Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry.

ABSTRACT: A vital point of convergence for many signaling pathways at cellular focal adhesions is the interaction of two nonreceptor tyrosine kinases, focal adhesion kinase (FAK) and Src. The binding of Src to FAK leads to the phosphorylation of Y(576) and Y(577), located within the activation loop domain of FAK. However, it has not been possible previously to determine the absolute quantitative relationship between phosphorylated and nonphosphorylated forms of this activation loop domain in cells undergoing normal metabolism. We have developed a stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) technique that allows such determinations to be made. Isotopically labeled and phosphorylated FAK protein standards were synthesized and used to control

SUBMITTER: Ciccimaro E 

PROVIDER: S-EPMC2706532 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature