Abstract
Cyclin‐dependent kinase 2 (Cdk2) phosphorylates Thr320 of protein phosphatase 1α (PP1α) in late G~1~, thereby inhibiting its activity. Phosphorylation‐resistant PP1αT320A, 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 PP1α‐mediated G~1~ arrest and PP1α phosphorylation in late G~1~ require the presence of pRb, indicating that PP1α is a crucial regulator of the pRb pathway, which is almost invariably mutated in human cancer. These findings prompted us to investigate whether PP1α 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 PP1αT320A, but not PP1α. Likewise, cells expressing PP1αT320A or PP1αT320A fused to green fluorescent protein (GFP) were unable to form colonies in soft agar, regardless of whether PP1α constructs were co‐transfected with ras/cyclin D1 or transfected into stably transformed cells. Overexpressed wild‐type (Wt) PP1α and GFP‐PP1α were phosphorylated in Thr320, most likely explaining its lack of effect. Expression of GFP‐PP1αT320A was associated with caspase‐cleaved pRb in Western blots (WB) and morphological signs of cell death. These findings demonstrate that PP1α activity can override oncogenic signaling by causing cell‐cycle arrest and/or apoptosis rather than restoring contact inhibition or anchorage dependence. © 2006 Wiley‐Liss, Inc.