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

Bernatík, Ondřej; Pejšková, Petra; Vysloužil, David; Hanáková, Kateřina; Zdráhal, Zbyněk; Čajánek, Lukáš

doi:10.1091/mbc.e19-06-0334

Cited by 32 publications

(48 citation statements)

References 79 publications

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“…Having characterized our model system, we examined cilia formation in those cells. Consistently with previous work, HEK293 cells form cilia less frequently than RPE-1 or NIH3T3 cells (Lancaster, Schroth, and Gleeson 2011; Bernatik et al 2020). We were able to detect about 5% of cells with Arl13b+ primary cilium in WT HEK293.…”

Section: Resultssupporting

confidence: 92%

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Primary cilia formation does not rely on WNT/β-catenin signaling

Bernatík

Paclíková

Kotrbová

et al. 2020

Preprint

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Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6 or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

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Section: Resultssupporting

confidence: 92%

“…WB was performed as previously described (Bernatik et al 2020). Antibodies used: LRP6 (Cell signaling, Cat.no.…”

Section: Western Blot Quantificationmentioning

confidence: 99%

Primary cilia formation does not rely on WNT/β-catenin signaling

Bernatík

Paclíková

Kotrbová

et al. 2020

Preprint

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“…Having characterized our model system, we examined cilia formation in those cells. Consistently with previous work, HEK293 cells form cilia less frequently than RPE-1 or NIH3T3 cells (Lancaster et al, 2011;Bernatik et al, 2020). We were able to detect about 5% of cells with Arl13b+ primary cilium in WT HEK293.…”

Section: Genetic Ablation Of Wnt/β-catenin Pathway Does Not Alter Prisupporting

confidence: 92%

“…Western blot was performed as previously described (Bernatik et al, 2020). Antibodies used: LRP6 (Cell signaling, Cat.no.…”

Section: Western Blot and Quantificationmentioning

confidence: 99%

Primary Cilia Formation Does Not Rely on WNT/β-Catenin Signaling

Bernatík

Paclíková

Kotrbová

et al. 2021

Front. Cell Dev. Biol.

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Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

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“…To ensure specificity of distal end uncapping and thereby cilium formation at the mother centriole, it is the distal appendage protein CEP164 that recruits TTBK2 (Schmidt et al, 2012;Čajánek and Nigg, 2014). TTBK2 has recently been shown to phosphorylate distal appendage proteins, such as CEP164 and CEP83 (Bernatik et al, 2020), which is required for efficient vesicle recruitment (Figure 2B; Lo et al, 2019). Notably, TTBK2 phosphorylates MPP9 resulting in the loss of MPP9 and the CEP97-CP110 complex from the distal centriolar end (Figure 2C; Huang et al, 2018).…”

Section: Cilium Dynamics Cilium Assembly-a Primary Cilium Is (Re)bornmentioning

confidence: 98%

Phosphorylation and Ubiquitylation Regulate Protein Trafficking, Signaling, and the Biogenesis of Primary Cilia

May

Sroka

Mick

2021

Front. Cell Dev. Biol.

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The primary cilium is a solitary, microtubule-based membrane protrusion extending from the surface of quiescent cells that senses the cellular environment and triggers specific cellular responses. The functions of primary cilia require not only numerous different components but also their regulated interplay. The cilium performs highly dynamic processes, such as cell cycle-dependent assembly and disassembly as well as delivery, modification, and removal of signaling components to perceive and process external signals. On a molecular level, these processes often rely on a stringent control of key modulatory proteins, of which the activity, localization, and stability are regulated by post-translational modifications (PTMs). While an increasing number of PTMs on ciliary components are being revealed, our knowledge on the identity of the modifying enzymes and their modulation is still limited. Here, we highlight recent findings on cilia-specific phosphorylation and ubiquitylation events. Shedding new light onto the molecular mechanisms that regulate the sensitive equilibrium required to maintain and remodel primary cilia functions, we discuss their implications for cilia biogenesis, protein trafficking, and cilia signaling processes.

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

Abstract: Our findings expand current knowledge of TTBK2 substrates and provide insight into extent of TTBK2-mediated phosphorylations in the context of basal body proteins and PC initiation. We further show that phosphorylation of CEP164 N-term by TTBK2 regulates ciliogenesis.

Cited by 32 publications

References 79 publications

Primary cilia formation does not rely on WNT/β-catenin signaling

Primary cilia formation does not rely on WNT/β-catenin signaling

Primary Cilia Formation Does Not Rely on WNT/β-Catenin Signaling

Phosphorylation and Ubiquitylation Regulate Protein Trafficking, Signaling, and the Biogenesis of Primary Cilia

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