Figure 4.

Specific phosphorylation of p150 by Cdc7-Dbf4 stabilizes p150 as a monomer. (A) Scheme: p150 was phosphorylated with ATPγ³²P using either hCdk2-hCyclin A or xCdc7-xDbf4, crosslinked with EGS and subjected to SDS–PAGE to resolve monomeric and dimeric forms of p150. (B) Monomer and dimer of p150 indicated by white and black arrowheads, respectively, are shown by autoradiography (top, ³²P) and western blot (bottom, total). As a control, kinase was omitted. Asterisk denotes degradation products, and the position of 150 and 250 kDa molecular weight (MW) marker is indicated. (C) Analysis as in (B) using either wild-type Cdc7-Dbf4 (WT) or the kinase-dead Cdc7(D165N) mutant (KD). Cdc7, cell division cycle 7 (xCdc7, Xenopus Cdc7); Dbf4, Dumb bell former 4 (xDbf4, Xenopus Dbf4); EGS, ethylene glycol bis[succinimidyl succinate]; hCdk2, human cyclin-dependent kinase 2; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.