Inhibitory phosphorylation of PP1α catalytic subunit during the G1/S transition

We have shown earlier that, in cells expressing the retinoblastoma protein (pRB), a protein phosphatase (PP) 1 alpha mutant (T320A) resistant to inhibitory phosphorylation by cyclin-dependent kinases (Cdks) causes G(1) arrest. In this study, we examined the cell cycle-dependent phosphorylation of PP1 alpha in, vivo using three different antibodies. PP1 alpha was phosphorylated at Thr-320 during M-phase and again in late G(1)- through early S-phase. Inhibition of Cdk2 led to a small increase in PP1 activity and also prevented PP1 alpha phosphorylation. In vitro, PP1 alpha was a substrate for Cdk2 but not Cdk4. In pRB-deficient cells, phosphorylation of PP1 alpha occurred in RI-phase but not at G(1)/S G(1)/S phosphorylation was at least partially 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. In conjunction with earlier studies, these results indicate that PP1 alpha may control PRE function throughout the cell cycle. In addition, our new results 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 inhibitory phosphorylation by G(1)-specific Cdks in order to prevent untimely reactivation of PRE and permit transition from G(1)- to S-phase and/or complete S-phase.