Rho-associated kinase phosphorylates MARCKS in human neuronal cells

Myristoylated alanine-rich C kinase substrate (MARCKS) is a filamentous act in bundling protein and has multiple sites for phosphorylation, by which th e bio chemical function is negatively regulated. However, the role of such phosphorylation in physiological functions, particularly in neuronal functi ons, is not well understood. Using a phosphorylation-site specific antibody , we detected the phosphorylation of MARCKS at Ser159 by various protein ki nases. Rho-kinase, protein kinase A and protein kinase C, could introduce P -32 into human recombinant MARCKS in vitro and the phosphorylation site was confirmed to be the Ser159 residue. In human neuronal teratoma (NT-2) cell s, lysophosphatidic acid (LPA) induced MARCKS phosphorylation dose- and tim e-dependently. This phosphorylation was sensitive to Rho-kinase inhibitor H A1077. However, the phosphorylation induced by PDBu was lesser sensitive. I n a skinned NTera-2 cell system, Ca2+-independent and GTP gammaS/ATP-stimul ated phosphorylation at Ser159 was also sensitive to pre-treatment C3 toxin and HA1077. These findings suggest that the Ser159 residue of MARCKS is a target of LPA-stimulated Rho-kinase in neuronal cells, (C) 2000 Academic Pr ess.