THROMBIN-INDUCED PHOSPHORYLATION OF THE MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE (MARCKS) PROTEIN IN BOVINE PULMONARY-ARTERY ENDOTHELIAL-CELLS

Myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate that is targeted to the plasma membra ne by an aminoterminal myristoyl group. In its nonphosphorylated form, MARCKS cross-links F-actin and binds calmodulin (CaM) reciprocally. H owever, upon phosphorylation by PKC, MARCKS releases the actin or CaM. MARCKS may therefore act as a CaM sink in resting cells and regulate CaM availability during cell activation. We have demonstrated previous ly that thrombin-induced myosin light chain (MLC) phosphorylation and increased monolayer permeability in bovine pulmonary artery endothelia l cells (BPAEC) require both PKC- and CaM-dependent pathways. We there fore decided to investigate the phosphorylation of MARCKS in BPAEC to ascertain whether this occurs in a temporally relevant manner to parti cipate in the thrombin-induced events. MARCKS is phosphorylated in res ponse to thrombin with a time course similar to that seen with MLC. As expected, MARCKS is also phosphorylated by phorbol 12-myristate 13 ac etate (PMA), a PKC activator, but with a slower onset and more prolong ed duration. Bradykinin also enhances MARCKS phosphorylation in BPAEC, but histamine does not. MARCKS is distributed evenly between the memb rane and cytosol in BPAEC, and neither thrombin nor PMA caused signifi cant translocation of the protein. Specific PKC inhibitors attenuated MARCKS phosphorylation by either thrombin or PMA. Since thrombin-induc ed MLC phosphorylation is also attenuated by these inhibitors, MARCKS may be involved in MLC kinase activation and subsequent BPAEC contract ion. W7, a CaM antagonist, enhances the phosphorylation of MARCKS. Thi s was expected since CaM binding to MARCKS has been shown to decrease MARCKS phosphorylation by PKC. On the other hand, tyrosine kinase inhi bitors, genistein and tyrphostin, attenuate MARCKS phosphorylation but have no effect on MLC phosphorylation, suggesting that MARCKS may be phosphorylated by kinases other than PKC. Phosphorylation of MARCKS ou tside the PKC phosphorylation domain would not be expected to induce t he release of CaM. These data provide support for the hypothesis that MARCKS may serve as a regulator of CaM availability in BPAEC. (C) 1996 Wiley-Liss, Inc.