Figure 5. Elevation of gankyrin triggers degradation of C/EBPα by UPS.

(A) Inhibition of proteasome blocks degradation of S193D C/EBPα in DEN-treated livers. Protein extracts of S193D untreated (Con), DEN-treated (for 25 weeks), and DEN-treated mice injected with MG132 were examined by Western blotting with Abs to C/EBPα and ubiquitin. Middle panel shows results of Western blotting of C/EBPα IP with antibodies to ubiquitin. Positions of C/EBPα isoforms and ubiquitinated conjugates are shown on the right. (B) Gankyrin preferentially interacts with S193D isoform of C/EBPα. GST-S193A and GST-S193D mutants were incubated with nuclear extracts isolated from livers of mice treated with DEN/PB for 25 weeks. (C) Expression of gankyrin is increased in livers of DEN/PB-treated mice. Upper image shows Western blotting with antibodies to C/EBPα and gankyrin. Middle image shows densitometric calculations of the levels of C/EBPα and gankyrin (gank). Bottom image shows levels of gankyrin mRNA determined by RT-PCR and calculated as a ratio to β-actin. Data shown are mean ± SD. (D) Gankyrin and cdk4 are associated with C/EBPα at early time points after DEN treatments. C/EBPα was IP from nuclear extracts and probed with antibodies to gankyrin and cdk4. (E) Expression of proteins that regulate activity of cdk4. Western blotting of nuclear extracts of DEN-treated mice was performed with antibodies shown on the right. (F) Gankyrin displaces p16 from cdk4 and activates cdk4. Cdk4 was IP from nuclear extracts and probed with Abs to gankyrin and to p16 (Western). The cdk4 IPs were examined in a kinase assay with WT C/EBPα and with S193A-C/EBPα as substrates (kinase assay).