FIG. 6.

Effect of Pkh on Pkc1. (A) Pkc1 kinase activity. Yeast strains 15Dau (wild type [WT]) and INA106-3B (pkh1^D398G pkh2Δ::LEU2 [D398G]) were transformed with pDL293 [GAL1p-PKC1-HA] or pDL295 [GAL1p-PKC1(K853R)-HA]. Transformants were grown at 23°C, treated with galactose for 1 h to induce the GAL1 promoter, and then shifted to 37°C for 1 h. Pkc1-HA (wild type [WT]) or Pkc1^K853R-HA (KN) was immunoprecipitated from cell extracts. In vitro protein kinase assays were conducted with a synthetic peptide substrate corresponding to the sequence surrounding Ser-939 of Bck1, a phosphorylation site for Pkc1 (top). A parallel set of immune complexes was subjected to immunoblotting (IB) for detection of Pkc1-HA or Pkc1^K853R-HA (bottom). (B) In vitro phosphorylation of Pkc1 by Pkh2. Yeast strain 15Dau (wild type) was transformed with pKT10-GAL-HA2-PKH2 (GAL1p-HA-PKH2) or pKT10-GAL-HA2-PKH2(KN) [GAL1p-HA-PKH2(K208R)]. Transformants were grown at 30°C and treated with galactose for 1 h to induce the GAL1 promoter. HA-Pkh2 (WT) or HA-Pkh2^K208R (KN) was immunoprecipitated from cell extracts. In vitro protein kinase assays were conducted with GST-Pkc1ΔN(787-1151, K853R) (983T) and GST-Pkc1ΔN(787-1151, K853R, T983A) (983A) as substrates (top). A parallel set of immune complexes was subjected to immunodetection of HA-Pkh2 or HA-Pkh2^K208R (KN) (bottom). (C) Mutation of Pkc1 phosphorylation site. Transformants of the yeast strain SYT11-12A (pkc1^ts stt1-1) carrying the indicated plasmids were streaked onto YPGlu medium and incubated at 25 or 34°C. Plasmids were YCplac33 (vector), pE722 (PKC1), and pE738 (pkc1^T983A). (D) Alignment of amino acid sequences of the protein kinase subdomains VII and VIII. The amino acid sequences surrounding the residues equivalent to Thr-308 of PKB are highly conserved in these protein kinases. The positions of sites in Ste7 that are phosphorylated by Ste11 are indicated by asterisks, and the position of the substituted Ser residue in Ste7^S368P is indicated by an arrow.