Constitutively active protein phosphatase 1alpha causes Rb-dependent G1 arrest in human cancer cells.
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, PP1alpha, and a constitutively active mutant of this subunit (PP1alphaT320A) on G1 progression, proliferation rates, and cell viability. In treated cells, PP1alpha levels were elevated 6-16-fold and remained stable for at least 48 hours. In Rb-positive cells, PP1alphaT320A, but not PP1alpha, 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 PP1alpha.


CONCLUSIONS
The difference between the effects of wild-type and mutant forms of PP1alpha suggests that PP1alpha 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 PP1alpha requires functional Rb to induce growth arrest, and that possibly another pool of PP1alpha induces cell death. This identifies PP1 as a potential target for therapeutic anti-proliferative strategies.
Berndt
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N
Division of Hematology/Oncology, Childrens Hospital Los Angeles, University of Southern California School of Medicine, 4650 Sunset Boulevard, Los Angeles, California 90027, USA. berndt@hsc.usc.edu
Dohadwala
M
M
Liu
C W
CW
D016923:Cell Death; D018274:Electroporation; D004789:Enzyme Activation; D016193:G1 Phase / Q000502:physiology; D018922:HL-60 Cells; D006801:Humans; D007399:Interphase / Q000502:physiology*; D009154:Mutation; D012516:Osteosarcoma; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D016160:Retinoblastoma Protein / Q000378:metabolism*; D016196:S Phase / Q000502:physiology; D012710:Serum Albumin, Bovine / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D016160:Retinoblastoma Protein; D012710:Serum Albumin, Bovine; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1