Figure 7.
S641 phosphorylation by CDK6 interrupts cilia base accumulation and RAB11 interaction of FIP5. (A) The putative CDK6 phosphorylation sites and cyclin binding motifs in FIP5. (B) Direct interaction between FIP5 and Cyclin D3 was detected by GST pull-down assay. GST-CDK6 (CDK6), GST-Cyclin-D3 (CycD3), and 6× his-tagged truncated FIP5 (129–653) (ΔC2-FIP5) proteins were used. The purity of recombinant proteins was assessed by Coomassie blue staining. IB: immunoblotting. The molecular weigth standards (kD) are labeled on the right. (C) Direct CDK6/cyclin D3-mediated FIP5 phosphorylation was detected by in vitro kinase reaction assay. Recombinant ΔC2-FIP5 and active Cyclin-D3/CDK6 (D3K6) proteins were used. GST-Cyclin D3 (CycD3) and His-CDK6 (CDK6) were also used. The phosphorylation of FIP5 was examined by anti-phosphorylated Serine antibody (p-Ser). (D) Indicated mCherry-tagged FIP5 and CyclinD3-CDK6-YFP were transfected in 293T cells. The phosphorylation of FIP5 was examined by anti-phosphorylated Serine antibody after immunoprecipitation (IP) of FIP5-mCherry. * indicates the positive band of D3K6-YFP. (E) mCherry or mCherry-tagged FIP5 mutants were transfected in RCTE cells. The length of the glutamylated axoneme in mCherry-positive cells was measured. Quantified data are presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post-hoc test for multiple comparisons. N ≥ 100 cilia. ***P < 0.001. n.s: not significant. (F) Subcellular localization of mCherry-tagged WT- or S641A-FIP5 in non-ciliated RCTE cells. The enlarged fields indicated by white squares and showed in the right panels. The line-scan fluorescence intensity profiles of mCherry at the positions marked by arrow lines are shown on the right. Scale bar: 10 μm. (G) The subcellular localization of mCherry-tagged FIP5 in indicated YFP-positive ciliated RCTE cells. The line-scan fluorescence intensity profiles of mCherry at the positions marked by arrow lines are shown on the right. Scale bar: 10 μm. (H and I) Indicated mCherry-tagged FIP5 and flag-tagged RAB11A were transfected in 293T cells. The interaction between FIP5 and RAB11A was examined by co-immunoprecipitation. (J) Indicated mCherry-tagged FIP5 were transfected in RCTE cells. The interaction between mCherry-tagged FIP5 and endogenous RAB11A was examined by co-immunoprecipitation. Source data are available for this figure: SourceData F7.

S641 phosphorylation by CDK6 interrupts cilia base accumulation and RAB11 interaction of FIP5. (A) The putative CDK6 phosphorylation sites and cyclin binding motifs in FIP5. (B) Direct interaction between FIP5 and Cyclin D3 was detected by GST pull-down assay. GST-CDK6 (CDK6), GST-Cyclin-D3 (CycD3), and 6× his-tagged truncated FIP5 (129–653) (ΔC2-FIP5) proteins were used. The purity of recombinant proteins was assessed by Coomassie blue staining. IB: immunoblotting. The molecular weigth standards (kD) are labeled on the right. (C) Direct CDK6/cyclin D3-mediated FIP5 phosphorylation was detected by in vitro kinase reaction assay. Recombinant ΔC2-FIP5 and active Cyclin-D3/CDK6 (D3K6) proteins were used. GST-Cyclin D3 (CycD3) and His-CDK6 (CDK6) were also used. The phosphorylation of FIP5 was examined by anti-phosphorylated Serine antibody (p-Ser). (D) Indicated mCherry-tagged FIP5 and CyclinD3-CDK6-YFP were transfected in 293T cells. The phosphorylation of FIP5 was examined by anti-phosphorylated Serine antibody after immunoprecipitation (IP) of FIP5-mCherry. * indicates the positive band of D3K6-YFP. (E) mCherry or mCherry-tagged FIP5 mutants were transfected in RCTE cells. The length of the glutamylated axoneme in mCherry-positive cells was measured. Quantified data are presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post-hoc test for multiple comparisons. N ≥ 100 cilia. ***P < 0.001. n.s: not significant. (F) Subcellular localization of mCherry-tagged WT- or S641A-FIP5 in non-ciliated RCTE cells. The enlarged fields indicated by white squares and showed in the right panels. The line-scan fluorescence intensity profiles of mCherry at the positions marked by arrow lines are shown on the right. Scale bar: 10 μm. (G) The subcellular localization of mCherry-tagged FIP5 in indicated YFP-positive ciliated RCTE cells. The line-scan fluorescence intensity profiles of mCherry at the positions marked by arrow lines are shown on the right. Scale bar: 10 μm. (H and I) Indicated mCherry-tagged FIP5 and flag-tagged RAB11A were transfected in 293T cells. The interaction between FIP5 and RAB11A was examined by co-immunoprecipitation. (J) Indicated mCherry-tagged FIP5 were transfected in RCTE cells. The interaction between mCherry-tagged FIP5 and endogenous RAB11A was examined by co-immunoprecipitation. Source data are available for this figure: SourceData F7.

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