Figure 5.

Msn2 PKA-dependent phosphorylation is not cell cycle regulated. (A) Wild-type cells were synchronized in G1 with mating pheromone (α-factor, 5 μg/mL) for 2 hr and released at 25°C. Samples were taken at the indicated time points and analyzed for Msn2 PKA-dependent phosphorylation by immunoblotting (top panel) and for nuclear division by microscopy (bottom panel). The Western blot was probed with P-Msn2 (α-CREB), α-Msn2, α-Clb2, or α-Pgk1. The bar graph (top right) is the relative ratio of P-Msn2/total Msn2 analyzed from two independent experiments. A value of 1 was assigned to the ratio of P-Msn2/total Msn2 in the wild-type cells. (B) Wild-type cells were grown to mid-logarithmic phase at 25°C in YPD medium and either kept in log phase (Log) or treated with 20 μg/mL nocodazole (Nz) for 2 hr at 25°C. Western blots were probed with P-Msn2 (α-CREB), α-Msn2, and α-Clb2 as a marker for cell-cycle arrest. The asterisk represents the significant difference between the P-Msn2 levels upon Nz treatment vs. log phase cells (P < 0.05).