Classical cell cycle kinase limits tubulin polyglutamylation and cilium stability

The primary cilium is a hair-like organelle that projects from the surface of nearly all vertebrate cells. Serving as the cell’s antenna, the primary cilium detects extracellular cues and converts them into intracellular signaling pathways, which ultimately determine the cellular response (1). Disruption of ciliary function leads to a wide spectrum of diseases, collectively known as ciliopathies (2). Multiple ciliopathies are associated with defects in the axoneme, the structural core of the primary cilium that contains nice microtubule doublets arranged into a ring structure.

The unit of microtubules in the axoneme, α/β-tubulin heterodimer, undergoes various conserved posttranslational modifications to adapt to a large diversity of functions. Polyglutamylation, for instance, is a major posttranslational modification of tubulins in the ciliary axoneme (3). Mutations in several ciliopathy genes are associated with defective axonemal polyglutamylation, resulting in disruption of ciliary stability and impaired transport of signaling transducers to the cilium (4, 5, 6). However, despite the importance of axonemal polyglutamylation in ciliary function, our understanding about its regulatory mechanism remains limited.

In a new study (7), He et al. uncovered a nonconventional role of cyclin-dependent kinase 6 (Cdk6) in this process. It is known that axoneme polyglutamylation is balanced by two enzyme systems, the cilium-specific tubulin tyrosine ligase-like glutamylases (TTLL5 and TTLL6) and the tubulin deglutamylase of the cytoplasmic carboxyl peptidase (CCP5). Additionally, the ciliary transport of TTLLs is controlled by the RAB11-FIP5–dependent vesicle-trafficking machinery (8) (Fig. 1 A). He et al. found that Cdk6 phosphorylates FIP5, thereby reducing the ciliary transport of TTLL5/6 (Fig. 1 B). Genetic and pharmacological inhibition of Cdk6 activity is sufficient to restore axoneme polyglutamylation and ciliary signaling in ciliopathy cell models (Fig. 1 C). These findings refined our understanding of the regulatory mechanisms of axonemal polyglutamylation and highlighted the therapeutic value of Cdk6 inhibitors for ciliopathies.

To identify key modulators in regulating axoneme polyglutamylation, He et al. performed a screening of kinases inhibitors. They found that Cdk7 inhibitor enhanced axoneme polyglutamylation, suggesting Cdk7 is a negative regulator in this process. This effect is specific to ciliary microtubules, as genetic inhibition of Cdk7 does not impact tubulin polyglutamylation in the cytoplasm. Cdk7 acts as the Cdk-activating kinase of multiple Cdks, including Cdk1, 2, 4, 6, and 9. To specify which of these Cdk is involved in axonemal polyglutamylation, He et al. performed an siRNA screen. They found that only Cdk6 knockdown significantly increased ciliary polyglutamylation. In addition, the Cdk6-selective inhibitors, abemaciclib and ribociclib, Food and Drug Administration (FDA)–approved agents for breast cancer treatment (9), significantly increased axoneme polyglutamylation. Hence, Cdk6 is specifically involved in this process.

Is the hyper-polyglutamylation mediated by elevated glutamylase activity or reduced deglutamylase activity? To address this, the authors targeted CCP5 to the cilium to block ciliary polyglutamylation. In these cells, inhibition of Cdk7 or Cdk6 partially restored the ciliary polyglutamylation levels. Therefore, the observed increase in ciliary polyglutamylation after Cdk7/Cdk6 inhibition is most likely due to reduced glutamylase activity in the cilium.

To determine how Cdk6 inhibits axoneme polyglutamylation, He et al. first determined its subcellular localization. They found that endogenous Cdk6 is enriched at the cilium base, besides its known nuclear localization. They then specifically targeted the constitutively active Cdk6 (cyclin D3–Cdk6 complex) to the cilium base by fusing the complex to CEP170C. This is sufficient to significantly reduce polyglutamylation levels in the cilium. Since polyglutamylation is facilitated by TTLLs, the authors examined the ciliary levels of the tubulin glutamylases TTLL5/6. They found that pharmacological inhibition or knockdown of either Cdk6 or Cdk7 significantly increased the ciliary level of TTLL5/6. Is this attributable to RAB11–FIP5, the transporting machinery that guides cargoes to the cilium base? To answer this question, the authors determined the levels of FIP5 at the cilium base. They found that inhibiting Cdk6/7 increased FIP5 levels, whereas enhancing centrosomal Cdk6 activity reduced FIP5 levels at the cilium base. Hence, Cdk6 at the basal body suppresses local FIP5 activities, thereby limiting the ciliary import of tubulin glutamylases.

Cdk modulation of protein activity occurs through phosphorylation of Cdk consensus sites. To elucidate the molecular interaction between Cdk6 and FIP5, He et al. performed a series of biochemistry experiments. They first identified that Cdk6 phosphorylates FIP5 at S641. The FIP5 S164A mutant, which prevents phosphorylation at this site, accumulates at the cilium base even in the presence of constitutively active Cdk6. This result indicates that, under normal conditions, Cdk6 phosphorylation of FIP5 reduces FIP5’s accumulation at the centrosome. Interestingly, the S164 phosphorylation site is located within the RAB11–FIP5 interaction site, and S164 phosphorylation disrupts RAB11–FIP5 interaction. Collectively, the data point to a model where Cdk6-mediated FIP5 phosphorylation abolishes RAB11–FIP5 interaction, thereby impeding the ciliary import of tubulin glutamylases.

Finally, the authors tested whether Cdk6 inhibition could restore ciliary polyglutamylation and ciliary signaling in ciliopathic cells. Knockdown of CEP41 or ARMC9, which models Joubert syndrome deficiencies, significantly reduced axoneme polyglutamylation. This in turn diminished multiple protein transport to the cilium, including the Hedgehog pathway transcription factors Gli2/3 and the calcium channel polycystin-2. Remarkably, all these defects were effectively restored by either abemaciclib treatment or knockdown of Cdk6. Hence, suppressing Cdk6 activity effectively restores the compromised ciliary glutamylation and ciliary signaling in ciliopathic cells.

This study uncovered a role for a classical cell cycle kinase in ciliary function. It elucidated a fundamental mechanism governing axoneme polyglutamylation and pinpointed an upstream regulatory pathway involving Cdk6-FIP5-TTLL5/6. Notably, the FDA-approved Cdk6 inhibitor abemaciclib restored axonemal polyglutamylation and ciliary signaling in cell models of Joubert syndrome (Fig. 1 C), suggesting the possibility of repurposing this anticancer agent for ciliopathy treatment. Encouragingly, enhancing axonemal polyglutamylation does not appear to negatively impact animal development, as genetic deletion of the deglutamylase CCP5 produces no obvious defects in mice (10). In the future, it is essential to validate the cell-based findings in animal models of Joubert syndrome to confirm their relevance in vivo.

This study also highlighted an intriguing coupling of cell cycle machinery with the primary cilium dynamics. Primacy cilia are assembled during G0/G1 phase and disassembled before mitosis. However, the precise events that initiate cilium disassembly during cell cycle remain elusive, and the molecular mechanisms underlying this process are not yet fully understood (11). Cdk6–cyclin D is known to drive cells past the G1 phase checkpoint to enter S phase. Does Cdk6 activation mark the initiation of cilium disassembly? How is Cdk6 activity at the basal body regulated? How is the cyclin D level at the basal body regulated? In addition to FIP5, what other Cdk6 substrates at the basal body contribute to cilium disassembly? Addressing these questions will offer new mechanistic insights into the link between cell cycle progression and cilium stability.