Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling

Shaoguang Li^1,2, Anthony D. Couvillon^3,4, Bradley B. Brasher⁵, and Richard A. Van Etten¹

Center for Blood Research, Department of Genetics, Harvard Medical School, Boston, MA 02115 and ³Division of Signal Transduction, Beth Israel-Deaconess Medical Center, Boston, MA, USA ²Present address: The Jackson Laboratory, Bar Harbor, ME, USA ⁴Present address: Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA ⁵Present address: Enanta Pharmaceuticals, Watertown, MA, USA ¹Corresponding authors e-mail: vanetten{at}cbr.med.harvard.edu

Abstract: Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identified as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and fibroblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.

Key Words: Keywords: Abelson/chronic myeloid leukemia/CML/ SH3