FIG. 8.

CksHs2 blocks the activation and phosphorylation of p34^cdc2 by Cak1p. In vitro-translated, ³⁵S-labeled p34^cdc2 was incubated with cyclin B and partially purified CksHs2 (lanes 4 to 10). Following incubation with added Cak1p, p34^cdc2-HA was immunoprecipitated and assayed for histone H1 kinase activity (top panel). In the bottom panel, in vitro-translated p34^cdc2-HA was immunoprecipitated and incubated with GST-cyclin B and partially purified CksHs2 (lanes 4 to 10) prior to phosphorylation by GST-Cak1p in the presence of [γ-³²P]ATP. The incorporation of ³²P into p34^cdc2 was assessed by SDS-PAGE and autoradiography. Control reactions were performed in the absence of p34^cdc2 (lane 1) or of Cak1p (lane 2). The final concentrations of CksHs2 after addition of Cak1p were as follows: lane 3, no CksHs2; lane 4, 26 μM; lane 5, 7 μM; lane 6, 1.6 μM; lane 7, 410 nM; lane 8, 100 nM; lane 9, 26 nM; lane 10, 6 nM.