Serine 123 phosphorylation enhances the ability of dog p21 to block S phase entry and subsequently to suppress cell proliferation.
A, Cf2Th cells uninduced or induced to express wild-type p21, dog p21(S123A), and dog p21(S123D) for 24 h. The levels of p21, Rb, PCNA, cyclin A, cyclin B, cdk1, and actin were determined by Western blot analysis. B, DNA histogram analysis of Cf2Th cells in the absence or presence of wild-type dog p21, dog p21(S123A), or dog p21(S123D). Cf2Th cells were uninduced or induced to express wild-type dog p21 (top panel), dog p21(S123D) (middle panel), and dog p21(S123A) (bottom panel) for 72 h, and the percentage of cells in each phase of the cell cycle was determined by DNA histogram analysis. DNA content was quantified using the data from at least three independent experiments. C–E, growth rates of Cf2Th cells in the presence or absence of wild-type dog p21 (C), dog p21(S123D) (D), or dog p21(S123A) (E) over a 9-day period. Error bars, S.E. F, upper panel, colony formation assay performed using Cf2Th cells uninduced or induced to express wild-type dog p21, dog p21(S123D), or dog p21(S123A) for 14 days. Lower panel, relative percentage of colony numbers shown in upper panel. Error bars, S.D.