BOMBESIN, ENDOTHELIN AND PLATELET-DERIVED GROWTH-FACTOR INDUCE RAPID TRANSLOCATION OF THE MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE IN SWISS 3T3 CELLS

We analyzed the effect of growth factors on the localization of the 80-kDa acidic myristoylated alanine-rich C-kinase substrate (80-kDa MARCKS), the major protein kinase C (PKC) substrate, in Swiss 3T3 fibroblasts. Virtually all 80-kDa MARCKS of quiescent cultures of these cells was membrane bound. However, within 40 min after addition of bombesin (10 nM) to these cells, the content of 80-kDa MARCKS in the cytoplasmic fraction increased 25-fold. Phosphorylated 80-kDa MARCKS was detectable in the cytoplasmic fraction as early as 30 s after addition of bombesin and the translocation was sustained for 6 h i.e. until 80-kDa MARCKS became down-regulated. The ability of bombesin to stimulate translocation of 80-kDa MARCKS was dose-dependent (concentration required to produce 50% of the effect was 0.6 nM bombesin) and was abolished by the specific antagonist [Leu(14,13)Psi(14)CH(2)NH]bombesin. Furthermore, platelet-derived growth factor (PDGF) stimulated a dose-dependent (concentration required to produce 50% of the effect was 3 ng/ml) translocation which was comparable to that induced by bombesin in terms of kinetics and magnitude. Translocation was independent of continuous protein synthesis, but dependent on active PKC. Depletion or inhibition of PKC activity abolished the 80-kDa MARCKS translocation induced by either bombesin or PDGE Furthermore, the neuropeptides beta-endothelin, bradykinin, and vasopressin, which are known to stimulate PKC activity, also promoted translocation. In contrast, epidermal growth factor, insulin and forskolin, which do not activate PKC, failed to cause such an effect. Translocation of 80-kDa MARCKS was also observed in Rat1 cells treated with phorbol ester, PDGF and beta-endothelin. We conclude that the translocation of 80-kDa MARCKS from the membrane to the cytosol is an early response to a variety of growth-promoting factors that stimulate PKC through different signal-transduction pathways.