TTBK2 phosphorylates CEP83. (A) TTBK2 coexpression promoted CEP83 phosphorylation in 293T cells was detected by immunoprecipitation followed by Western blots with anti-phospho-(Ser/Thr) antibody. (B) FH-TTBK2^FL or FH-TTBK2^KD was expressed and purified from 293T cells. The TTBK2 in vitro kinase assay was performed with TTBK2 purified from 293T cells and recombinant His-CEP83 purified from bacteria in the presence of ATP-γ-S for 30 min at RT. CEP83 phosphorylation was revealed by Western blot using an antibody against thiophosphate ester. CBB staining indicated the equal amount of His-CEP83^WT. (C) Recombinant GST-TTBK2 (1–622)^WT, TTBK2(1–622)^KD, and His-CEP83^WT were expressed and purified from bacteria to perform the TTBK2 in vitro kinase assay. CEP83 phosphorylation was revealed by Western blot using anti-thiophosphate ester antibody. CBB staining indicated the purified GST-TTBK2 (1–622)^WT (arrow), GST-TTBK2 (1–622)^KD (arrowhead), and His-CEP83^WT. (D) FH-CEP83^WT or FH-CEP83^4A was coexpressed with Myc-TTBK2 in 293T cells. FH-CEP83^WT or FH-CEP83^4A phosphorylation was detected by immunoprecipitation (IP) followed by Western blots with anti-phospho-(Ser/Thr) antibody. (E) TTBK2 was purified from 293T cells and incubated with purified CEP83^WT or CEP83^4A from bacteria to perform the TTBK2 in vitro kinase assay. CEP83 phosphorylation was detected by Western blot using an anti-thiophosphate ester antibody. CBB staining indicated the His-tagged CEP83^WT and CEP83^WT. (F) FH-CEP83 was stably expressed in WT and TTBK2^−/− cells. Cells were serum starved for 24 h, and CEP83 phosphorylation was detected by immunoprecipitation followed by Western blots with an anti-phospho-(Ser/Thr) antibody. All of Western blots (A–F) are representative blots from at least three independent experiments. Gel bands were quantified. n.s., not significant. ***, P < 0.001 (Student’s t test). TCL, total cell lysates. (G) RPE1 cells were stained with antibodies against Arl13b, phospho-CEP83^Ser29 (CEP83^pS29), and phospho-CEP83^Thr292 (CEP83^pT292). (H) Quantification of phosphorylated CEP83^Ser29 and CEP83^Thr292 signals at the centrioles in ciliated and nonciliated cells. (I) WT and TTBK2^−/− RPE1 cells were serum starved for 2 d and stained with antibodies against centrin, phospho-CEP83^Ser29, and phospho-CEP83^Thr292. Scale bar, 1 µm. (J) The phosphorylated CEP83^Ser29 and CEP83^Thr292 signals at the centrioles were quantified. In H and J, 200 cells were analyzed for each independent experiment. Error bars represent mean ± SEM; n = 3. n.s., not significant. ***, P < 0.001 (Student’s t test).