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 protei n (80-kDa MARCKS) is the major protein-kinase-C substrate in rodent fi broblasts. To elucidate its function, we transfected the cDNA coding f or 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 level s of 80-kDa MARCKS protein under quiescent and growing conditions, res pectively, compared to mock or untransfected control cells. Southern-b lot and Northern-blot analyses of Rat1-80K showed intact integration a nd correct transcription of the introduced 80-kDa MARCKS gene. The ove rexpressed 80-kDa MARCKS protein was phosphorylated and translocated f rom 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-aminohexy l)-5-chloro-1-naphthalenes and triflouperazine on the entry into the S -phase of the cell cycle in intact cells. DNA synthesis by Rat1-80K ce lls was more sensitive to either N-(6-aminohexyl)-5-chloro-1-naphthale nesulfonamide or triflouperazine than that of control cells. Our resul ts suggest that overexpression of the 80-kDa MARCKS protein reduces th e free concentration of calmodulin in the cell.