FIG. 2.

cdk4 and cdk6 phosphorylate Ser193 of C/EBPα in vitro and in vivo. A. Cyclin D3-cdk4 and cyclin D3-cdk6 phosphorylate C/EBPα in liver. cdk4, cdk6 and cyclin D3 were immunoprecipitated from young and old livers and examined in a kinase assay with GST-C/EBPα substrate. Ag, agarose control for absorption. The bottom part shows a Coomassie stain of the filter to verify loading of the GST-C/EBPα substrate. B. Baculovirus-expressed, purified cdk4-cyclin D1 phosphorylates C/EBPα. Full-length GST-C/EBPα or GST-140-200 (growth-inhibitory region of C/EBPα) was incubated with the cyclin D1-cdk4 complex in a kinase assay mixture containing [γ-³²P]ATP. Proteins were transferred on the membrane and exposed. The bottom image shows Western blotting of the same membrane with antibodies to cdk4. C. C/EBPα forms a stable complex with cyclin D1-cdk4 in the kinase reactions. C/EBPα was incubated with cdk4-cyclin D1 in a kinase reaction, cyclin D1 was immunoprecipitated, and cdk4 and C/EBPα in IPs were examined by Western blotting. A control experiment was performed with bovine serum albumin (BSA) instead of C/EBPα. An aliquot from each reaction mixture was incubated with the GST-Rb substrate in the presence of [γ-³²P]ATP (bottom part). D. Baculovirus-expressed, purified cdk4-cyclin D1 and cdk6-cyclin D3 phosphorylate Ser193 of C/EBPα. GST-WT-C/EBPα (W) and a C/EBPα-S193A mutant (S193) were incubated with cdk4-cyclin D1 and with cdk6-cyclin D3 in a kinase assay. Positions of C/EBPα and a nonspecific band (NS for cdk4-cyclin D1) are shown. The filter was reprobed with Abs to C/EBPα, cdk4, and cdk6 (Western). E. cdk4-cyclin D3 phosphorylates Ser193 of C/EBPα. WT and S193A mutant GST-C/EBPα were incubated with cdk4 and cyclin D3 IPs from old livers. The bottom image shows GST-C/EBPα stained with Coomassie. F. cdk6-cyclin D3 phosphorylates Ser193 of C/EBPα. WT and S193A mutant GST C/EBPα were incubated with cdk6 and cyclin D3 IPs from old livers. The bottom image shows GST-C/EBPα stained with Coomassie. G. Examination of efficiency of phosphorylation of C/EBPα by cdk4 relative to phosphorylation of Rb. GST-Rb (0.5 μg) and increasing amounts (0.5, 1, and 2 μg) of the growth-inhibitory region of C/EBPα (GST-C/EBPα-140-200) were added into kinase reaction mixtures with purified cyclin D1-cdk4 and [γ-³²P]ATP. The reaction mixtures were loaded on a denaturing PAAG, transferred on membrane, and exposed to X rays. The bottom part shows a Coomassie stain of the gel loaded with the corresponding ratios of the proteins.