FIGURE 7.

Dual TPPP1 phosphorylation by Cdk and ROCK inhibits its regulation of cell proliferation. A, U2OS cells stably expressing wild-type TPPP1, TPPP1^3Ala/4Ala, TPPP1^3Ala/4Glu, TPPP1^3Glu/4Ala, TPPP1^3Glu/4Glu, or vector were synchronized in the G₂/M-phase by nocodazole treatment (200 ng/ml) and the mitotic population was collected by shake-off. After nocodazole washout and re-plating, cells were harvested at the indicated time points fixed in 70% ethanol, stained with PI, and analyzed by flow cytometry. 1N represents the G₁-phase cell population, with only one copy of the genome, whereas 2N is the G₂/M-phase population with two copies of the genome. B, proliferation assays of the cells described in A were performed as described under “Experimental Procedures.”