Stimulation of protein kinase C during Ca(2+)-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 (TPA) 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 Ca(2+)- 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 elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. Lopez-Barahona, 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 Ca2+ did not. Furthermore, Ca(2+)-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 Ca2+.CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca2+.Ca2+.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 Ca2+ to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca2+.calmodulin complexes, formed by the internal Ca2+ surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
B R
BR
Chakravarthy
R J
RJ
Isaacs
P
P
Morley
J P
JP
Durkin
J F
JF
Whitfield
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D004789:Enzyme Activation; D047908:Intracellular Signaling Peptides and Proteins; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects*; D008565:Membrane Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium