title: Grb2 regulates Stat3 activation negatively in epidermal growth factor signalling.
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.
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors*; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000502:physiology*; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D015850:Interleukin-6; D011506:Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; D017027:Protein Tyrosine Phosphatases