Abemaciclib rescues glutamylation and ciliary signaling in JBTS mutant cells. (A–E) Abemaciclib (200 nM, 24 h) restores axoneme polyglutamylation and Shh signaling in Armc9 and Cep41 knocked-down normal or Halo-mGLI2 Flp-In NIH-3T3 cells, examined by mean intensity of GT335 in cilia (A and B), and SAG (100 nM, 24 h) induced cilia tip accumulation of GLI3 (A and C) and GLI2 (D and E). T: Cilia tip. B: Cilia base. (F–H) Abemaciclib (200 nM, 24 h) restores axoneme polyglutamylation (F and G) and ciliary localization of PC2 (F and H) in Armc9 and Cep41 knocked-down IMCD3 cells. 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 ≥ 50 cilia. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 2 μm. (I) Schematic model illustrating the CDK6-FIP5 phosphorylation cascade-regulated axoneme polyglutamylation. In cells with inactivated CDK6, FIP5 interacts with RAB11 to promote the ciliary import of TTLL5/6 and subsequent axoneme polyglutamylation. Glutamylated axoneme supports the cilia localization of polycystins and signaling molecules such as Hedgehog component GLI2/3. In cells with activated CDK6, CDK6 at the cilia base phosphorylates FIP5 at S641, disrupting the FIP5-RAB11 interaction and subsequent ciliary import of TTLL5/6. This leads to axoneme hypoglutamylation and defective ciliary localization of polycystins and GLI2/3. Image created with https://BioRender.com.