Figure 5.

Dephosphorylation blocks the interactions of C/EBPα with cdks and Brm in vitro and in vivo. (A) C/EBPα is phosphorylated in livers. 2D gel electrophoresis was performed with C/EBPα precipitated from NE of young mouse livers. (CIP) 2D gel separation of C/EBPα treated with CIP; (Control) C/EBPα precipitated from 3T3-L1 cells transfected with pcDNA–C/EBPα. Five isoforms (a, b, c, d, and e) of C/EBPα are observed in mouse liver. (B) Dephosphorylation of C/EBPα in liver extracts destroys C/EBPα–cdk2 complexes. Untreated (control) and CIP-treated nuclear extracts were fractionated by size exclusion chromatography (HPLC). Positions of C/EBPα and cdk2 within the fractions were determined by Western blotting. To detect C/EBPα–cdk2 complexes, we precipitated C/EBPα from each fraction and examined cdk2 in C/EBPα IPs. (IgG) Heavy chains of immunoglobulins. (C) Insulin signaling leads to a dephosphorylation of C/EBPα on Ser 193. Wild-type C/EBPα or C/EBPα-S193A mutant were transfected in 3T3-L1, cells were treated with insulin, and C/EBPα was isolated and examined by 2D gel electrophoresis. Acidic isoforms (a and b) are not detectable in C/EBPα-S193A mutant and are dramatically reduced in wild-type C/EBPα after treatment with insulin. (D) Insulin-mediated dephosphorylation of C/EBPα blocks the interaction of C/EBPα with cdks and Brm. (Left image) C/EBPα was transfected into 3T3-L1 cells, cells were treated with insulin, and C/EBPα was precipitated with specific antibodies. Cdk2, cdk4, Brm, and C/EBPα were examined in C/EBPα IPs. (Right image) Insulin does not affect the interaction of p21 with cdk2. 3T3-L1 cells were transfected with p21, treated with insulin, and p21 was precipitated with specific Abs. cdk2 and p21 were examined in p21 IPs.