FIG. 7.

In vitro phosphorylation of p34^cdc2 by CAK stimulates binding of CksHs2. (A) Histone H1 kinase assays to assess Cak1p activation of p34^cdc2. Purified yeast Cak1p was incubated in the presence of cyclin B with in vitro-translated, ³⁵S-labeled wild-type (wt) p34^cdc2-HA (lanes 3 and 4) or p34^cdc2-HA (T161A) (lanes 5 and 6). p34^cdc2-HA was immunoprecipitated, and histone H1 kinase assays were performed on the immunoprecipitates. Controls for background histone H1 kinase activity include substituting unprogrammed reticulocyte lysate for the p34^cdc2 translation (lane 1) and substituting buffer for cyclin B (lane 2). The histone H1 kinase activity seen in lane 3 is 6% of that in lane 4 (by phosphorimage quantitation) and is due to a low level of CAK activity in the reticulocyte lysate. (B) Cak1p phosphorylation of p34^cdc2 stimulates association with CksHs2. Cak1p was incubated in the presence of cyclin B with in vitro-translated, ³⁵S-labeled wild-type p34^cdc2-HA (lanes 2 to 4) or p34^cdc2-HA (T161A) (lanes 5 and 6). In vitro-translated, ³⁵S-labeled CksHs2 was then added, and the p34^cdc2 and associated CksHs2 were immunoprecipitated and examined by SDS-PAGE and fluorography. Controls include wild-type cyclin B-p34^cdc2 and cyclin B-p34^cdc2 (T161A) that were not incubated with Cak1p (lanes 3 and 5) and controls for background association of CksHs2 with the protein A-agarose beads, substituting unprogrammed reticulocyte lysate for the p34^cdc2 translation (lane 1) or performing the experiment as for lane 4 but precipitating with protein A-agarose beads in the absence of antibody (Ab) (lane 2). (C) Both cyclin binding and Cak1p phosphorylation of p33^cdk2 are required to stimulate association with CksHs2. Monomeric HA-p33^cdk2 (lanes 3 and 4) or GST-cyclin A-p33^cdk2 (lanes 5 and 6) was incubated with GST-Cak1p (lanes 4 and 6) or a buffer control (lanes 3 and 5) and then incubated with in vitro-translated, ³⁵S-labeled CksHs2. The p33^cdk2 and associated CksHs2 were immunoprecipitated and analyzed by SDS-PAGE and fluorography. Buffer was substituted for p33^cdk2 to control for background immunoprecipitation of CksHs2 (lanes 1 and 2). (D) Cak1p phosphorylation of monomeric p33^cdk2 and GST-cyclin A-p33^cdk2. Phosphorylation of HA-p33^cdk2 (lane 2) or GST-cyclin A-p33^cdk2 (lane 3) was performed as for panel C in the presence of [γ-³²P]ATP. The incorporation of ³²P into p33^cdk2 was assessed by SDS-PAGE and autoradiography. A control reaction contained only GST-Cak1p (lane 1).