FIGURE 5.

Phosphorylation of CREB by CDK1 reduced its DNA binding activity in vitro. A, mitotic HeLa extracts (see “Experimental Procedures”) were immunoprecipitated (IP) with α-CCNB1 or IgG antibodies, and the resulting immune complexes were incubated with His-CREB^WT or His-CREB^S270A/S271A in the presence of unlabeled ATP. The in vitro phosphorylated His-CREB proteins were then incubated with a ³²P-labeled CRE oligonucleotides and an EMSA performed as described under “Experimental Procedures.” Input of His-CREB proteins was resolved on SDS-polyacrylamide gel and immunoblotted with α-CREB. B, densitometric quantification of the EMSA data (n = 4) was performed.