PT  - JOURNAL ARTICLE
AU  - Eugene Ciccimaro
AU  - Steven K. Hanks
AU  - Ian A. Blair
TI  - Quantification of Focal Adhesion Kinase Activation Loop Phosphorylation as a Biomarker of Src Activity
AID  - 10.1124/mol.108.052464
DP  - 2009 Mar 01
TA  - Molecular Pharmacology
PG  - 658--666
VI  - 75
IP  - 3
4099  - http://molpharm.aspetjournals.org/content/75/3/658.short
4100  - http://molpharm.aspetjournals.org/content/75/3/658.full
SO  - Mol Pharmacol2009 Mar 01; 75
AB  - A recently developed stable isotope dilution liquid chromatography-multiple reaction/mass spectrometry method to quantify focal adhesion kinase (FAK) activation loop phosphorylation was used to study endogenous Src kinase activity. This revealed that bis-phosphorylated pTyr576/Tyr577-FAK was a biomarker of Src activity and inactivation in vitro and in cell culture. Mouse embryonic fibroblasts (MEFs) expressing endogenous Src family kinases contained 65% unmodified Tyr576/Tyr577, 33% mono-phosphorylated-pTyr576-FAK, and 6% bis-phosphorylated-pTyr576/pTyr577-FAK. In contrast, MEFs expressing oncogenic Y529FSrc contained 38% unmodified Tyr576/Tyr577-FAK, 29% mono-phosphorylated-pTyr576-FAK, and 19% bis-phosphorylated-pTyr576/pTyr577-FAK. This new method has made it possible to accurately determine the absolute amounts of FAK phosphorylation that occur after Src inhibition in cell culture and in vitro with increasing concentrations of the Src inhibitor N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530). Phosphorylation of FAK at Tyr576/Tyr577 was inhibited by AZD0530 in a dose-dependent manner both in cell culture and in vitro. However, there was a substantial difference in the ability of AZD0530 to inhibit Src that was constitutively activated in a cellular context (IC50 = 2.12 μM) compared with the isolated enzyme (IC50 = 0.14 μM). When normal MEFs and Y529FSrc-expressing MEFs were treated with pervanadate (a global phosphatase inhibitor), pTyr576/pTyr577-FAK accounted for almost 60% of the total FAK present in the cells. This suggests that activation loop phosphorylation is regulated by tyrosine phosphatases. These results confirm that FAK phosphorylation is a useful biomarker of Src inhibition in vivo. The accuracy and specificity of stable isotope dilution liquid chromatography-mass spectrometry methodology offers significant advantages over current immunochemical approaches for monitoring Src activity. The American Society for Pharmacology and Experimental Therapeutics