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- Title
Function of activation loop tyrosine phosphorylation in the mechanism of c-Kit auto-activation and its implication in sunitinib resistance.
- Authors
DiNitto, Jonathan P; Deshmukh, Gayatri D; Zhang, Yan; Jacques, Suzanne L; Coli, Rocco; Worrall, Joseph W; Diehl, Wade; English, Jessie M; Wu, Joe C
- Abstract
The activation of receptor tyrosine kinases (RTKs) is tightly regulated through a variety of mechanisms. Kinetic studies show that activation of c-Kit RTK occurs through an inter-molecular autophosphorylation. Phosphopeptide mapping of c-Kit reveals that 14-22 phosphates are added to each mol of wild-type (WT) c-Kit during the activation. Phosphorylation sites are found on the JM, kinase insert (KID), c-terminal domains and the activation loop (A-loop), but only the sites on the JM domain contribute to the kinase activation. The A-loop tyrosine (Y(823)) is not phosphorylated until very late in the activation (>90% completion), indicating that the A-loop phosphorylation is not required for c-Kit activation. A sunitinib-resistant mutant D816H that accelerates auto-activation by 184-fold shows no phosphorylation on the A-loop tyrosine after full activation. A loss-of-phosphorylation mutation Y823F remains fully competent in auto-activation. Similar to WT and D816H, the unactivated Y823F mutant binds sunitinib and imatinib with high affinity (K(D) = 5.9 nM). But unlike the WT and D816H where the activated enzymes lose the ability to bind the two drugs, activated Y823F binds the two inhibitors effectively. These observations suggest that the A-loop of activated Y823F remains flexible and can readily adopt unactivated conformations to accommodate DFG-out binders.
- Publication
Journal of biochemistry, 2010, Vol 147, Issue 4, p601
- ISSN
1756-2651
- Publication type
Journal Article
- DOI
10.1093/jb/mvq015