bacon C.M. Sudarshan C. Rees R.C. Finbloom D. Pine R. O'Shea J.J. J. Immunol. 1996; 157: 4781-4789PubMed Google Scholar). In reconstitution experiments, it has been recently shown that for STAT1 to elicit IFN-γ-mediated antigrowth activity, the serine phosphorylation site (Ser-727) is required (27Bromberg J.F. Horvath C.M. Zilong W. Schreiber R. Darnell J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7673-7678Crossref PubMed Scopus (445) Google Scholar). We have also recently shown that IL-2 regulation of STAT5 is mediated by both tyrosine and serine/threonine kinase pathways (28Beadling C. Ng J. Babbage J.W. Cantrell D.A. EMBO J. 1996; 15: 1902-1913Crossref PubMed Scopus (161) Google Scholar). STAT5 serine phosphorylation in T cells is regulated by an as yet uncharacterized kinase whose activity is required for STAT5 transcriptional activity. IL-2 induces the tyrosine phosphorylation and DNA binding of STAT3. The phosphorylation of serine 727 in the STAT3 carboxyl terminus is important for STAT3 transcriptional function, but neither has there been any characterization of STAT3 serine phosphorylation nor any study of the identity of STAT3 serine kinases in T cells. Recent debates have focused particularly on the role of MEK/ERK kinase pathways in STAT activation (29Ihle J.N. BioEssays. 1996; 18: 95-98Crossref PubMed Scopus (73) Google Scholar). However, the identity of the STAT3 serine 727 kinase has not been fully resolved in any cellular system. This site forms anin vitro substrate for the MAP kinase ERKs which raised the possibility that STAT3 integrates signals from the MAP kinases. This hypothesis has not been fully explored in vivo, in particular, for certain cell systems phosphorylation of serine 727 is constitutive and in others, subject to regulation by extracellular stimuli (25Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1755) Google Scholar). Herein we present evidence that IL-2 induces tyrosine phosphorylation and DNA binding of two different forms of STAT3: 90-kDa STAT3α and 83-kDa STAT3β. IL-2 also induces serine phosphorylation of serine 727 in STAT3α via a MEK/ERK pathway. The present data also show that STAT3 tyrosine and serine phosphorylation can be independently regulated: activation of MAP kinases by phorbol esters or T cell antigen receptor cross-linking induces phosphorylation of STAT3α on serine 727 without concurrently inducing STAT3 DNA binding or tyrosine phosphorylation. The phosphorylation of serine 727 is not obligatory for DNA binding or tyrosine phosphorylation of STAT3. Nevertheless, we find a H-7-sensitive kinase pathway that does not regulate STAT3 tyrosine phosphorylation or phosphorylation of serine 727 in STAT3α is able to modulate STAT3 DNA binding. These data collectively indicate the role of multiple T cell signaling pathways in mediating differential STAT activation. cells T. Uchiyama T. Tsudo M. H. H. S. K. H. PubMed Google Scholar) in with and 1 IL-2 IL-2 receptor T and as C. D. A. Ihle J.N. Kerr I.M. Cantrell D.A. EMBO J. 1994; PubMed Scopus Google Scholar). by in and in with in the of IL-2 for The and of L. with cells in for to cytokine IL-2 or phorbol at a from I. from cell by of in a of 1 1 1 1 1 1 The used from the of the gene proteins from of cells in the by at for 1 with 1 of to of a of affinity of proteins binding to the the to at 5 and used to binding proteins from 1 of cell at for 1 and complexes in to by in by on and to with to and 1 and or STAT or with the the of on the raised carboxyl-terminal raised and carboxyl-terminal raised protein and a from I. the of STAT3 tyrosine phosphorylation by raised to the tyrosine phosphorylation used a tyrosine or the from The to STAT3 at site with serine 727 as a or a These then to via the antisera to serine 727 by for antigen by and the 727 antisera used at a of for and at a of for in the used for affinity experiments, and proteins by with of from 1 to 3 cells by the gel for and Raf-1 and for MAP kinase to and by with the for STAT5 and antisera Raf-1 or STAT3 the sis-inducible element used to affinity STAT proteins from cell of quiescent or T The data in A show that IL-2 induces DNA binding of STAT3 and STAT5 in T cells. IL-2 induced DNA binding of STAT3 and STAT5 is 1 of to the cytokine and by a 90-kDa and a 83-kDa form of STAT3 in both cells and T A and Previous studies have not STAT3 in T cells. However, two different STAT3 proteins of and in the IL-2 complexes in both T cells and cells A and different forms of STAT3 have been STAT3α and a carboxyl-terminal truncated of a STAT3 gene D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google E. T. R. J. L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the and 83-kDa IL-2 induced STAT3 proteins as STAT3α and antisera raised different of STAT3 used in of the STAT3 proteins in T cell and in DNA binding complexes 1 a tyrosine the site of the receptor used to an affinity for the of STAT3 from cell The data in 1 show that antisera raised an STAT3 protein and antisera a protein residues of STAT3 two STAT3 proteins in T cell and in the also both STAT3 proteins in DNA binding In antisera raised a STAT3 residues the 90-kDa STAT3 and not the 83-kDa protein 1 The 83-kDa protein in T cells thus to the recently carboxyl-terminal truncated of STAT3 D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google E. T. R. J. L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Activated and tyrosine phosphorylated STAT proteins in STAT3 from IL-2 T cells raised to the tyrosine phosphorylation site of STAT3 phosphorylation of STAT3 is STAT3 in IL-2 and cells. from and IL-2 and or and cells and to an raised to residues of STAT3. by and by with STAT3 STAT3 and STAT5 a in the of STAT3α but not from IL-2-activated cells and in IL-2 T A and data not forms of STAT3α and for the different can DNA IL-2 activation and are both The of STAT3α to an in response to IL-2 serine phosphorylation of STAT3. is a serine phosphorylation site at residue 727 in STAT3α that is required for transcriptional activation (25Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1755) Google Scholar). of phosphorylation has been Chen J. J. P. 1991; PubMed Scopus Google Scholar) and been shown to be in the of of a of proteins J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, K. J. 1994; Scopus Google Scholar, S.S. Mol. Biol. Cell. PubMed Scopus Google Scholar), transcription factors H. Science. PubMed Scopus Google Scholar). to and to IL-2 is inducing the phosphorylation of STAT3α on serine we antisera to a to the serine 727 site in STAT3. The of this antisera for the phosphorylated serine 727 STAT3 with the is shown by 3 The 727 STAT3 antisera STAT3α proteins from quiescent T cells but of STAT3α with the 727 STAT3 antisera in cells from IL-2 T cells The carboxyl-terminal truncated protein the serine 727 site not be with the 727 STAT3 antisera from quiescent or IL-2-activated cells. IL-2 induced STAT3α DNA proteins also with the 727 STAT3 antisera The of the 727 STAT3 antisera with STAT3α proteins from IL-2-activated T cells be by with the phosphorylated serine 727 STAT3 but not the not STAT3 affinity the tyrosine for with the 727 STAT3 a of 727 STAT3 antisera with STAT3 proteins from quiescent cells but this IL-2 of 727 STAT3 antisera with be These data collectively show that IL-2 induces serine phosphorylation of STAT3α on serine 727 in T MAP kinase can vitro at the carboxyl terminus phosphorylation site (25Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1755) Google Scholar). The in vitro ERK site in STAT3 is serine 727. the role of the MEK/ERK pathway in STAT3 regulation by IL-2 we used an of the ERK MAP kinase kinase The of as a has been D. L. S. A. A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar, D. A. P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). monitor the of the in T we the phosphorylation of ERK induced by IL-2 in T cells with an to monitor ERK activation in of ERK also for ERK in T as in include the p21ras and the protein A.D. J.K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, M. P. T. 1995; 11: Google Scholar). These proteins are both of the MAP but are phosphorylated by the MAP kinases signaling and have by The data in A show that IL-2 regulated of and Raf-1 are by the that this is at the MEK/ERK pathway in T cells. of STAT3α and in DNA complexes 5 that IL-2 induced in STAT3α by the this not IL-2 induced DNA binding of STAT3. the of on IL-2 induced phosphorylation of serine 727 of STAT3α we used the 727 STAT3 The data in 5 C a in of the 727 STAT3 antisera with from IL-2-activated T cells. This in STAT3α proteins from T cells that been with the Hence, of the MEK/ERK pathway the phosphorylation of serine 727 in The phorbol protein kinase C and the kinase pathway independently of IL-2 receptor activation. STAT3α is regulated by MEK/ERK then possibility is that PDBu, which ERK via a protein kinase pathway of IL-2, also the phosphorylation of serine 727 in This is that serine phosphorylation of STAT proteins can independently of tyrosine phosphorylation. The data in A show that not in the of STAT5 but to IL-2, induced in the of STAT3α but not STAT3β. and of STAT3 proteins that induced STAT3α with the 727 STAT3 antisera a not DNA binding of STAT3 of the of phorbol esters to STAT3 tyrosine phosphorylation C and data not The data thus show that phosphorylation of STAT3α on serine 727 can independently of DNA binding or tyrosine phosphorylation. esters are of the ERK pathway and to a for ERK activation can phosphorylation of STAT3 proteins in the of STAT3 tyrosine we the of triggering the T cell antigen receptor complex on STAT3. Previous studies have shown that triggering of the T cell antigen receptor complex does not tyrosine phosphorylation of STAT3 C. D. A. Ihle J.N. Kerr I.M. Cantrell D.A. EMBO J. 1994; PubMed Scopus Google Scholar). However, the data in show that STAT3α proteins from T cells are with the 727 STAT3 antisera with the in STAT3α proteins from quiescent T cells. These results show that STAT3α can be phosphorylated on serine 727 in response to triggering the complex. This is by T cells with the with a in which regulation of the phosphorylation of serine 727 mediated by the MEK/ERK pathway The data show that IL-2 regulates a serine kinase that is on serine 727 in the terminus of The activity of this pathway is not or for induction of STAT3α DNA binding 5 and the STAT3α serine 727 phosphorylation 5 the data in A show that of T cells with the does not IL-2 induced tyrosine phosphorylation 2 the pathway can STAT3α phosphorylation on serine activity is not required for IL-2 induced DNA binding of STAT3α or or tyrosine phosphorylation. In certain cytokine induction of STAT3 DNA binding can be regulated by a serine kinase pathway to the serine/threonine kinase X. Blenis J. Li H.-C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (525) Google Scholar). does not the activation of the MEK/ERK pathway in T cells (28Beadling C. Ng J. Babbage J.W. Cantrell D.A. EMBO J. 1996; 15: 1902-1913Crossref PubMed Scopus (161) Google Scholar) but the role of H-7-sensitive kinases in regulation in T cells not been We the possibility that STAT3 be regulated by two different serine kinases. We the of of T cells on IL-2 regulation of STAT3. The data in that IL-2 induced DNA binding in T cells with not tyrosine phosphorylation of 2 The of on STAT3 DNA binding this not IL-2 induced tyrosine phosphorylation of STAT3 or STAT5 DNA binding of STAT5 proteins by as the of the STAT5 form as shown by the of on of STAT5 (28Beadling C. Ng J. Babbage J.W. Cantrell D.A. EMBO J. 1996; 15: 1902-1913Crossref PubMed Scopus (161) Google Scholar) The also not the of STAT3α in IL-2-activated cells in to the of the A STAT3α proteins from IL-2 T cells also with 727 STAT3 antisera that in T cells does not phosphorylation of serine 727 in IL-2 induction of STAT3 DNA complexes without tyrosine phosphorylation or phosphorylation of serine 727 in The present study that IL-2 DNA binding of two forms of STAT3 in T 90-kDa STAT3α and an 83-kDa carboxyl-terminal truncated STAT3β. We show that STAT3 proteins are both and in IL-2-activated T cells. we a specific with phosphorylated serine 727 in STAT3α and show that STAT3α is regulated by a regulated pathway that is not required for STAT3 DNA binding but which targets the site serine 727 required for STAT3α transcriptional activity (25Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1755) Google Scholar). The present results also that phosphorylation of serine 727 in STAT3α is not regulated by IL-2 but can be regulated by the or phorbol esters IL-2 activation of STAT3 concurrently induces phosphorylation on serine 727 and STAT3 tyrosine phosphorylation. However, STAT3 phosphorylation on serine 727 in response to or phorbol esters without induction of tyrosine phosphorylation or DNA binding of STAT3. the serine and tyrosine phosphorylation pathways that the STATs can be independently by extracellular A in T cell is to the that regulate the of T cell of serine 727 in STAT3α is required for STAT3 transcriptional activity (25Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1755) Google Scholar). The of T cells to to triggering by inducing phosphorylation of STAT3 on a residue that is for STAT3 transcriptional activity that in T cells there is regulation of the STATs by members of the antigen receptor This allows the transcriptional activity of STAT3α in T cells to be to the activation of the a which the of T cell The has been with which to STAT serine kinases in a of cell the regulation of STAT3 by H-7-sensitive kinases has been of with we to regulate the phosphorylation of serine 727 in The present study that STAT3 DNA binding can be regulated by a H-7-sensitive pathway. Nevertheless, the H-7-sensitive pathway does not regulate STAT3α serine 727 phosphorylation. The is but is of the of on STAT3 DNA binding. The of the data is that STAT3α integrates signals from two serine kinase pathways in T a pathway and a H-7-sensitive pathway. These pathways are as does not IL-2 activation of the pathway (28Beadling C. Ng J. Babbage J.W. Cantrell D.A. EMBO J. 1996; 15: 1902-1913Crossref PubMed Scopus (161) Google Scholar) or the phosphorylation of serine 727 in does IL-2-activated STAT3 DNA binding in T cells. the pathway regulates phosphorylation of serine 727 in STAT3α but is not for IL-2 induced STAT3 DNA binding. The regulation of both STAT3α and DNA binding that the H-7-sensitive pathway regulate a site the carboxyl of the two forms of STAT3. is that a H-7-sensitive phosphorylation or the DNA binding affinity of STAT3 Nevertheless, it is also that the on STAT3 proteins is and mediated with transcription factors or phosphorylation of factors to a in STAT3 DNA binding We have shown that IL-2 activation of STAT5 involves the of both tyrosine and serine/threonine kinases (28Beadling C. Ng J. Babbage J.W. Cantrell D.A. EMBO J. 1996; 15: 1902-1913Crossref PubMed Scopus (161) Google Scholar). The STAT5 serine kinase is and not regulated by and be by of the MAP kinase (28Beadling C. Ng J. Babbage J.W. Cantrell D.A. EMBO J. 1996; 15: 1902-1913Crossref PubMed Scopus (161) Google Scholar). Herein we show that the STAT3 727 serine kinase is to a and can be by receptors that the independently of STAT tyrosine phosphorylation. The of the pathway IL-2 regulation of STAT3α and STAT5 and a cytokine in the cell can serine kinase pathways to regulate different has been as to the identity of STAT serine kinases. The present results show that the identity of STAT serine kinases that can STATs involving multiple and different serine kinase The MEK/ERK pathway is to have an role in the activation of T cells and in the of from to cells activity as does of J. K.A. K.A. Perlmutter R.M. J. 1996; PubMed Scopus Google Scholar, T. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the of this regulated pathways have been identified in T We now show that serine a residue critical for STAT3 function, is a substrate for the of the in T cells. Interestingly, serine 727 in STAT3α can be phosphorylated by the MEK/ERK pathway not in response to the cytokine IL-2 but also in response to triggering of the antigen receptor complex. The present results serine phosphorylation of residue 727 can independently of STAT3α tyrosine that the regulation of STAT3 DNA binding and transcriptional activity are These data collectively the of STAT regulation and show that STATs are not targets for members of the cytokine receptor in T cells but can also integrate MAP kinase signals signals from antigen receptors. We and N. and E. Li for and K. for We also and members of the for