Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling.
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.
Li
S
S
Center for Blood Research, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Couvillon
A D
AD
Brasher
B B
BB
Van Etten
R A
RA
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002461:Cell Line, Transformed; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020014:K562 Cells; D020935:MAP Kinase Signaling System; D013058:Mass Spectrometry; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction; D020839:Son of Sevenless Protein, Drosophila / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D010748:Phosphopeptides; D011506:Proteins; D020839:Son of Sevenless Protein, Drosophila; D014443:Tyrosine; D066246:ErbB Receptors; D016044:Fusion Proteins, bcr-abl; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins