Analysis of lipid kinases in a cdc37^S14A strain. (A) Western blot analysis of TAP-tagged Pik1 (arrow) and Pgk1 in wild-type (ser14) and cdc37^S14A (ala14) strains. (B) Analysis of Vps34; details are as described in A. (C and D) Analysis of Guk1 and Prs5 steady-state levels by Western blotting. In each case, the arrow indicates the position of the TAP-tagged protein.

Pulse-chase analysis of kinases in the cdc37^S14A mutant. (A) Pulse-chase analysis of TAP-tagged Tpk2 in wild-type (WT) and cdc37^S14A mutant (S14A) cells. Chase times are indicated in minutes. (B) Pulse-chase analysis of TAP-tagged Cmk2 in wild-type and cdc37^S14A mutant (S14A) cells. (C) Pulse labeling of Tpk2 in wild-type cells and cdc37^S14A mutant (S14A) cells in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 100 μM MG132 added 30 min before pulse labeling (5-min pulse). (D) Pulse-chase experiment of Tpk2 in the presence of 50 μM geldanamycin (+GA) or DMSO (−GA) added 1 h before labeling. (C and D) Vertical lines denote nonconsecutive lanes from the same gel. (E) Pulse labeling and immunoprecipitation of Pho85 in wild-type (lanes 1 and 2) and cdc37^S14A mutant (S14A) cells in the absence (lanes 1 and 3) or presence of 100 μM MG132 (lanes 2 and 4) added 30 min before labeling. (F) As in E except that Rim11 kinase was immunoprecipitated. (G) Pulse-chase analysis of untagged Cdc28 immunoprecipitated with anti-PSTAIRE in wild-type (lanes 1–7) and cdc37^S14A mutant (S14A; lanes 7–12) cells. The band labeled with an asterisk is nonspecific. Chase times are given in minutes. The strain background for the experiments shown in C–G is erg6Δ, which improves MG132 permeability (Lee and Goldberg, 1996; Lee et al., 1996).

Sky1 dependence on Cdc37 is temperature dependent. Activity of Sky1 after the isolation of wild-type (ser14) and cdc37^S14A mutant (ala14) cells grown at 30 and 26°C as indicated. Bars represent SEM (n = 3).

Effect of CDC37 mutation on protein kinase levels. (A) Schematic of the CDC37 locus showing the position where URA3 was inserted as well as the serine 14 to alanine mutation (S14A) in CDC37. Arrows denote the direction of transcription. (B) Four examples of unstable kinases identified with anti-TAP (cells grown at 30°C). Western blots of the same samples with anti-Pgk1 are shown as loading controls. (C) Four examples of kinases that were unchanged after culturing wild type and cdc37^S14A at 30°C. (D) Phylogenetic analysis of protein kinases used in this study. Kinases that accumulated to wild-type levels in the cdc37^S14A mutant are shown in red. Kinases in black accumulated at less than twofold compared with wild-type levels.

Regulation of kinase levels by other kinases and Cdc37. (A) Schematic of the pheromone-responsive mitogen-activated protein kinase pathway is shown at the left. Levels of Fus3 in wild-type (WT) cells (lane 1), ste11Δ cells (lane 2), and ste11Δ/cdc37^S14A cells (lane 3) are shown at the right. (B) Schematic at left shows the PKC signaling pathway. Levels of the Slt2 mitogen-activated protein kinase in wild-type cells (lane 1), bck1Δ cells (lane 2), and bck1Δ/cdc37^S14A cells (lane 3) are shown at the right. (A and B) Pgk1 levels are shown as a loading control. Vertical lines denote nonconsecutive lanes from the same gel and Western blots.

Effect of temperature on kinase accumulation and activity. (A) Western blot analysis of Rim11, Tpk2, and Yck2 (arrows) after growth of the relevant TAP-tagged cdc37^S14A strains at the indicated temperatures. Asterisks denote Pgk1, which was used as a loading control. (B) Comparison of Rim11 (lanes 1 and 2), Tpk2 (lanes 3 and 4), and Yck2 (lanes 5 and 6) levels by Western blotting in whole cell extracts from wild-type (ser14) and cdc37^S14A (ala14) cells grown at 26°C. The asterisk denotes Pgk1. (C) Activity of protein kinases in the cdc37 mutant strain. Activity of Rim11 and Tpk2 after growth of the relevant TAP-tagged wild-type (WT) and cdc37^S14A cells at 26°C. The blots above the graph show Western blot analysis of protein levels of the kinases used in the assays. Error bars represent SEM. (D) Binding of HA-Bcy1 to Tpk2 in wild-type (ser14; lanes 1 and 3) and cdc37^S14A (ala14; lanes 2 and 4) cells grown at 30 and 26°C as indicated. Top panels show Western blots of Tpk2; middle panels show HA-Bcy1 (Bcy1) after immunoprecipitation (IP) of TAP-Tpk2 using IgG–Sepharose beads; bottom panel shows levels of HA-Bcy1 in cell lysates. Note that two bands appear in the anti-HA Western blots, but only the slower migrating band coimmunoprecipitated with TAP-Tpk2 (not depicted).