Figure 1.

Cdk5 phosphorylates FOXO1 at serine 249. A, B, Lysates of 293T cells transfected with Cdk5/p35, Cdk5/p25, Cdk5-KD/p35, Cdk5-KD/p25 (Cdk5 kinase-dead plasmid), Cdk1/Cyclin B, Cdk2/Cyclin A, or control vector, together with GFP-FOXO1 or the GFP-FOXO1–S249A mutant, were analyzed by immunoblotting with antibodies to pS249-FOXO1, FOXO1, Cdk5, p35/p25, GFP, or GAPGH as shown. C, D, In vitro kinase assays with immunoprecipitated (IP) Cdk5. Recombinant GST-FOXO1 or GST-FOXO1–S249A proteins were subjected to an in vitro kinase assay with IP GFP fusion proteins from 293T cells expressing the indicated constructs. Kinase reaction products were immunoblotted with the pS249-FOXO1 antibody. FOXO1 proteins levels were evaluated by Coomassie Blue staining. E, The interactions among FOXO1, Cdk5, and p35 were investigated in 293T cells. The 293T cells were cotransfected Flag-FOXO1 with GFP-Cdk5 and/or GFP-p35. The antibodies used for IP are indicated in the top of the gel. After immunoprecipitation, the gels were blotted with indicated antibodies. F, Primary cortical neurons were transfected with the GFP-FOXO1-WT or GFP-FOXO1–S249A plasmid together with p25/Cdk5 or p25/Cdk5-KD or control vector in combination with the 3xIRS luciferase reporter system and subjected to luciferase assay. Data are mean ± SEM; n = 5. *p < 0.05. G, Primary cortical neurons were transfected with different Cdk/cyclin pairs together with the 3xIRS-luciferase reporter gene and subjected to luciferase assays. Data are mean ± SEM; n = 5. *p < 0.05.