Cyclins but not Cdc28 are competent to form active Cdk complexes in the cdc37-1 mutant. Crude insect cell lysate containing recombinant Cln2-HA₃ was added to increasing amounts of wild-type (WT; RD488) or cdc37-1 (RD249-2D) lysate. Formation of an active Cdc28–Cln2-HA₃ complex was measured by immunoprecipitation of Cln2-HA₃ with 12CA5, followed by histone H1 phosphorylation (A) and by immunoblotting for Cdc28 protein associated with Cln2-HA₃ (B). (C) Crude insect cell lysate containing Cdc28-HA was incubated with increasing amounts of wild-type (WT) or cdc37-1 lysate to assess the ability of endogenous cyclins to bind and activate Cdc28. Cdc28-HA was immunopurified, and the formation of active kinase complexes was determined by histone H1 kinase assay.

Assembly of active Cdc28-cyclin complexes in vitro is independent of Cdc37. The indicated combinations of purified Cdc28-HA, GST-Clb2, human CAK, and Cdc37 were incubated for 15 min on ice and then for 15 min at 23°C. The resulting histone H1 kinase activity was measured in solution. In the final lane, Cdc37 was not included in the incubation step but added immediately prior to the histone H1 kinase assay.

Cdc28 does not bind a salt-perturbable inhibitor in cdc37-1 cells. cdc37-1 (RD249-2D p86-1) lysate (1.5 mg) was injected onto a Superose 12 sizing column, and the elution of Cdc28-HA at increasing salt concentrations (150 mM, 300 mM, 500 mM, and 1 M NaCl) was monitored by immunoprecipitation of Cdc28-HA from each fraction, followed by immunoblotting against the HA epitope. Fractions in which proteins of 669, 232, 160, 67, and 14 kDa elute are marked with arrows. Cdc28 monomer in wild-type cells normally migrates in fractions 28 to 29 (data not shown).

Cak1 protein and activity are reduced in the cdc37-1 mutant. (A) Wild-type (WT; AF42) and cdc37-1 (AF40) strains expressing HA-tagged Cak1 were incubated at 23 or 37°C for 3 h. Cells were lysed by bead beating, and Cak1 levels were compared by immunoblotting with 12CA5 against the HA epitope. (B) Wild-type (WT; AF23) and cdc37-1 (AF22) cells were incubated at 23 or 37°C for 3 h and lysed by freeze-thaw. HA epitope-tagged Cak1 was immunoprecipitated, and analysis of its kinase activity was performed by direct phosphorylation of Cdc28. (C) Wild-type and cdc37-1 lysates from the experiment in panel B were incubated with 1 μg of purified Cdc37 for 20 min at 23°C prior to immunoprecipitation of Cak1. Purified Cdc28 and GST-Clb2 were added to the Cak1 immunoprecipitate, and Cak1-dependent activation of the Cdk complex was determined by histone H1 phosphorylation. (D) Wild-type (WT; AL13) and cdc37-1 (AL14) strains expressing CAK1-HA under the control of the GAL1 promoter were incubated in galactose-containing medium at 23 or 37°C for 3 h. Cells were lysed, and Cak1 was immunoprecipitated with anti-HA monoclonal antibody. Cak1 activity was measured indirectly by its ability to phosphorylate and activate an exogenous Cdc28–GST-Clb2 complex. Activation of Cdc28–GST-Clb2 was measured by histone H1 kinase assay.

Pulse-chase analysis of the half-lives of Cdc28 and Cak1 in wild-type and cdc37-1 cells. (A) Wild-type (WT; RD270-8B) and cdc37-1 (RD249-2D p86-1) strains expressing HA epitope-tagged Cdc28 (Cdc28HA) were pulsed with [³⁵S]methionine at 23 or 37°C for 15 min and chased at the same temperature with unlabeled medium for the indicated times. Similar studies were performed with wild-type (WT; AF42) and cdc37-1 (AF40) strains expressing HA epitope-tagged Cak1. Cell lysates were subjected to immunoprecipitation with an anti-HA monoclonal antibody. (B) Wild-type (WT; RD270-8B) and cdc37-1 (RD249-2D p86-1) strains expressing HA epitope-tagged Cdc28 were pulsed with [³⁵S]methionine at 23°C for 30 min and chased with unlabeled medium at 37°C for the indicated times. Similar studies were performed with wild-type (WT; AF42) and cdc37-1 (AF40) strains expressing HA epitope-tagged Cak1. Cell lysates were subjected to immunoprecipitation with an anti-HA monoclonal antibody.

Cdc37 upregulates Cak1 expression in insect cells. Insect cells were infected with baculoviruses encoding the following kinases: Cdc28 with a C-terminal six-His tag (Cdc28-C6His), Cak1 with an N-terminal six-His tag (N6His-Cak1), and Cak1 with a C-terminal six-His tag (Cak1-C6His). Cells were infected either singly with each kinase virus (left lanes) or simultaneously with a baculovirus encoding Cdc37 (right lanes). The infected insect cells were lysed, and total cell protein was resolved by SDS-PAGE and stained with Coomassie brilliant blue. Sizes are indicated in kilodaltons.