Identification of physiological CLASP2 GSK3β phosphorylation sites. (A) Immunoblot of HeLa cell lysates probed with a polyclonal CLASP antibody (Hannak and Heald, 2006). Treatment with GSK3β inhibitors (20 µM SB216763 or 10 mM LiCl) results in dephosphorylation (downshift) of endogenous CLASP and transfected EGFP-CLASP2(340–1,084) that contains all potential GSK3β sites. (B) Mutation of GSK3β motifs within the MT plus end tracking domain identifies the motif between S594 and S614 as responsible for the GSK3β inhibitor–induced gel shift. (C) Diagram of constructs and phosphorylation site mutants used in this study. Bolded letters indicate serine residues identified to be phosphorylated by GSK3β. Predicted Cdk consensus motifs are underlined. Arrowheads indicate phosphorylated residues identified by mass spectrometry phosphoproteomics (Trinidad et al., 2006; Matsuoka et al., 2007; Dephoure et al., 2008; Imami et al., 2008). The numbering of amino acid positions corresponds to the old National Center for Biotechnology Information reference sequence XP_291057.5. This record has been replaced by NP_055912, which has a longer N terminus. However, we have kept the numbering for consistency with our previous paper (Wittmann and Waterman-Storer, 2005). The asterisk indicates an eight–amino acid difference between the CLASP2 clone used in this study and XP_291057.5. Black bars indicate functionally defined domains: MT#1, MT plus end tracking and EB1-binding domain; MT#2, region required for binding along lamella MTs; CLIP, C-terminal domain required for association with CLIP-170 and LL5β. Gray bars indicate putative TOG-like domains (Slep and Vale, 2007).