Figure 2.
Figure 2. TTBK2 phosphorylates the MT depolymerase KIF2A. (A) Schematic diagram of KIF2A. The domain organization of KIF2A and its fragments is indicated. (B) Phosphorylation of KIF2A by TTBK2 in vitro. (Left) KIF2A-FL and control RhoGDI from Sf9 cells were incubated with GST–TTBK2-cat. TTBK2 effectively phosphorylated KIF2A at a stoichiometry of 2.54 ± 0.13. The asterisk indicates the autophosphorylation of TTBK2-cat. (Right) GST-KIF2A fragments from bacteria were incubated with TTBK2-FL. TTBK2 predominantly phosphorylated KIF2A-N. The intact bands of the KIF2A fragments are labeled with red dots. (C) Schematic diagram of KIF2A and multiple sequence alignment of KIF2A from several organisms. A TTBK2-mediated phosphorylation site in KIF2A (S135 in human KIF2A) is located in the N-terminal region of KIF2A. S135 on KIF2A is conserved among vertebrates, and those are highlighted in red. Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Gg, Gallus gallus; XI, Xenopus laevis. (D) Purified GST–KIF2A-N-WT or -S135A was incubated with TTBK2 in the presence or absence of ATP followed by silver staining or immunoblot analysis using an anti–pS135-KIF2A antibody. The anti–pS135-KIF2A antibody only detected KIF2A-N-WT incubated in the presence of ATP, indicating that the antibody specifically recognizes KIF2A phosphorylated at S135. The red dot indicates the intact band of GST–KIF2A-N. (E) HeLa cells were transfected with the indicated siRNAs and cultured for 48 h. The phosphorylation of KIF2A at S135 was visualized by immunoblot analysis using an anti–pS135-KIF2A antibody. TTBK2 depletion reduced the phosphorylation of endogenous KIF2A at S135. (F) GST-KIF2A was coexpressed with the indicated HA-fused proteins in COS-7 cells. Coexpression of TTBK2-WT increased the phosphorylation of KIF2A at S135. All of the results are representative of more than four independent experiments. See Fig. S2 for the determination of TTBK2 substrates and Table S2 for identification of the TTBK2-mediated phosphorylation sites of KIF2A. IB, immunoblotting.

TTBK2 phosphorylates the MT depolymerase KIF2A. (A) Schematic diagram of KIF2A. The domain organization of KIF2A and its fragments is indicated. (B) Phosphorylation of KIF2A by TTBK2 in vitro. (Left) KIF2A-FL and control RhoGDI from Sf9 cells were incubated with GST–TTBK2-cat. TTBK2 effectively phosphorylated KIF2A at a stoichiometry of 2.54 ± 0.13. The asterisk indicates the autophosphorylation of TTBK2-cat. (Right) GST-KIF2A fragments from bacteria were incubated with TTBK2-FL. TTBK2 predominantly phosphorylated KIF2A-N. The intact bands of the KIF2A fragments are labeled with red dots. (C) Schematic diagram of KIF2A and multiple sequence alignment of KIF2A from several organisms. A TTBK2-mediated phosphorylation site in KIF2A (S135 in human KIF2A) is located in the N-terminal region of KIF2A. S135 on KIF2A is conserved among vertebrates, and those are highlighted in red. Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Gg, Gallus gallus; XI, Xenopus laevis. (D) Purified GST–KIF2A-N-WT or -S135A was incubated with TTBK2 in the presence or absence of ATP followed by silver staining or immunoblot analysis using an anti–pS135-KIF2A antibody. The anti–pS135-KIF2A antibody only detected KIF2A-N-WT incubated in the presence of ATP, indicating that the antibody specifically recognizes KIF2A phosphorylated at S135. The red dot indicates the intact band of GST–KIF2A-N. (E) HeLa cells were transfected with the indicated siRNAs and cultured for 48 h. The phosphorylation of KIF2A at S135 was visualized by immunoblot analysis using an anti–pS135-KIF2A antibody. TTBK2 depletion reduced the phosphorylation of endogenous KIF2A at S135. (F) GST-KIF2A was coexpressed with the indicated HA-fused proteins in COS-7 cells. Coexpression of TTBK2-WT increased the phosphorylation of KIF2A at S135. All of the results are representative of more than four independent experiments. See Fig. S2 for the determination of TTBK2 substrates and Table S2 for identification of the TTBK2-mediated phosphorylation sites of KIF2A. IB, immunoblotting.

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