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

Abstract

We have shown earlier that, in cells expressing the retinoblastoma protein (pRB), a protein phosphatase (PP) 1α mutant (T320A) resistant to inhibitory phosphorylation by cyclin-dependent kinases (Cdks) causes G1 arrest. In this study, we examined the cell cycle-dependent phosphorylation of PP1α in vivo using three different antibodies. PP1α was phosphorylated at Thr-320 during M-phase and again in late G1- through early S-phase. Inhibition of Cdk2 led to a small increase in PP1 activity and also prevented PP1α phosphorylation. In vitro, PP1α was a substrate for Cdk2 but not Cdk4. In pRB-deficient cells, phosphorylation of PP1α occurred in M-phase but not at G1/S. G1/S phosphorylation was at least partially restored after reintroduction of pRB into these cells. Consistent with this result, PP1α phosphorylated at Thr-320 co-precipitated with pRB during G1/S but was found in extracts immunodepleted of pRB in M-phase. In conjunction with earlier studies, these results indicate that PP1α may control pRB function throughout the cell cycle. In addition, our new results suggest that different subpopulations of PP1α regulate the G1/S and G2/M transitions and that PP1α complexed to pRB requires inhibitory phosphorylation by G1- specific Cdks in order to prevent untimely reactivation of pRB and permit transition from G1- to S-phase and/or complete S-phase.