FIGURE 4.

Identification of the 12-amino acid BIN2-DM. A and B, convenient restriction sites were introduced into the C82 fragment of the GST-M:C82 fusion protein by PCR-based site-directed mutagenesis at indicated positions. Two series of deletion constructs were created from the N terminus (ΔN) or the C terminus (ΔC) of the C82 domain by double enzyme digestion (with one enzyme cutting a terminal end and the other cutting a newly created restriction site at the indicated positions) followed by re-ligation. Pattern bars represent regions carrying predicted GSK3 phosphorylation motifs, and the names of serially deleted GST-BZR1 proteins are shown on the left. C and D, similar to Fig. 2D, testing the in vitro phosphorylation of the serially deleted GST-BZR1 proteins by the MBP-tagged BIN2. The GST-M:C82 (M:C82) and GST-M (M) fusion proteins were used as the positive and negative control, respectively. E, three consecutive amino acids were simultaneously changed to three alanine residues for the 24-amino acid (Gly²⁹⁷–Val³²⁰) segment, and a total eight mutated forms (m1 to m8) of the GST-M:C82 fusion protein were created (left panel). Asterisks indicate nonmutated amino acids. The mutated proteins were subject to the in vitro phosphorylation assay described in Fig. 2D. C–E, the upper panels show Coomassie Blue staining of the fusion proteins used in the kinase assays, and the lower panels are autoradiographs showing the intensity of ³²P-labeled GST-BZR1 fusions.