Inhibitory phosphorylation of PP1 alpha catalytic subunit during the G(1)/S transition

We have shown earlier that, in cells expressing the retinoblastoma protein (pRB), a protein phosphatase (PP) 1 alpha mutant (T320A) resistant to inhib itory phosphorylation by cyclin-dependent kinases (Cdks) causes G(1) arrest . In this study, we examined the cell cycle-dependent phosphorylation of PP 1 alpha in, vivo using three different antibodies. PP1 alpha was phosphoryl ated at Thr-320 during M-phase and again in late G(1)- through early S-phas e. Inhibition of Cdk2 led to a small increase in PP1 activity and also prev ented PP1 alpha phosphorylation. In vitro, PP1 alpha was a substrate for Cd k2 but not Cdk4. In pRB-deficient cells, phosphorylation of PP1 alpha occur red in RI-phase but not at G(1)/S G(1)/S phosphorylation was at least parti ally restored after reintroduction of pRB into these cells. Consistent with this result, PP1 alpha phosphorylated at Thr-320 co-precipitated with pRB during G(1)/S but was found in extracts immunodepleted of pRB in M-phase. I n conjunction with earlier studies, these results indicate that PP1 alpha m ay control PRE function throughout the cell cycle. In addition, our new res ults suggest that different subpopulations of PP1 alpha regulate the G(1)/S and G(2)/M transitions and that PP1 alpha complexed to pRB requires inhibi tory phosphorylation by G(1)-specific Cdks in order to prevent untimely rea ctivation of PRE and permit transition from G(1)- to S-phase and/or complet e S-phase.