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.
Guadagno
S N
SN
Department of Pharmacology, Columbia University, New York, New York 10032.
Borner
C
C
Weinstein
I B
IB
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