FIG. 5.

Pak4-induced cell cycle arrest and premature senescence are mediated by the ERK pathway. A. ERK kinase activity is elevated in primary MEFs infected with activated Pak4. ERK kinase activity was assayed in cell populations infected with empty vector (E.V.), activated PAK4 (PAK4*), or RasV12. ERK kinase activity was measured by immunocomplex kinase assay at day 2 postselection using MBP as a substrate. Levels of endogenous ERK were assessed by Western blotting using an anti-ERK antibody. B. Inhibition of MEK abrogates the growth inhibition triggered by Pak4. Growth curves of MEFs infected with empty vector (pLPC), activated PAK4 (PAK4*), or RasV12 are indicated. Cells were either treated with 50 μM of the MEK inhibitor PD 98059 (PD) or left untreated (NO). Open bars indicate empty vector, black bars indicate activated Pak4, and gray bars indicate activated Ras. d4 is day 4 postselection; d6 is day 6 postselection. For each condition, the percentage of cells relative to the empty vector control is indicated. C. Inhibition of MEK abrogates the effects of Pak4 on BrdU incorporation. The percentages of BrdU-incorporating cells at day 6 postselection in cell populations infected with empty vector, activated PAK4 (PAK4*), or RasV12 are shown (open bars indicate untreated cells; checked bars indicate cells treated with 50 μM PD98059). D and E. Inhibition of MEK abrogates the induction of SA-β-Gal expression in response to Pak4. Percentages of cells positive for SA-β-Gal (at pH 6.0) at day 4 (open bars) and at day 6 (closed bars) postselection are shown. NO is untreated; PD is PD98059-treated cells. Cells were scored as in Fig. 1F. Typical fields of cells are shown in panel E. SA-β-Gal-positive cells stain blue. F. Inhibition of MEK abrogates Pak4-induced transformation. NIH 3T3 cells stably transfected with either empty vector (E.V.) or activated PAK4 (PAK4*) were grown in soft agar for 2 weeks in the absence or presence of the indicated drugs. Colonies are shown at magnification ×4.