Altered regulation of a major substrate of protein kinase C in rat 6 fibroblasts overproducing PKC beta I.
We have shown previously that the stable overproduction of protein kinase C beta I (cPKC beta I) in rat 6 (R6) embryo fibroblasts results in multiple cellular growth abnormalities. To characterize the pathways through which cPKC beta I acts to exert its effects, we have undertaken a biochemical analysis of the cell line R6-PKC3. The subcellular distribution of cPKC beta I in unstimulated R6-PKC3 cells was approximately 80% cytosolic and approximately 20% membrane bound, and treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in translocation and down-regulation of an appreciable fraction of the cPKC beta I enzyme. However, long term TPA treatment was not sufficient to down-regulate all of the overproduced enzyme from both the cytosolic and membrane fractions. Two-dimensional gel analysis of 32P-labeled cellular phosphoproteins from either untreated or TPA-treated cultures revealed only minor qualitative differences between R6-PKC3 cells and a vector control cell line, R6-C1. On the other hand, several quantitative differences in the level of phosphorylation of discrete protein spots were seen. The most prominent phosphoprotein was a previously described 80/87-kDa protein designated MARCKS (myristoylated alanine-rich C kinase substrate). Compared with R6-C1 cells, R6-PKC3 cells exhibited a 2-3-fold increase in the basal level of phosphorylation of MARCKS and after treatment with TPA, displayed a dramatic prolongation in phosphorylation of this protein. Additionally, treatment of R6-PKC3 cells with TPA led to a prolonged increase in both the cytosolic and total cellular level of the MARCKS protein and a pronounced decrease in the level of MARCKS mRNA. Taken together, these results indicate that overproduction of cPKC beta I markedly alters several parameters of the MARCKS protein which may be responsible, at least in part, for the altered phenotype of these cells.
Department of Pharmacology, Columbia University, New York, New York 10032.
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Guadagno
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C
Borner
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Weinstein
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002460:Cell Line; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000032:analysis; D051381:Rats; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D012313:RNA; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate