STIMULATION OF PROTEIN-KINASE-C DURING CA2-INDUCED KERATINOCYTE DIFFERENTIATION - SELECTIVE BLOCKADE OF MARCKS PHOSPHORYLATION BY CALMODULIN()

Raising the external Ca2+ concentration from 0.05 to 1.8 mM stimulated membrane-associated protein kinase Cs (PKCs) activity as strongly as the specific PKCs activator, 12-O-tetradecanoyl phorbol-13-acetate (TP A) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH2, by membranes isolated from the Ca2+- or TPA-stimulated keratinocytes. Raising the external Ca2+ concentration to 1.8 mM also triggered a 4- fold rise in the intracellular free Ca2+ concentration. As reported el sewhere (Moscat, J. Fleming, T. P., Molloy, C. J. Lopez-Barahona, M., and Aaronson, S. A. (1989) J. Biol. Chem. 264, 11228-11235), TPA stimu lated the phosphorylation of the PKCs substrate, the 85-kDa myristoyla ted alanine-rich kinase C substrate (MARCKS) protein, in intact kerati nocytes, but Ca2+ did not. Furthermore, Ca2+-pretreatment reduced the TPA-induced phosphorylation of the 85-kDa protein in intact cells. The re was no significant increase in MARCKS phosphorylation when keratino cytes were treated with a Ca2+.CaM-dependent phosphatase inhibitor, cy closporin A, before stimulation with 1.8 mM Ca2+.Ca2+.calmodulin suppr essed the ability of isolated membranes to phosphorylate the 85-kDa MA RCKS holoprotein in vitro in the presence of phosphatase inhibitors su ch as fluoride, pyrophosphate, and vanadate, and this inhibition was o vercome by a calmodulin antagonist, the calmodulin-binding domain pept ide. Thus, the ability of 1.8 mM Ca2+ to strongly stimulate the membra ne PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca2+.c almodulin complexes, formed by the internal Ca2+ surge, binding to, an d blocking the phosphorylation of, this PKC protein substrate.