Regulation of tyrosine phosphorylation of PYK2 in vascular endothelial cells by lysophosphatidylcholine

Lysophosphatidylcholine (LPC), a component of oxidized low density lipoprot ein, exerts various biological effects on vascular endothelial cells. Howev er, the intracellular signaling of LPC is poorly understood. In this study, we investigated the involvement of proline-rich tyrosine kinase (PYK2) in LPC signaling in cultured bovine aortic endothelial cells by immunoprecipit ation and Western blotting assays. Treatment of cells with LPC promoted a r apid increase in tyrosine phosphorylation of PYK2. LPC-stimulated PYK2 phos phorylation was inhibited by calcium chelators, 1,2-bis(2-amino-phenoxy) et hane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, EGTA, protein kinase C (PKC) inhibitor, GF-109203X, or PKC depletion by phorbol esters. PYK2 phos phorylation was inhibited by treatment with cytochalasin D but with neither botulinum C3 transferase nor overexpression of a dominant negative mutant of Rho A. LPC stimulated the association of Shc with PYK2, Shc tyrosine pho sphorylation, and Grb2 binding to Shc and induced Ras activation. These res ults provide evidence that 1) LPC tyrosine phosphorylates PYK2 by calcium- and PKC-dependent mechanisms, 2) the intact cytoskeleton is required for LP C-stimulated PYK2 phosphorylation, and 3) LPC-activated Ras via the PYK2/Sh c/Grb2 signaling.