Conventional PKC-α, Novel PKC-ε and PKC-θ, but Not Atypical PKC-λ Are MARCKS Kinases in Intact NIH 3T3 Fibroblasts

• Published in: The Journal of biological chemistry Vol. 272; no. 7; pp. 4072 - 4078
• Main Authors: Überall, Florian, Giselbrecht, Sabine, Hellbert, Karina, Fresser, Friedrich, Bauer, Birgit, Gschwendt, Michael, Grunicke, Hans H., Baier, Gottfried
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14-02-1997
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Enzyme Induction; Enzyme Inhibitors - pharmacology; Indoles - pharmacology; Intracellular Signaling Peptides and Proteins; Isoenzymes - metabolism; Maleimides - pharmacology; Membrane Proteins; Mice; Molecular Sequence Data; Myristoylated Alanine-Rich C Kinase Substrate; Phorbol 12,13-Dibutyrate - pharmacology; Phosphorylation; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.7.4072

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-α or novel nPKC-ε increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-η significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-η as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-α, nPKC-ε, and nPKC-η in vitro Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-α K368R or (dominant negative) PKC-ε K436R. The fact, that the constitutively active PKC-λ A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-α and novel nPKC-ε, but not atypical aPKC-λ are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.