Differential mass spectrometry and immuno-analysis of FAK and paxillin following inhibition of FA kinase activity with PF-562,271

GRIGERA, PR. , PINTO, A., BORGMAN C., SHERMAN,N, FOX, J. AND J.T.PARSONS

Dulles, VA, EE.UU.

Workshop; Cell Migration Consortium Annual Meeting; 2009

Cell Migration Consortium/NIH/NIGMS

SUMMARY: We have used differential mass spectrometric approaches (e.g., labeling with 16O/18O) and immunoblotting with phospho-site specific antibodies to  18 defined  tyrosine and serine phosphorylation sites to assess the changes in phosphorylation of Focal Adhesion Kinase (FAK) and paxillin (Pax) following inhibition of FAK catalytic activity with the highly specific inhibitor PF-562,271.  We have previously mapped the utilized sites of phosphorylation of FAK (18 sites) and paxillin (38 sites).  In this study we determine how the phosphorylation of these sites is changed when cells adhere to fibronectin in the absence or presence of a highly specific inhibitor of FAK, PF-562,271.  We describe the changes in phosphorylation of a total of 11 sites in FAK and Pax using differential MS approaches and assess the changes in phosphorylation of 18 sites using antibodies to phospho-sites in FAK and Pax.