Characterization of paxillin LIM domain-associated serine threonine kinases: Activation by angiotensin II in vascular smooth muscle cells

Recently we reported a novel means of regulating LIM domain protein functio n. Paxillin LIM zinc-finger phosphorylation in response to cell adhesion re gulates the subscellular localization of th is cytoskeletal adaptor protein to focal adhesions, and also modulates cell adhesion to fibronectin (Brown et al. [1998] Mol. Biol. Cell 9:1803-1816). In the present study, we chara cterize further the protein kinases that phosphorylate paxillin LIM2 on thr eonine and LIM3 on serine. Analysis of the subcellular distribution of the LIM kinases demonstrated that the LIM3 protein kinase, but not the LIM2 kin ase, resides within a detergent-insoluble fraction. The activities of the p axillin LIM domain kinases are differentially regulated during embryogenesi s, and analysis of tissue distribution indicated a specificity in expressio n patterns between the LIM2 and LIM3 kinases. In addition, these protein ki nases were refractory to inhibition by a panel of broad-spectrum serine/thr eonine kinase inhibitors, suggesting a novel derivation. The paxillin prote in kinase activities were stimulated in serum-starved CHO.K1 cells by the m itogen phorbol myristate acetate (PMA), and by PMA and angiotensin II in ra t aortic smooth muscle cells. In vivo labeling, phosphoamino acid analysis, and phosphopeptide mapping of paxillin immunoprecipitated from angiotensin Ii-stimulated smooth muscle cells confirmed an induction of paxillin serin e/threonine phosphorylation and supports the contention that these newly id entified paxillin kinases are dynamic components of growth factor signaling through the cytoskeleton. J. Cell. Biochem. 76:99-108, 1999. (C) 1999 Wile y-Liss, Inc.