Analysis of CLASP2 phosphorylation. (A–D) Metabolic labeling of tissue culture cells with [³²P]-labeled phosphate. EGFP-tagged CLASP2 constructs were immunoprecipitated and analyzed by SDS-PAGE. Top panels show autoradiograph, and bottom panels show the corresponding Coomassie-stained gel as loading control. Quantification of radioactivity incorporation by densitometry is shown below the gel images. (A) In both HeLa and HaCaT cells, GSK3β inhibition with 20 µM SB216763 decreases CLASP2(340–1,084) phosphorylation. Mutation of the GSK3β motif between S594 to S614 (6×S/A) eliminates GSK3β-dependent phosphorylation. (B) Mutation of individual serine residues between S594 and S614 shows that S614 is not part of the motif and reveals hierarchical phosphorylation by GSK3β. (C) The domain required for CLASP2 association along lamella MTs (875–1,084) is not required for efficient phosphorylation by GSK3β. (D) Combined mutation of the GSK3β motifs between S568 to S576 and S594 to S614 (9×S/A) is required to completely abolish phosphorylation of the MT plus end tracking domain CLASP2(512–650) by constitutively active GSK3β(S9A). (E) In vitro phosphorylation of immunoprecipitated EGFP-CLASP2(512–650) by purified GSK3β in the presence of γ-[³²P]ATP.