Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk.
We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.
Lennartsson
Johan
J
Ludwig Institute for Cancer Research, Biomedical Centre, P.O. Box 595, SE-751 24 Uppsala, Sweden.
Wernstedt
Christer
C
Engström
Ulla
U
Hellman
Ulf
U
Rönnstrand
Lars
L
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001665:Binding Sites; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D014443:Tyrosine; D019009:Proto-Oncogene Proteins c-kit; D019061:src-Family Kinases