OKADAIC ACID REGULATION OF THE RETINOBLASTOMA GENE-PRODUCT IS CORRELATED WITH THE INHIBITION OF GROWTH FACTOR-INDUCED CELL-PROLIFERATION INMOUSE FIBROBLASTS

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, w as used to study the mechanism of action of transforming growth factor beta (TGF-beta) on cell cycle progression in C3H/10T1/2 mouse embryon ic fibroblasts, where TGF-beta exerts a growth-stimulatory effect. Con centrations of okadaic acid as low as 5 nM inhibited TGF-beta (5 ng/ml )- or 10% serum-induced [H-3]thymidine incorporation into postconfluen t, quiescent cells. Further, these inhibitory effects were observed wh en okadaic acid was added as late as 10 hr after TGF-beta or serum sti mulation. Since C3H/10T1/2 fibroblasts undergo the G1/S transition at 10-14 hr after TGF-beta and 8-12 hr after serum stimulation, these obs ervations indicate that a phosphatase activity may be required for S-p hase entry. In a parallel experiment, okadaic acid partially inhibited TGF-beta-induced [C-14]leucine incorporation by 20-65%, depending upo n the okadaic acid concentration. In conjunction with the effect of ok adaic acid on DNA and protein synthesis, Western blot analysis indicat ed that okadaic acid inhibited phosphorylation of the retinoblastoma g ene product and decreased its protein level, even when added 10 hr aft er TGF-beta or 8 hr after serum stimulation. These findings strongly s uggest that protein phosphatases play a pivotal role for S-phase entry in mouse fibroblasts. Moreover, protein phosphatases may be required in the intermediate steps of TGF-beta or serum growth factor signal-tr ansduction pathways for the stimulation of phosphorylation of the reti noblastoma protein, especially in late G1.