Cak1 is required for Kin28 phosphorylation and activation in vivo

Abstract

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.

FIG. 1

Growth rates and morphologies of temperature-sensitive cak1, cdc28, and kin28 mutants. (A) Isogenic strains carrying the indicated mutations were grown to mid-log phase at 25°C and switched to 37°C at time zero. At the indicated times, the cells were counted with a hemocytometer. (B) The indicated strains were grown for 3 h at 37°C and analyzed by Nomarski microscopy.

FIG. 2

Cdc28 and Kin28 are phosphorylated in vivo. HA epitope-tagged Cdc28, Kin28, and Pho85, under the control of their own promoters, were immunoprecipitated from cell lysates with the 12CA5 antibody and treated with phosphatase buffer alone (lanes 1, 4, and 7), λ-phosphatase (λ PP’ase) (lanes 2, 5, and 8), or λ-phosphatase in the presence of phosphatase inhibitors (lanes 3, 6, and 9). The immunoprecipitates were then subjected to immunoblotting with 12CA5.

FIG. 3

Mutation of T162 abolishes Kin28 phosphorylation, activity, and function. (A) Alignment of activating loop regions in Cdc28, Kin28, and Kin28A. The activating phosphorylation site (T169) in Cdc28 is indicated by an asterisk. (B) HA epitope-tagged wild-type Kin28 (WT) or Kin28A (A) was expressed under the control of the GAL1-10 promoter in wild-type or kin28-3 cells at 25°C. Anti-HA immunoprecipitates were prepared from cell lysates and subjected to immunoblotting to detect Kin28 (top). Kin28-associated CTD kinase activity was measured in parallel immunoprecipitates (bottom). Lane C contains a control immunoprecipitation from cells lacking epitope-tagged Kin28. The asterisk indicates a background band phosphorylated in CTD kinase assays in the absence of Kin28. (C) Growth of wild-type or kin28-3 strains containing KIN28 (WT) or kin28A (A) under the control of the GAL1-10 promoter at 25°C (top) or 37°C (bottom) on plates containing dextrose (DEX) or galactose (GAL).

FIG. 4

Phosphorylation and activity of yeast CDKs in cak1 mutants. The indicated mutant strains were grown to mid-log phase at 25°C and shifted to 37°C for 3 h. Epitope-tagged Cdc28 (A), Kin28 (B and C), Pho85 (D), and Srb10 (E) were immunoprecipitated from cell lysates and analyzed by immunoblotting with anti-HA antibodies (top panels) or kinase activity toward the indicated substrate (bottom panels). Cdc28 phosphorylation does not correlate with kinase activity in panel A, presumably because only a small fraction of Cdc28 is associated with cyclin and is active in asynchronous cells, and kinase activity should be normal even if only this fraction is phosphorylated.

FIG. 5

Kin28 phosphorylation and activity in wild-type and cak1Δ cells carrying Cak1-independent Cdc28 mutants. Myc-tagged Kin28 mobility on Western blots (top) and CTD kinase activity in immunoprecipitates (bottom) were measured in wild-type (wt) cells (lane 1), cak1-23 cells at room temperature (lane 2), and cells carrying Cak1-independent Cdc28-4324 (lanes 3 and 4) or Cdc28-5331 (lanes 5 and 6), in the presence (lanes 3 and 5) or absence (lanes 4 and 6) of the CAK1 gene. Lane C is a control sample from cells lacking epitope-tagged Kin28.

FIG. 6

Cak1-dependent activation of Kin28 in insect cells. Sf9 cells were coinfected with recombinant baculoviruses encoding the indicated combinations of Kin28, Ccl1, Tfb3, and Cak1. Kin28 was immunoprecipitated from cell lysates 2 days after infection and analyzed by Western blotting (top) and CTD kinase activity (bottom) assays. All the cells in this experiment were also coinfected with a virus encoding yeast Cdc37 (17), which increases Cak1 expression in insect cells.

FIG. 7

Regulatory pathways governing the activities of Kin28 and Cdc28 in S. cerevisiae (left), and homologous pathways in higher eukaryotes (right).