FIGURE 4.

Identification of GSK-3β phosphorylation sites and a C-terminal destruction motif in GCM1. A, Ser³²² and Ser³²⁶ are required for GCM1 ubiquitination. 293T cells were transfected with 1 μg of pHA-Ub and wild-type or mutant pGCM1-FLAG, treated with MG132, and subject to ubiquitination analysis as described in the legend to Fig. 3A. B, Ser³²² and Ser³²⁶ are involved in regulation of GCM1 stability. 293T cells were transfected with the indicated pGCM1-FLAG expression plasmid for protein stability analysis as described in the legend to Fig. 3B. C and D, FBW2 interacts with the C-terminal TpSWPCpS (where pS represents phosphoserine) destruction motif in GCM1. GST- or GST-FBW2-loaded glutathione beads were incubated with 100 μg of the cell lysate prepared from 293T cells transfected with wild-type or mutant pGCM1-FLAG expression plasmids for pull-down analysis, followed by immunoblotting with FLAG mAb. Biotinylated unmodified peptides or phosphopeptides covering amino acids 313–333 were incubated with 100 μg of cell lysate prepared from 293T cells transfected with pFBW2-Myc, pβTrcp-Myc, or pSKP2-Myc for pull-down analysis, followed by immunoblotting with Myc mAb. IP, immunoprecipitation; IB, immunoblot.