S1691 Endogenous Protein Kinase D Contributes to Gq-Coupled Receptor Induction of DNA Synthesis in Swiss 3T3 Cells

Abstract

overexpressing prostaglandin dehydrogenase (18.PGDH) comprise a model with abolished basal PG production. Pyk2 expression, nearly absent in these cells, was reintroduced by transient transfection either as wild type or a kinase deficient (KD) mutant form. Pyk2 immunoprecipitates were used to phosphorylate a FAK regulatory domain segment with either wild type Y397 or mutant Y397F fused to GST. Total 32P incorporation In Vitro from 32P-ATP was measured by autoradiography. Y397 phosphorylation of GST-FAK In Vitro, and endogenous FAK in cell extracts, was detected by Western blot analysis with phosphospecific FAK Y397 antibody. Results: IEC-18 cells with siRNA-reduced Pyk2 expression exhibited similar levels of total FAK protein, but very low basal FAK Y397 phosphorylation in cell extracts. 18.PGDH cells, in comparison with parental IEC-18 cells, also exhibited selectively reduced constitutive FAK Tyr397 phosphorylation. Transfected Pyk2, but not Pyk2-KD, partially restored constitutive FAK Y397 phosphorylation in these cells. Pyk2, but not Pyk2-KD immunoprecipitates incorporated 32P into both GST-FAK and, to a lesser extent, GST-FAK(Y397F), but not GST. In particular, phospho-Y397Western blot confirmed that Pyk2 catalyzed dramatically increased FAK Y397 phosphorylation in GST-FAK. Conclusions: Taken together, these studies extend the observed correlation between Pyk2 downregulation and FAK Y397 phosphorylation in intestinal cells deprived of basal PG, indicating that Pyk2 can directly phosphorylate FAK Y397 as well as additional, as yet unidentified site(s) within the FAK regulatory domain.