Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes.
The Csk homologous kinase (CHK), formerly MATK, has previously been shown to bind to activated c-KIT. In this report, we characterize the binding of SH2(CHK) to specific phosphotyrosine sites on the c-KIT protein sequence. Phosphopeptide inhibition of the in vitro interaction of SH2(CHK)-glutathione S-transferase fusion protein/c-KIT from SCF/KL-treated Mo7e megakaryocytic cells indicated that two sites on c-KIT were able to bind SH2(CHK). These sites were the Tyr568/570 diphosphorylated sequence and the monophosphorylated Tyr721 sequence. To confirm this, we precipitated native CHK from cellular extracts using phosphorylated peptides linked to Affi-Gel 15. In addition, purified SH2(CHK)-glutathione S-transferase fusion protein was precipitated with the same peptide beads. All of the peptide bead-binding studies were consistent with the direct binding of SH2(CHK) to phosphorylated Tyr568/570 and Tyr721 sites. Binding of FYN and SHC to the diphosphorylated Tyr568/570 site was observed, while binding of Csk to this site was not observed. The SH2(CHK) binding to the two sites is direct and not through phosphorylated intermediates such as FYN or SHC. Site-directed mutagenesis of the full-length c-KIT cDNA followed by transient transfection indicated that only the Tyr568/570, and not the Tyr721, is able to bind SH2(CHK). This indicates that CHK binds to the same site on c-KIT to which FYN binds, possibly bringing the two into proximity on associated c-KIT subunits and leading to the down-regulation of FYN by CHK.
Price
D J
DJ
Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
Rivnay
B
B
Fu
Y
Y
Jiang
S
S
Avraham
S
S
Avraham
H
H
D000818:Animals; D004789:Enzyme Activation; D008533:Megakaryocytes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src); D019089:Stem Cell Factor / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D019089:Stem Cell Factor; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; C085377:MATK protein, human; D016392:Proto-Oncogene Proteins pp60(c-src)