Stimulation of Protein Kinase C during Ca-induced Keratinocyte Differentiation

Raising the external Ca 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 (TPA) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH 2 , by membranes isolated from the Ca - or TPA-stimulated keratinocytes. Raising the external Ca concentration to 1.8 mM also triggered a 4-fold rise in the intracellular free Ca concentration. As reported elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. LopezBarahona, M., and Aaronson, S. A.(1989) J. Biol. Chem. 264, 11228-11235), TPA stimulated the phosphorylation of the PKCs substrate, the 85-kDa myristoylated alanine-rich kinase C substrate (MARCKS) protein, in intact keratinocytes, but Ca did not. Furthermore, Ca -pretreatment reduced the TPA-induced phosphorylation of the 85-kDa protein in intact cells. There was no significant increase in MARCKS phosphorylation when keratinocytes were treated with a Ca •CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca . Ca •calmodulin suppressed the ability of isolated membranes to phosphorylate the 85-kDa MARCKS holoprotein in vitro in the presence of phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate, and this inhibition was overcome by a calmodulin antagonist, the calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca •calmodulin complexes, formed by the internal Ca surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.