CONVENTIONAL PKC-ALPHA, NOVEL PKC-EPSILON AND PKC-THETA, BUT NOT ATYPICAL PKC-LAMBDA ARE MARCKS KINASES IN INTACT NIH 3T3 FIBROBLASTS

Phosphorylation of myristoylated alanine-rich protein kinase C substra te (MARCKS) in intact cells has been employed as an indicator for acti vation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, re mained 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-alpha or novel nPKC-epsilon increased phosphorylation of endogenous MARCKS in the absence of phor bol 12,13-dibutyrate in intact mouse fibroblasts, implicating that eac h of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation, Similarly, ectopic expression of a constitutively ac tive mutant of PKC-theta significantly increased MARCKS phosphorylatio n compared to vector controls, identifying PKC-theta as a MARCKS kinas e, The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduc ed MARCKS phosphorylation in intact cells at a similar dose-response a s enzymatic activity of recombinant isoenzymes cPKC-alpha, nPKC-epsilo n, and nPKC-theta in vitro, Consistently, phorbol 12,13-dibutyrate-dep endent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-alpha K368R or (dom inant negative) PKC-epsilon R436R, The fact, that the constitutively a ctive PKC-lambda A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involve d in this process, We conclude that under physiological conditions, co nventional cPKC-alpha and novel nPKC-epsilon, but not atypical aPKC-la mbda are responsible for MARCKS phosphorylation in intact NIH 3T3 fibr oblasts.