Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Dec 1;376(Pt 2):457–464. doi: 10.1042/BJ20030668

Grb2 regulates Stat3 activation negatively in epidermal growth factor signalling.

Tong Zhang 1, Jing Ma 1, Xinmin Cao 1
PMCID: PMC1223792  PMID: 14498832

Abstract

EGF (epidermal growth factor) binding to its receptor (EGFR) induces dimerization and autophosphorylation of the receptor at multiple tyrosine residues, which serve as docking sites for recruitment of proteins with SH2 (Src homology 2) domains that activate multiple downstream signalling pathways. The adaptor protein Grb2 (growth factor receptor-binding protein 2) binds to EGFR, which leads to activation of Ras-MAPK (mitogen-activated protein kinase) cascade. The latent transcription factors, STAT (signal transduction and activator of transcription), can also be activated by EGF in certain cell types. Since Ras-MAPK and STAT pathways are simultaneously stimulated by EGF, and Tyr-1086 and Tyr-1068 of EGFR are reported to be the binding sites for both Grb2 and Stat3, we investigated the possible regulatory role of Grb2 in STAT activation. In the present study, we report that transient expression of Grb2 specifically down-regulates EGF-stimulated tyrosine phosphorylation of Stat3, which leads to a repression of Stat3 transcriptional activity. In contrast, depletion of Grb2 by RNA interference substantially increases Stat3 tyrosine phosphorylation induced by EGF. The inhibition is neither mediated by a direct interaction between Grb2 and Stat3 nor via activation of tyrosine phosphatases. However, the repression was abolished by a mutation in the SH2 domain, but not the SH3 domains of Grb2, suggesting that inhibition involves binding of the receptor. Indeed, Grb2 inhibits the interaction between Stat3 and EGFR by competitive binding to the EGFR. On the other hand, Grb2 does not interact with the same sites as Stat3 on the interleukin-6 receptor and, therefore, has no effect on interleukin-6-induced tyrosine phosphorylation of Stat3. Taken together, our results demonstrate that, in EGF signalling, Grb2 regulates Stat3 activation negatively at the receptor level.

Full Text

The Full Text of this article is available as a PDF (232.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akira S., Nishio Y., Inoue M., Wang X. J., Wei S., Matsusaka T., Yoshida K., Sudo T., Naruto M., Kishimoto T. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell. 1994 Apr 8;77(1):63–71. doi: 10.1016/0092-8674(94)90235-6. [DOI] [PubMed] [Google Scholar]
  2. Batzer A. G., Rotin D., Ureña J. M., Skolnik E. Y., Schlessinger J. Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol. 1994 Aug;14(8):5192–5201. doi: 10.1128/mcb.14.8.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cao X., Tay A., Guy G. R., Tan Y. H. Activation and association of Stat3 with Src in v-Src-transformed cell lines. Mol Cell Biol. 1996 Apr;16(4):1595–1603. doi: 10.1128/mcb.16.4.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carpenter G., King L., Jr, Cohen S. Epidermal growth factor stimulates phosphorylation in membrane preparations in vitro. Nature. 1978 Nov 23;276(5686):409–410. doi: 10.1038/276409a0. [DOI] [PubMed] [Google Scholar]
  5. Chung J., Uchida E., Grammer T. C., Blenis J. STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation. Mol Cell Biol. 1997 Nov;17(11):6508–6516. doi: 10.1128/mcb.17.11.6508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coffer P. J., Kruijer W. EGF receptor deletions define a region specifically mediating STAT transcription factor activation. Biochem Biophys Res Commun. 1995 May 5;210(1):74–81. doi: 10.1006/bbrc.1995.1629. [DOI] [PubMed] [Google Scholar]
  7. Darnell J. E., Jr, Kerr I. M., Stark G. R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994 Jun 3;264(5164):1415–1421. doi: 10.1126/science.8197455. [DOI] [PubMed] [Google Scholar]
  8. Elbashir S. M., Harborth J., Lendeckel W., Yalcin A., Weber K., Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494–498. doi: 10.1038/35078107. [DOI] [PubMed] [Google Scholar]
  9. Fu X. Y., Zhang J. J. Transcription factor p91 interacts with the epidermal growth factor receptor and mediates activation of the c-fos gene promoter. Cell. 1993 Sep 24;74(6):1135–1145. doi: 10.1016/0092-8674(93)90734-8. [DOI] [PubMed] [Google Scholar]
  10. Giordano V., De Falco G., Chiari R., Quinto I., Pelicci P. G., Bartholomew L., Delmastro P., Gadina M., Scala G. Shc mediates IL-6 signaling by interacting with gp130 and Jak2 kinase. J Immunol. 1997 May 1;158(9):4097–4103. [PubMed] [Google Scholar]
  11. Gupta R. W., Mayer B. J. Dominant-negative mutants of the SH2/SH3 adapters Nck and Grb2 inhibit MAP kinase activation and mesoderm-specific gene induction by eFGF in Xenopus. Oncogene. 1998 Oct 29;17(17):2155–2165. doi: 10.1038/sj.onc.1202158. [DOI] [PubMed] [Google Scholar]
  12. Hemmann U., Gerhartz C., Heesel B., Sasse J., Kurapkat G., Grötzinger J., Wollmer A., Zhong Z., Darnell J. E., Jr, Graeve L. Differential activation of acute phase response factor/Stat3 and Stat1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. II. Src homology SH2 domains define the specificity of stat factor activation. J Biol Chem. 1996 May 31;271(22):12999–13007. doi: 10.1074/jbc.271.22.12999. [DOI] [PubMed] [Google Scholar]
  13. Jain N., Zhang T., Fong S. L., Lim C. P., Cao X. Repression of Stat3 activity by activation of mitogen-activated protein kinase (MAPK). Oncogene. 1998 Dec 17;17(24):3157–3167. doi: 10.1038/sj.onc.1202238. [DOI] [PubMed] [Google Scholar]
  14. Levy David E., Darnell J. E., Jr Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002 Sep;3(9):651–662. doi: 10.1038/nrm909. [DOI] [PubMed] [Google Scholar]
  15. Lim C. P., Cao X. Regulation of Stat3 activation by MEK kinase 1. J Biol Chem. 2001 Mar 16;276(24):21004–21011. doi: 10.1074/jbc.M007592200. [DOI] [PubMed] [Google Scholar]
  16. Lim C. P., Cao X. Serine phosphorylation and negative regulation of Stat3 by JNK. J Biol Chem. 1999 Oct 22;274(43):31055–31061. doi: 10.1074/jbc.274.43.31055. [DOI] [PubMed] [Google Scholar]
  17. Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
  18. Lufei Chengchen, Ma Jing, Huang Guochang, Zhang Tong, Novotny-Diermayr Veronica, Ong Chin Thing, Cao Xinmin. GRIM-19, a death-regulatory gene product, suppresses Stat3 activity via functional interaction. EMBO J. 2003 Mar 17;22(6):1325–1335. doi: 10.1093/emboj/cdg135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ma Jing, Zhang Tong, Novotny-Diermayr Veronica, Tan Agnes L. C., Cao Xinmin. A novel sequence in the coiled-coil domain of Stat3 essential for its nuclear translocation. J Biol Chem. 2003 May 13;278(31):29252–29260. doi: 10.1074/jbc.M304196200. [DOI] [PubMed] [Google Scholar]
  20. Marshall C. J. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev. 1994 Feb;4(1):82–89. doi: 10.1016/0959-437x(94)90095-7. [DOI] [PubMed] [Google Scholar]
  21. Olayioye M. A., Beuvink I., Horsch K., Daly J. M., Hynes N. E. ErbB receptor-induced activation of stat transcription factors is mediated by Src tyrosine kinases. J Biol Chem. 1999 Jun 11;274(24):17209–17218. doi: 10.1074/jbc.274.24.17209. [DOI] [PubMed] [Google Scholar]
  22. Ruff-Jamison S., Chen K., Cohen S. Induction by EGF and interferon-gamma of tyrosine phosphorylated DNA binding proteins in mouse liver nuclei. Science. 1993 Sep 24;261(5129):1733–1736. doi: 10.1126/science.8378774. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Sadowski H. B., Gilman M. Z. Cell-free activation of a DNA-binding protein by epidermal growth factor. Nature. 1993 Mar 4;362(6415):79–83. doi: 10.1038/362079a0. [DOI] [PubMed] [Google Scholar]
  25. Sano S., Itami S., Takeda K., Tarutani M., Yamaguchi Y., Miura H., Yoshikawa K., Akira S., Takeda J. Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis. EMBO J. 1999 Sep 1;18(17):4657–4668. doi: 10.1093/emboj/18.17.4657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000 Oct 13;103(2):211–225. doi: 10.1016/s0092-8674(00)00114-8. [DOI] [PubMed] [Google Scholar]
  27. Sengupta T. K., Talbot E. S., Scherle P. A., Ivashkiv L. B. Rapid inhibition of interleukin-6 signaling and Stat3 activation mediated by mitogen-activated protein kinases. Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11107–11112. doi: 10.1073/pnas.95.19.11107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shao Huang, Cheng Haiyun Y., Cook Richard G., Tweardy David J. Identification and characterization of signal transducer and activator of transcription 3 recruitment sites within the epidermal growth factor receptor. Cancer Res. 2003 Jul 15;63(14):3923–3930. [PubMed] [Google Scholar]
  29. Tanaka M., Gupta R., Mayer B. J. Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins. Mol Cell Biol. 1995 Dec;15(12):6829–6837. doi: 10.1128/mcb.15.12.6829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Xia Ling, Wang Lijuan, Chung Alicia S., Ivanov Stanimir S., Ling Mike Y., Dragoi Ana M., Platt Adam, Gilmer Tona M., Fu Xin-Yuan, Chin Y. Eugene. Identification of both positive and negative domains within the epidermal growth factor receptor COOH-terminal region for signal transducer and activator of transcription (STAT) activation. J Biol Chem. 2002 Jun 17;277(34):30716–30723. doi: 10.1074/jbc.M202823200. [DOI] [PubMed] [Google Scholar]
  32. Yamanaka Y., Nakajima K., Fukada T., Hibi M., Hirano T. Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation. EMBO J. 1996 Apr 1;15(7):1557–1565. [PMC free article] [PubMed] [Google Scholar]
  33. Zhang T., Kee W. H., Seow K. T., Fung W., Cao X. The coiled-coil domain of Stat3 is essential for its SH2 domain-mediated receptor binding and subsequent activation induced by epidermal growth factor and interleukin-6. Mol Cell Biol. 2000 Oct;20(19):7132–7139. doi: 10.1128/mcb.20.19.7132-7139.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES