Protein phosphatase 1alpha activity prevents oncogenic transformation.
Cyclin-dependent kinase 2 (Cdk2) phosphorylates Thr320 of protein phosphatase 1alpha (PP1alpha) in late G(1), thereby inhibiting its activity. Phosphorylation-resistant PP1alphaT320A, acting as a constitutively active (CA) mutant, causes a late G(1) arrest by preventing the phosphorylation and inactivation of the retinoblastoma protein (pRb). Both PP1alpha-mediated G(1) arrest and PP1alpha phosphorylation in late G(1) require the presence of pRb, indicating that PP1alpha is a crucial regulator of the pRb pathway, which is almost invariably mutated in human cancer. These findings prompted us to investigate whether PP1alpha interferes with oncogenic transformation. The ability of NIH 3T3 cells to form foci after transformation with ras/cyclin D1 was significantly inhibited by co-transfection with PP1alphaT320A, but not PP1alpha. Likewise, cells expressing PP1alphaT320A or PP1alphaT320A fused to green fluorescent protein (GFP) were unable to form colonies in soft agar, regardless of whether PP1alpha constructs were co-transfected with ras/cyclin D1 or transfected into stably transformed cells. Overexpressed wild-type (Wt) PP1alpha and GFP-PP1alpha were phosphorylated in Thr320, most likely explaining its lack of effect. Expression of GFP-PP1alphaT320A was associated with caspase-cleaved pRb in Western blots (WB) and morphological signs of cell death. These findings demonstrate that PP1alpha activity can override oncogenic signaling by causing cell-cycle arrest and/or apoptosis rather than restoring contact inhibition or anchorage dependence.
Division of Hematology/Oncology, Childrens Hospital Los Angeles, The University of Southern California Keck School of Medicine, Los Angeles, 90027, USA.
Cathy W Y
CW
Liu
Rui-Hong
RH
Wang
Norbert
N
Berndt
D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D019938:Cyclin D1 / Q000037:antagonists & inhibitors / Q000235:genetics; D006801:Humans; D051379:Mice; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism*; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000235:genetics
D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D013912:Threonine; D010749:Phosphoprotein Phosphatases; D020169:Caspases; D018631:ras Proteins