FIGURE 6.

The PIPP·CRMP2 complex is regulated by GSK3β. A, GSK3β sequentially phosphorylates CRMP2 at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ following priming phosphorylation of Ser⁵²² by Cdk5. B, COS-1 cells expressing GST or GST-PIPP and either FLAG-CRMP2 wild type (WT) or FLAG-CRMP2(S522A) were captured on glutathione-Sepharose, washed, and immunoblotted with FLAG (upper panel) or GST antibodies (middle panel). Lysates were immunoblotted (WB) with FLAG antibodies (lower panel). C, relative FLAG-CRMP2 WT or S522A mutant binding to GST-PIPP was quantitated using densitometry, standardized to GST-PIPP levels in pulldowns. Bars = mean ± S.E., where the FLAG-CRMP2 WT is designated 1 (n = 4). D, COS-1 cells were transfected with FLAG-CRMP2 and GST or GST-PIPP and increasing amounts of Myc-GSK3β(S9A) (0–5 μg) and lysates were incubated with glutathione-Sepharose, pellets were washed and immunoblotted with FLAG (upper panel) or GST antibodies (middle panel). Lysates were immunoblotted with FLAG and Myc antibodies (lower panels). E, relative FLAG-CRMP2 detected in PIPP pulldowns in the presence or absence of Myc-GSK3β(S9A) (5 μg) was quantitated using densitometry, standardized to GST-PIPP levels in pulldowns. Bars = mean ± S.E., where the no GSK3β(S9A) control is designated 1 (n = 6, **, p < 0.001). F, PC12 cells were transfected with the indicated constructs and GST pulldown was performed followed by immunoblot with FLAG or GST antibodies. WB, Western blot.