Fig. 4.

Phosphorylation by either MAPK or cdk2/cyclinA primes C/EBPβ for phosphorylation by GSK3β. (A) rC/EBPβ was incubated without or with MAPK or cdk2/cyclinA or/and in combination with GSK3β in the presence of [γ-³²P]ATP. After SDS/PAGE [³²P]C/EBPβ was detected by autoradiography (Upper), and protein was detected by Coommassie blue staining (Lower). Similar experiments were performed but with cold ATP. (B) The C/EBPβ band was excised from the gel, digested with trypsin, and analyzed by nanoLC-MS/MS. A doubly phosphorylated peptide species was observed (m/z 1,121.0) with C/EBPβ incubated with MAPK plus GSK3β (Middle) or cdk2/cyclinA plus GSK3β (Bottom). No phosphorylation of the peptide (m/z 1,067.5) occurred without kinase (Top).