CONSTITUTIVELY ACTIVE PROTEIN PHOSPHATASE 1-ALPHA CAUSES RB-DEPENDENTG1 ARREST IN HUMAN CANCER-CELLS

Background: The retinoblastoma protein (Rb) needs to be phosphorylated by cyclin-dependent kinases (CDKs) before mammalian cells can enter t he S phase of the cell cycle. As protein phosphatase 1 (PP1) activates Rb and is itself a target for inhibitory phosphorylation by CDKs in v itro, we asked whether any effects of PP1 on cell cycle progression de pend on its phosphorylation and are mediated through Rb. Results: Usin g electrotransfer of recombinant protein into Rb-positive and Rb-negat ive cells, we have compared the effects of a wild-type PP1 catalytic s ubunit, PP1 alpha, and a constitutively active mutant of this subunit (PP1 alpha T320A) on G1 progression, proliferation rates, and cell via bility. In treated cells, PP1 alpha levels were elevated 6-16-fold and remained stable for at least 48 hours. In Rb-positive cells, PP1 alph a T320A, but not PP1 alpha, caused cell cycle arrest in late G1, which was associated with a lack of Rb phosphorylation. In Rb-negative cell s, neither wild-type nor mutant phosphatase caused any change in cell cycle progression. Increased cell death was observed in both Rb-positi ve and Rb-negative cells, however, upon introduction of excess PP1 alp ha. Conclusions: The difference between the effects of wild-type and m utant forms of PP1 alpha suggests that PP1 alpha has the potential to arrest cell growth in G1 unless it is inactivated by periodic phosphor ylation at Thr320, presumably by CDKs that regulate passage through th e G1-S cell cycle transition. Together, the effects in both cell types suggest that PP1 alpha requires functional Rb to induce growth arrest , and that possibly another pool of PP1 alpha induces cell death. This identifies PP1 as a potential target for therapeutic anti-proliferati ve strategies.