Mapping of Stat3 serine phosphorylation to a single residue (727) and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3 (original) (raw)

Abstract

During their polypeptide ligand-induced activation Stats (signaltransducers andactivators oftranscription) 1 and 3 acquire, in addition to an obligatory tyrosine phosphorylation, phosphorylation on serine which boosts their transactivating potential [Wen, Z., Zhong, Z. and Darnell, J. E. Jr. (1995) Cell 82, 241-250]. By examining phosphopeptide maps of wild-type and mutant protein we show here that the Stat3 serine phosphorylation, like the Stat1 serine phosphorylation, occurs on a single residue, serine 727. Neither the DNA binding of Stat1 nor Stat3 is demonstrably affected by the presence or absence of the serine phosphorylation. Thus the earlier demonstration that transcription is enhanced by the presence of the serine 727 residue likely occurs after DNA binding. These findings do not agree with earlier claims of excess serine to tyrosine phosphorylation in activated Stats 1 and 3 or to claims of more stable DNA binding of serine phosphorylated Stat dimers.

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Selected References

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  1. Beadling C., Ng J., Babbage J. W., Cantrell D. A. Interleukin-2 activation of STAT5 requires the convergent action of tyrosine kinases and a serine/threonine kinase pathway distinct from the Raf1/ERK2 MAP kinase pathway. EMBO J. 1996 Apr 15;15(8):1902–1913. [PMC free article] [PubMed] [Google Scholar]
  2. Benore-Parsons M., Seidah N. G., Wennogle L. P. Substrate phosphorylation can inhibit proteolysis by trypsin-like enzymes. Arch Biochem Biophys. 1989 Aug 1;272(2):274–280. doi: 10.1016/0003-9861(89)90220-8. [DOI] [PubMed] [Google Scholar]
  3. Boulton T. G., Zhong Z., Wen Z., Darnell J. E., Jr, Stahl N., Yancopoulos G. D. STAT3 activation by cytokines utilizing gp130 and related transducers involves a secondary modification requiring an H7-sensitive kinase. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6915–6919. doi: 10.1073/pnas.92.15.6915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bromberg J. F., Horvath C. M., Wen Z., Schreiber R. D., Darnell J. E., Jr Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7673–7678. doi: 10.1073/pnas.93.15.7673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Decker T., Lew D. J., Mirkovitch J., Darnell J. E., Jr Cytoplasmic activation of GAF, an IFN-gamma-regulated DNA-binding factor. EMBO J. 1991 Apr;10(4):927–932. doi: 10.1002/j.1460-2075.1991.tb08026.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eilers A., Georgellis D., Klose B., Schindler C., Ziemiecki A., Harpur A. G., Wilks A. F., Decker T. Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages. Mol Cell Biol. 1995 Jul;15(7):3579–3586. doi: 10.1128/mcb.15.7.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hattori M., Abraham L. J., Northemann W., Fey G. H. Acute-phase reaction induces a specific complex between hepatic nuclear proteins and the interleukin 6 response element of the rat alpha 2-macroglobulin gene. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2364–2368. doi: 10.1073/pnas.87.6.2364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Horvath C. M., Darnell J. E., Jr The antiviral state induced by alpha interferon and gamma interferon requires transcriptionally active Stat1 protein. J Virol. 1996 Jan;70(1):647–650. doi: 10.1128/jvi.70.1.647-650.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ito T., Tanahashi H., Misumi Y., Sakaki Y. Nuclear factors interacting with an interleukin-6 responsive element of rat alpha 2-macroglobulin gene. Nucleic Acids Res. 1989 Nov 25;17(22):9425–9435. doi: 10.1093/nar/17.22.9425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Khan K. D., Shuai K., Lindwall G., Maher S. E., Darnell J. E., Jr, Bothwell A. L. Induction of the Ly-6A/E gene by interferon alpha/beta and gamma requires a DNA element to which a tyrosine-phosphorylated 91-kDa protein binds. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6806–6810. doi: 10.1073/pnas.90.14.6806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lütticken C., Coffer P., Yuan J., Schwartz C., Caldenhoven E., Schindler C., Kruijer W., Heinrich P. C., Horn F. Interleukin-6-induced serine phosphorylation of transcription factor APRF: evidence for a role in interleukin-6 target gene induction. FEBS Lett. 1995 Feb 27;360(2):137–143. doi: 10.1016/0014-5793(95)00076-l. [DOI] [PubMed] [Google Scholar]
  13. Mikita T., Campbell D., Wu P., Williamson K., Schindler U. Requirements for interleukin-4-induced gene expression and functional characterization of Stat6. Mol Cell Biol. 1996 Oct;16(10):5811–5820. doi: 10.1128/mcb.16.10.5811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Moriggl R., Gouilleux-Gruart V., Jähne R., Berchtold S., Gartmann C., Liu X., Hennighausen L., Sotiropoulos A., Groner B., Gouilleux F. Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype. Mol Cell Biol. 1996 Oct;16(10):5691–5700. doi: 10.1128/mcb.16.10.5691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Müller M., Laxton C., Briscoe J., Schindler C., Improta T., Darnell J. E., Jr, Stark G. R., Kerr I. M. Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways. EMBO J. 1993 Nov;12(11):4221–4228. doi: 10.1002/j.1460-2075.1993.tb06106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pellegrini S., John J., Shearer M., Kerr I. M., Stark G. R. Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway. Mol Cell Biol. 1989 Nov;9(11):4605–4612. doi: 10.1128/mcb.9.11.4605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Qureshi S. A., Leung S., Kerr I. M., Stark G. R., Darnell J. E., Jr Function of Stat2 protein in transcriptional activation by alpha interferon. Mol Cell Biol. 1996 Jan;16(1):288–293. doi: 10.1128/mcb.16.1.288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ruff-Jamison S., Zhong Z., Wen Z., Chen K., Darnell J. E., Jr, Cohen S. Epidermal growth factor and lipopolysaccharide activate Stat3 transcription factor in mouse liver. J Biol Chem. 1994 Sep 2;269(35):21933–21935. [PubMed] [Google Scholar]
  19. Sadowski H. B., Shuai K., Darnell J. E., Jr, Gilman M. Z. A common nuclear signal transduction pathway activated by growth factor and cytokine receptors. Science. 1993 Sep 24;261(5129):1739–1744. doi: 10.1126/science.8397445. [DOI] [PubMed] [Google Scholar]
  20. Schindler C., Darnell J. E., Jr Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem. 1995;64:621–651. doi: 10.1146/annurev.bi.64.070195.003201. [DOI] [PubMed] [Google Scholar]
  21. Schindler C., Fu X. Y., Improta T., Aebersold R., Darnell J. E., Jr Proteins of transcription factor ISGF-3: one gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon alpha. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7836–7839. doi: 10.1073/pnas.89.16.7836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shuai K., Schindler C., Prezioso V. R., Darnell J. E., Jr Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. Science. 1992 Dec 11;258(5089):1808–1812. doi: 10.1126/science.1281555. [DOI] [PubMed] [Google Scholar]
  23. Shuai K., Stark G. R., Kerr I. M., Darnell J. E., Jr A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma. Science. 1993 Sep 24;261(5129):1744–1746. doi: 10.1126/science.7690989. [DOI] [PubMed] [Google Scholar]
  24. Sims S. H., Cha Y., Romine M. F., Gao P. Q., Gottlieb K., Deisseroth A. B. A novel interferon-inducible domain: structural and functional analysis of the human interferon regulatory factor 1 gene promoter. Mol Cell Biol. 1993 Jan;13(1):690–702. doi: 10.1128/mcb.13.1.690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wagner B. J., Hayes T. E., Hoban C. J., Cochran B. H. The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter. EMBO J. 1990 Dec;9(13):4477–4484. doi: 10.1002/j.1460-2075.1990.tb07898.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Walaas S. I., Nairn A. C. Multisite phosphorylation of microtubule-associated protein 2 (MAP-2) in rat brain: peptide mapping distinguishes between cyclic AMP-, calcium/calmodulin-, and calcium/phospholipid-regulated phosphorylation mechanisms. J Mol Neurosci. 1989;1(2):117–127. doi: 10.1007/BF02896895. [DOI] [PubMed] [Google Scholar]
  27. Wen Z., Zhong Z., Darnell J. E., Jr Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell. 1995 Jul 28;82(2):241–250. doi: 10.1016/0092-8674(95)90311-9. [DOI] [PubMed] [Google Scholar]
  28. Yuan J., Wegenka U. M., Lütticken C., Buschmann J., Decker T., Schindler C., Heinrich P. C., Horn F. The signalling pathways of interleukin-6 and gamma interferon converge by the activation of different transcription factors which bind to common responsive DNA elements. Mol Cell Biol. 1994 Mar;14(3):1657–1668. doi: 10.1128/mcb.14.3.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhang X., Blenis J., Li H. C., Schindler C., Chen-Kiang S. Requirement of serine phosphorylation for formation of STAT-promoter complexes. Science. 1995 Mar 31;267(5206):1990–1994. doi: 10.1126/science.7701321. [DOI] [PubMed] [Google Scholar]
  30. Zhong Z., Wen Z., Darnell J. E., Jr Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science. 1994 Apr 1;264(5155):95–98. doi: 10.1126/science.8140422. [DOI] [PubMed] [Google Scholar]