FIG 3.

C. albicans Gyp1 is a PKA substrate. (A) Gyp1 is phosphorylated by PKA upon hyphal induction. Log-phase yeast cells expressing Gyp1-GFP (HZX202) were subjected to hyphal induction by switching from GMM at 30°C to prewarmed GMM–20% serum at 37°C. Aliquots were harvested at the indicated times after hyphal induction for preparation of cell lysates. PKA phosphorylation of Gyp1 was detected by Western blot analysis using the anti-PKA substrate antibody, and Gyp1 was probed with anti-GFP antibody. (B) Gyp1 can be phosphorylated by bovine PKA in vitro. Gyp1-GFP immunoprecipitated from yeast cells (HZX202) was subjected to in vitro kinase assay in the presence (+) or absence (−) of bovine PKA and then subjected to Western blot analysis using anti-PKA substrate antibody and anti-GFP antibody. (C and D) Gyp1 PKA phosphorylation levels are decreased in cyr1Δ/Δ and tpk2Δ/Δ cells upon hyphal induction. WT (HZX202), cyr1Δ/Δ (HZX232), tpk1Δ/Δ (HZX226), and tpk2Δ/Δ (HZX227) cells expressing Gyp1-GFP were processed and PKA phosphorylation of Gyp1 was analyzed as described for panel A.