Constitutively active protein phosphatase 1α causes Rb-dependent G1 arrest in human cancer cells

Abstract

Background: The retinoblastoma protein (Rb) needs to be phosphorylated by cyclin-dependent kinases (CDKs) before mammalian cells can enter the S phase of the cell cycle. As protein phosphatase 1 (PP1) activates Rb and is itself a target for inhibitory phosphorylation by CDKs in vitro, we asked whether any effects of PP1 on cell cycle progression depend on its phosphorylation and are mediated through Rb.Results: Using electrotransfer of recombinant protein into Rb-positive and Rb-negative cells, we have compared the effects of a wild-type PP1 catalytic subunit, PP1α, and a constitutively active mutant of this subunit (PP1αT320A) on G1 progression, proliferation rates, and cell viability. In treated cells, PP1α levels were elevated 6–16-fold and remained stable for at least 48 hours. In Rb-positive cells, PP1αT320A, but not PP1α, caused cell cycle arrest in late G1, which was associated with a lack of Rb phosphorylation. In Rb-negative cells, neither wild-type nor mutant phosphatase caused any change in cell cycle progression. Increased cell death was observed in both Rb-positive and Rb-negative cells, however, upon introduction of excess PP1α.Conclusions: The difference between the effects of wild-type and mutant forms of PP1α suggests that PP1α has the potential to arrest cell growth in G1 unless it is inactivated by periodic phosphorylation at Thr320, presumably by CDKs that regulate passage through the G1–S cell cycle transition. Together, the effects in both cell types suggest that PP1α requires functional Rb to induce growth arrest, and that possibly another pool of PP1α induces cell death. This identifies PP1 as a potential target for therapeutic anti-proliferative strategies.