Overexpression of the myristoylated alanine-rich C-kinase substrate in Rat1 cells increases sensitivity to calmodulin antagonists.
The acidic 80-kDa myristoylated alanine-rich C-kinase substrate protein (80-kDa MARCKS) is the major protein-kinase-C substrate in rodent fibroblasts. To elucidate its function, we transfected the cDNA coding for the 80-kDa MARCKS protein into Rat1 fibroblasts. One clone, called Rat1-80K, expressed 4.5 +/- 0.8-fold and 9.5 +/- 1.5-fold higher levels of 80-kDa MARCKS protein under quiescent and growing conditions, respectively, compared to mock or untransfected control cells. Southern-blot and Northern-blot analyses of Rat1-80K showed intact integration and correct transcription of the introduced 80-kDa MARCKS gene. The overexpressed 80-kDa MARCKS protein was phosphorylated and translocated from the membrane to the cytoplasmic fraction. Since 80-kDa MARCKS has been described as a calmodulin-binding protein in in vitro studies, we investigated the effects of the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and triflouperazine on the entry into the S-phase of the cell cycle in intact cells. DNA synthesis by Rat1-80K cells was more sensitive to either N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or triflouperazine than that of control cells. Our results suggest that overexpression of the 80-kDa MARCKS protein reduces the free concentration of calmodulin in the cell.
Imperial Cancer Research Fund, London, England.
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Rozengurt
D000818:Animals; D001692:Biological Transport; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002453:Cell Cycle / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D005347:Fibroblasts; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D013449:Sulfonamides; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D014268:Trifluoperazine; C017967:W 7; D004247:DNA; D011493:Protein Kinase C