Phosphorylation of multiple proteins involved in ciliogenesis by Tau Tubulin kinase 2

Abstract

Primary cilia (PC) are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling final step of cilia initiation. The function of TTBK2 in ciliogenesisis is critically dependent on its kinase activity, however, precise mechanism of TTBK2 action is so far incompletely understood, due to very limited information about its relevant substrates. In this study we identify CEP83, CEP89, CCDC92, Rabin8 and DVL3 as substrates of TTBK2 kinase activity. Further, we characterise a set of phosphosites of the newly identified substrates and CEP164, induced by TTBK2 in vitro and in vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we address functional relevance of selected phosphorylations of CEP164 and provide evidence that the examined TTBK2-induced phosphorylations of CEP164 are relevant for the process of cilia formation. In summary, our work provides important insight into substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.

A hierarchical pathway for assembly of the distal appendages that organize primary cilia

“…Loss of TTBK2 resulted in the decrease in the localization of several distal appendage proteins, including the most upstream CEP83 (Figure 2M and N). This is consistent with the previous finding that TTBK2 phosphorylates distal appendage proteins, such as CEP83 and CEP89 (Bernatik et al, 2020;Lo et al, 2019). Further investigations are needed to assess whether other distal appendage proteins are also phosphorylated by TTBK2 and how the phosphorylation affects the structure and function of the substrates.…”

“…The decrease of CEP164 intensity in TTBK2 knockout cells is consistent with the observation that CEP164 is a substrate of TTBK2 and that centriolar CEP164 localization was markedly increased upon overexpression of wild type but not kinase-dead TTBK2 (Cajanek & Nigg, 2014). Interestingly, localization of another TTBK2 substrate, CEP83 (Bernatik et al, 2020;Lo et al, 2019), was also affected by TTBK2 depletion (Figure 2C). Since only the outer ring of CEP83 was affected by TTBK2, we predict that CEP83 phosphorylation by TTBK2 induces conformational change of CEP83 to help enable the protein as a backbone of the distal appendage.…”

Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

“…In contrast, the feedback complex CEP164-TTBK2 (see (Tomoharu Kanie et al, 2023) for the detail) was required for proper centriolar localization of NCS1 (Figure 2-figure supplement 3D) but not for CEP89 (Figure 2F of (Tomoharu Kanie et al, 2023)). Given that CEP89 is a substrate of TTBK2 (Bernatik et al, 2020;Lo et al, 2019), this might suggest that phosphorylation of TTBK2 could affect the interaction between CEP89 and NCS1. It is also possible that NCS1 may be a phosphorylation target of TTBK2.…”

Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT)

“…S1B). CEP164-TTBK2 interaction facilitates TTBK2-mediated phosphorylation of both CEP83 [8,9] and MPHOSP9 (M-Phase Phosphoprotein 9, MPP9) at the distal end of the mother centriole [10]. MPHOSP9 and the associated CP110-CEP97 complex that caps the distal ends of mother centrioles are removed subsequently to promote ciliary axoneme growth [10][11][12].…”