FIGURE 3.

C-terminal 82 amino acids carry a portable BIN2-docking motif critical for the BIN2-BZR1 interaction. A, schematic structures of three truncated/fused BZR1 proteins as follows: ΔC82 and C82 lacking and containing the C-terminal 82 amino acids, respectively, whereas M:C82 is a fusion protein between the M fragment (Ser¹⁰²–Ala²⁰³) and the C82 fragment (Ser²⁵⁵–Gly³³⁶). B, similar to Fig. 2C but using different GST-BZR1 fusion proteins. C and D, testing in vitro phosphorylation of different GST fusion proteins by MBP-BIN2. The listed GST fusion proteins, including fusion proteins between a Drosophila GSK3 substrate Arm with C82 or our identified BIN2-docking motif (DM, see Fig. 5A), were incubated with MBP-BIN2 in 30 μl of GSK3 assay buffer at 25 °C for 30 min, and the reactions were stopped by adding SDS sample buffer and 5 min of boiling at 95 °C. The denatured samples were separated by SDS-PAGE and analyzed by Coomassie Blue staining (upper panels) and autoradiography (lower panels).