CA2-CALMODULIN PREVENTS MYRISTOYLATED ALANINE-RICH KINASE-C SUBSTRATEPROTEIN-PHOSPHORYLATION BY PROTEIN-KINASE CS IN C6 RAT GLIOMA-CELLS()

Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) acti vity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O -tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expect ed, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myri stoylated alanine-rich protein kinase C substrate (MARCKS) protein, io nomycin unexpectedly did not. Instead, ionomycin reduced the basal MAR CKS phosphorylation. Pretreating the glioma cells with ionomycin preve nted TPA-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS pho sphorylation by ionomycin required external Ca2+ because they were bot h abolished by adding 5 mM EGTA to the culture medium, Recently (Chakr avarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and Whitfiel d, J. F. (1995) J, Biol. Chem, 270, 1362-1368), we proposed that Ca2calmodulin complexes block MARCKS phosphorylation by the activated PKC s in keratinocytes stimulated by raising the external Ca2+ concentrati on. In the present experiments calmodulin prevented MARCKS phosphoryla tion by TPA-stimulated PKCs in glioma cell lysates, and this blockade was lifted by a calmodulin antagonist, the calmodulin-binding domain p eptide. But, physiologically more significant, pretreating intact glio ma cells with a cell-permeable calmodulin antagonist, calmidazolium, p revented ionomycin from blocking MARCKS phosphorylation by PKCs in uns timulated and TPA-stimulated cells. The effect of ionomycin on MARCKS phosphorylation was not due to the stimulation of Ca2+ calmodulin-depe ndent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, did not stop ionomycin from pr eventing MARCKS phosphorylation. The ability of ionomycin to prevent T PA-stimulated PKCs hom phosphorylating MARCKS depended on whether iono mycin was added before, with, or after TPA. Maximum blockade occurred when ionomycin was added before TPA but was less effective when added with or after TPA. These results indicate that Ca2+ calmodulin can pro foundly affect PKCs' signaling at the substrate level.