Cla4 binds and phosphorylates Vac17. (A) Purified recombinant GST-Cla4 but not GST alone binds Vac17-TAP from dma1Δ dma2Δ VAC17-TAP lysates. (B) The anti-pS222 antibody recognizes wild-type Vac17-GFP but not the vac17-S222A-GFP mutant. (C) λ-Phosphatase treatment causes an increase in the electrophoretic mobility of Vac17-GFP and ablates detection by the anti-pS222 antibody. (D) Inactivation of PAKs decreases the phosphorylation of Vac17-S222 and to a lesser extent, Vac17-T240. Vac17 phosphorylation was monitored using anti-pS222 or anti-pT240 antibodies in the cla4^ts ste20Δ mutant transformed with Vac17-GFP. Cells were grown at 24°C or shifted to 37°C for 3 h before immunoprecipitation (IP) of Vac17-GFP using anti-GFP antibodies. As a positive control, Vac17-GFP immunoprecipitated from a dma1Δ mutant was phosphorylated at both sites. (E) Levels of pT240 or pS222 were normalized to GFP, and those ratios were normalized to dma1Δ. Error bars indicate SEM. n = 2. *, P < 0.05; two-tailed Student’s t test. AU, arbitrary unit. (F) 6×HIS-Vac17 (96–355) but not 6×HIS-vac17-S222A (96–355) was phosphorylated by Cla4 but not the kinase-dead cla4-K594A mutant nor in the absence of ATP. Phosphorylation was analyzed via immunoblotting with the anti-pS222 antibody. SA indicates the vac17-S222A-GFP mutant, and kd indicates the Cla4 kinase-dead mutant. Molecular mass is indicated in kilodaltons.