FIG.2.

GST-hCRMP4 requires priming at Ser⁵²² for subsequent phosphorylation by GSK-3β. A, GST-hCRMP4 was incubated with DYRK2 and [γ-³²P]ATP for the times indicated, prior to SDS-PAGE and autoradiography. Stoichiometry of phosphorylation at each time point is presented below the autoradiograph. B, GST-hCRMP4 was incubated with (upper panel) or without (lower panel) DYRK2 and non-radioactive ATP for 30 min. DYRK2 was removed using Ni²⁺-agarose and the supernatant incubated with His₆-GSK-3β and [γ-³²P]ATP. Aliquots were removed and analyzed as described for A. The stiochiometry of phosphorylation is presented below the autoradiograph (dashed line in the absence of DYRK2 preincubation). C, HEK293 cells were transfected with expression vectors as indicated and then incubated with the GSK-3 inhibitor CT-99021 (lane 3) or Me₂SO. FLAG-tagged hCRMP2 was immunoprecipitated, subjected to SDS-PAGE, and phosphorylated proteins detected using ProQ Diamond phospho-specific stain (upper panel). Gels were subsequently stained with CBR-250 to detect protein content (lower panel). D, HEK293 cells were transfected with the equivalent constructs for hCRMP4 and analyzed as described for C. E, HEK293 cells transfected with the indicated expression vectors were lysed and hCRMP2 proteins immunoprecipitated subjected to Western blotting using an antibody that specifically recognizes hCRMP2 only when phosphorylated at both Thr⁵⁰⁹ and Thr⁵¹⁴. hCRMP proteins in C, D, and E are indicated by arrows.