Primary cilia 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 the final step of cilia initiation. The function of TTBK2 in ciliogenesis is critically dependent on its kinase activity; however, the precise mechanism of TTBK2 action has so far not been fully understood due to the very limited information about its relevant substrates. In this study, we demonstrate that CEP83, CEP89, CCDC92, Rabin8, and DVL3 are substrates of TTBK2 kinase activity. Further, we characterize a set of phosphosites of those 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 show that TTBK2 is also required for efficient phosphorylation of many S/T sites in CEP164 and provide evidence that TTBK2-induced phosphorylations of CEP164 modulate its function, which in turn seems relevant for the process of cilia formation. In summary, our work provides important insight into the 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.

Central European Institute of Technology Masaryk University 62500 Brno Czech Republic

Department of Histology and Embryology Faculty of Medicine Masaryk University 62500 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University 62500 Brno Czech Republic

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Bah A, Forman-Kay JD. (2016). Modulation of intrinsically disordered protein function by post-translational modifications. J Biol Chem , 6696–6705. PubMed PMC

Bah A, Vernon RM, Siddiqui Z, Krzeminski M, Muhandiram R, Zhao C, Sonenberg N, Kay LE, Forman-Kay JD. (2015). Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch. Nature , 106–109. PubMed

Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. (2009). MEME Suite: Tools for motif discovery and searching. Nucleic Acids Res , 10.1093/nar/gkp335. PubMed DOI PMC

Bauer P, Stevanin G, Beetz C, Synofzik M, Schmitz-Hübsch T, Wüllner U, Berthier E, Ollagnon-Roman E, Riess O, Forlani S, et al (2010). Spinocerebellar ataxia type 11 (SCA11) is an uncommon cause of dominant ataxia among French and German kindreds. J Neurol Neurosurg Psychiatry , 1229–1232. PubMed

Bernatík O, Šedová K, Schille C, Ganji RS, Cˇervenka I, Trantírek L, Schambony A, Zdráhal Z, Bryja V. (2014). Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1ε and Frizzled5. J Biol Chem , 23520–23533. PubMed PMC

Borgal L, Rinschen MM, Dafinger C, Hoff S, Reinert MJ, Lamkemeyer T, Lienkamp SS, Benzing T, Schermer B. (2014). Casein kinase 1 α phosphorylates the Wnt regulator Jade-1 and modulates its activity. J Biol Chem , 26344–26356. PubMed PMC

Bouskila M, Esoof N, Gay L, Fang EH, Deak M, Begley MJ, Cantley LC, Prescott A, Storey KG, Alessi DR. (2011). TTBK2 kinase substrate specificity and the impact of spinocerebellar- ataxia-causing mutations on expression, activity, localization and development. Biochem J , 157–167. PubMed PMC

Bowie E, Norris R, Anderson KV, Goetz SC. (2018). Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability. PLoS Genet , 10.1371/journal.pgen.1007844. PubMed DOI PMC

Bowler M, Kong D, Sun S, Nanjundappa R, Evans L, Farmer V, Holland A, Mahjoub MR, Sui H, Loncarek J. (2019). High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy. Nat Commun , 993. PubMed PMC

Bryja V, Cˇervenka I, Cˇajánek L. (2017). The connections of Wnt pathway components with cell cycle and centrosome: side effects or a hidden logic? Crit Rev Biochem Mol Biol , 614–637. PubMed PMC

Cˇajánek L, Nigg EA. (2014). Cep164 triggers ciliogenesis by recruiting Tau tubulin kinase 2 to the mother centriole. Proc Natl Acad Sci USA , E2841–E2850. PubMed PMC

Cegielska A, Gietzen KF, Rivers A, Virshup DM. (1998). Autoinhibition of casein kinase I ε (CKIε) is relieved by protein phosphatases and limited proteolysis. J Biol Chem , 1357–1364. PubMed

Cervenka I, Valnohova J, Bernatik O, Harnos J, Radsetoulal M, Sedova K, Hanakova K, Potesil D, Sedlackova M, Salasova A, et al (2016). Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins. Proc Natl Acad Sci USA , 9304–9309. PubMed PMC

Chaki M, Airik R, Ghosh AK, Giles RH, Chen R, Slaats GG, Wang H, Hurd TW, Zhou W, Cluckey A, et al (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling. Cell , 533–548. PubMed PMC

Chang J, Seo SG, Lee KH, Nagashima K, Bang JK, Kim BY, Erikson RL, Lee KW, Lee HJ, Park JE, et al (2013). Essential role of Cenexin1, but not Odf2, in ciliogenesis. Cell Cycle , 655–662. PubMed PMC

Cheng A, Grant C, Bailey TL, Noble W. (2017). MoMo: Discovery of post-translational modification motifs. BioRxiv . PubMed PMC

Cheong JK, Virshup DM. (2011). Casein kinase 1: Complexity in the family. Int J Biochem Cell Biol , 465–469. PubMed

Chou MF, Schwartz D. (2011). Biological sequence motif discovery sing motif-x. Curr Protoc Bioinforma, 10.1002/0471250953.bi1315s35. PubMed DOI

Cohen P. (2000). The regulation of protein function by multisite phosphorylation - A 25 year update. Trends Biochem Sci , 596–601. PubMed

Dyson HJ, Wright PE. (2005). Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol , 197–208. PubMed

Ferrarese A, Marin O, Bustos VH, Venerando A, Antonelli M, Allende JE, Pinna LA. (2007). Chemical dissection of the APC repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3. Biochemistry , 11902–11910. PubMed

Flotow H, Graves PR, Wang A, Fiol CJ, Roeske RW, Roach PJ. (1990). Phosphate groups as substrate determinants for casein kinase I action. J Biol Chem , 14264–14269. PubMed

Flotow H, Roach PJ. (1991). Role of acidic residues as substrate determinants for casein kinase I. J Biol Chem , 3724–3727. PubMed

Goetz SC, Liem KF, Anderson KV. (2012). The spinocerebellar ataxia-associated gene tau tubulin kinase 2 controls the initiation of ciliogenesis. Cell , 847–858. PubMed PMC

Graser S, Stierhof YD, Lavoie SB, Gassner OS, Lamla S, Le Clech M, Nigg EA. (2007). Cep164, a novel centriole appendage protein required for primary cilium formation. J Cell Biol , 321–330. PubMed PMC

Hanáková K, Bernatík O, Ovesná P, Kravec M, Micka M, Rádsetoulal M, Poteˇšil D, Cˇajánek L, Zdráhal Z, Bryja V. (2019). Comparative phosphorylation map of Dishevelled3 (DVL3). BioRxiv . PubMed PMC

Hegyi H, Schad E, Tompa P. (2007). Structural disorder promotes assembly of protein complexes. BMC Struct Biol , 10.1186/1472-6807-7-65. PubMed DOI PMC

Hehnly H, Chen CT, Powers CM, Liu HL, Doxsey S. (2012). The centrosome regulates the Rab11- dependent recycling endosome pathway at appendages of the mother centriole. Curr Biol , 1944–1950. PubMed PMC

Houlden H, Johnson J, Gardner-Thorpe C, Lashley T, Hernandez D, Wort P, Singleton AB, Hilton DA, Holton J, Revesz T, et al (2007). Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11. Nat Genet , 1434–1436. PubMed

Huang N, Zhang D, Li F, Chai P, Wang S, Teng J, Chen J. (2018). M-Phase Phosphoprotein 9 regulates ciliogenesis by modulating CP110-CEP97 complex localization at the mother centriole. Nat Commun , 4511. PubMed PMC

Hunter T. (1995). Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling. Cell , 225–236. PubMed

Iakoucheva LM, Radivojac P, Brown CJ, O’Connor TR, Sikes JG, Obradovic Z, Dunker AK. (2004). The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res , 1037–1049. PubMed PMC

Ikezu S, Ikezu T. (2014). Tau-tubulin kinase. Front Mol Neurosci , 10.3389/fnmol.2014.00033. PubMed DOI PMC

Ishikawa H, Marshall WF. (2011). Ciliogenesis: Building the cell’s antenna. Nat Rev Mol Cell Biol , 222–234. PubMed

Jay J, Hammer A, Nestor-Kalinoski A, Diakonova M. (2015). JAK2 tyrosine kinase phosphorylates and is negatively regulated by centrosomal protein ninein. Mol Cell Biol , 111–131. PubMed PMC

Jenks AD, Vyse S, Wong JP, Kostaras E, Keller D, Burgoyne T, Shoemark A, Tsalikis A, de la Roche M, Michaelis M, et al (2018). Primary cilia mediate diverse kinase inhibitor resistance mechanisms in cancer. Cell Rep , 3042–3055. PubMed PMC

Joo K, Kim CG, Lee MS, Moon HY, Lee SH, Kim MJ, Kweon HS, Park WY, Kim CH, Gleeson JG, et al (2013). CCDC41 is required for ciliary vesicle docking to the mother centriole. Proc Natl Acad Sci USA , 5987–5992. PubMed PMC

Kim J, Lee JE, Heynen-Genel S, Suyama E, Ono K, Lee K, Ideker T, Aza-Blanc P, Gleeson JG. (2010). Functional genomic screen for modulators of ciliogenesis and cilium length. Nature , 1048–1051. PubMed PMC

Kitano-Takahashi M, Morita H, Kondo S, Tomizawa K, Kato R, Tanio M, Shirota Y, Takahashi H, Sugio S, Kohno T. (2007). Expression, purification and crystallization of a human tau-tubulin kinase 2 that phosphorylates tau protein. Acta Crystallogr Sect F Struct Biol Cryst Commun , 602–604. PubMed PMC

Knippschild U, Gocht A, Wolff S, Huber N, Löhler J, Stöter M. (2005). The casein kinase 1 family: Participation in multiple cellular processes in eukaryotes. Cell Signal , 675–689. PubMed

Knödler A, Feng S, Zhang J, Zhang X, Das A, Peränen J, Guo W. (2010). Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc Natl Acad Sci USA , 6346–6351. PubMed PMC

Kuhns S, Schmidt KN, Reymann J, Gilbert DF, Neuner A, Hub B, Carvalho R, Wiedemann P, Zentgraf H, Erfle H, et al (2013). The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis. J Cell Biol , 505–522. PubMed PMC

Kurtulmus B, Yuan C, Schuy J, Neuner A, Hata S, Kalamakis G, Martin-Villalba A, Pereira G. (2018). LRRC45 contributes to early steps of axoneme extension. J Cell Sci , 10.1242/jcs.223594. PubMed DOI

Liao JC, Yang TT, Weng RR, Kuo CTe, Chang CW. (2015). TTBK2: A tau protein kinase beyond tau phosphorylation. Biomed Res Int , 10.1155/2015/575170. PubMed DOI PMC

Lindquist SG, Møller LB, Dali CI, Marner L, Kamsteeg EJ, Nielsen JE, Hjermind LE. (2017). A novel TTBK2 de novo mutation in a danish family with early-onset spinocerebellar ataxia. Cerebellum , 268–271. PubMed

Lo CH, Lin IH, Yang TT, Huang YC, Tanos BE, Chou PC, Chang CW, Tsay YG, Liao JC, Wang WJ. (2019). Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation. J Cell Biol , 3489–3505. PubMed PMC

Lu Q, Insinna C, Ott C, Stauffer J, Pintado PA, Rahajeng J, Baxa U, Walia V, Cuenca A, Hwang YS, et al (2015). Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat Cell Biol , 228–240. PubMed PMC

Marin O, Bustos VH, Cesaro L, Meggio F, Pagano MA, Antonelli M, Allende CC, Pinna LA, Allende JE. (2003). A noncanonical sequence phosphorylated by casein kinase 1 in β-catenin may play a role in casein kinase 1 targeting of important signaling proteins. Proc Natl Acad Sci USA , 10193–10200. PubMed PMC

Mitchison HM, Valente EM. (2017). Motile and non-motile cilia in human pathology: from function to phenotypes. J Pathol , 294–309. PubMed

Mlodzik M. (2016). The dishevelled protein family: still rather a mystery after over 20 years of molecular studies. Curr Top Dev Biol, 75–91. PubMed PMC

Nigg EA, Stearns T. (2011). The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries. Nat Cell Biol , 1154–1160. PubMed PMC

Oda T, Chiba S, Nagai T, Mizuno K. (2014). Binding to Cep164, but not EB1, is essential for centriolar localization of TTBK2 and its function in ciliogenesis. Genes to Cells , 927–940. PubMed

Peng K, Vucetic S, Radivojac P, Brown CJ, Dunker AK, Obradovic Z. (2005). Optimizing long intrinsic disorder predictors with protein evolutionary information. J Bioinform Comput Biol , 35–60. PubMed

Peng QY, Zhang QF. (2006). Precise positions of Phoebe determined with CCD image-overlapping calibration. Mon Not R Astron Soc , 208–212.

Perez-Riverol Y, et al (2019). The PRIDE database and related tools and resources in 2019: Improving support for quantification data. Nucleic Acids Res , D442–D450. PubMed PMC

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc , 2281–2308. PubMed PMC

Reiter JF, Leroux MR. (2017). Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol , 533–547. PubMed PMC

Schindelin J, et al (2012). Fiji: An open-source platform for biological-image analysis. Nat Methods , 676–682. PubMed PMC

Schmidt KN, Kuhns S, Neuner A, Hub B, Zentgraf H, Pereira G. (2012). Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis. J Cell Biol , 1083–1101. PubMed PMC

Schwarz-Romond T, Fiedler M, Shibata N, Butler PJG, Kikuchi A, Higuchi Y, Bienz M. (2007). The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization. Nat Struct Mol Biol , 484–492. PubMed

Seeley ES, Nachury MV. (2010). The perennial organelle: Assembly and disassembly of the primary cilium. J Cell Sci , 511–518. PubMed PMC

Sieracki NA, Komarova YA. (2013). Studying cell signal transduction with biomimetic point mutations. InTech, 10.5772/35029. DOI

Sillibourne JE, Hurbain I, Grand-Perret T, Goud B, Tran P, Bornens M. (2013). Primary ciliogenesis requires the distal appendage component Cep123. Biol Open , 535–545. PubMed PMC

Sorokin S. (1962). Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol , 363–377. PubMed PMC

Spektor A, Tsang WY, Khoo D, Dynlacht BD. (2007). Cep97 and CP110 suppress a cilia assembly program. Cell , 678–690. PubMed

Takahashi M, Tomizawa K, Sato K, Ohtake A, Omori A. (1995). A novel tau-tubulin kinase from bovine brain. FEBS Lett , 59–64. PubMed

Tanos BE, Yang HJ, Soni R, Wang WJ, Macaluso FP, Asara JM, Tsou MFB. (2013). Centriole distal appendages promote membrane docking, leading to cilia initiation. Genes Dev , 163–168. PubMed PMC

Tomizawa K, Omori A, Ohtake A, Sato K, Takahashi M. (2001). Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau. FEBS Lett , 221–227. PubMed

Tsun A, Qureshi I, Stinchcombe JC, Jenkins MR, De La Roche M, Kleczkowska J, Zamoyska R, Griffiths GM. (2011). Centrosome docking at the immunological synapse is controlled by Lck signaling. J Cell Biol , 663–674. PubMed PMC

Wang L, Dynlacht BD. (2018). The regulation of cilium assembly and disassembly in development and disease. Dev . PubMed PMC

Watanabe T, Kakeno M, Matsui T, Sugiyama I, Arimura N, Matsuzawa K, Shirahige A, Ishidate F, Nishioka T, Taya S, et al (2015). TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation. J Cell Biol , 737–751. PubMed PMC

Wei Q, Xu Q, Zhang Y, Li Y, Zhang Q, Hu Z, Harris PC, Torres VE, Ling K, Hu J. (2013). Transition fibre protein FBF1 is required for the ciliary entry of assembled intraflagellar transport complexes. Nat Commun , 10.1038/ncomms3750. PubMed DOI PMC

Westlake CJ, Baye LM, Nachury MV, Wright KJ, Ervin KE, Phu L, Chalouni C, Beck JS, Kirkpatrick DS, Slusarski DC, et al (2011). Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. Proc Natl Acad Sci USA , 2759–2764. PubMed PMC

Wong SY, Seol AD, So PL, Ermilov AN, Bichakjian CK, Epstein EH, Dlugosz AA, Reiter JF. (2009). Primary cilia can both mediate and suppress Hedgehog pathway-dependent tumorigenesis. Nat Med , 1055–1061. PubMed PMC

Wong YL, Anzola JV, Davis RL, Yoon M, Motamedi A, Kroll A, Seo CP, Hsia JE, Kim SK, Mitchell JW, et al (2015). Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science (80-) , 1155–1160. PubMed PMC

Wu CT, Chen HY, Tang TK. (2018). Myosin-Va is required for preciliary vesicle transportation to the mother centriole during ciliogenesis. Nat Cell Biol , 175–185. PubMed

Xie H, Vucetic S, Iakoucheva LM, Oldfield CJ, Dunker AK, Obradovic Z, Uversky VN. (2007). Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins. J Proteome Res , 1917–1932. PubMed PMC

Xu Q, Zhang Y, Wei Q, Huang Y, Hu J, Ling K. (2016). Phosphatidylinositol phosphate kinase PIPKIγ and phosphatase INPP5E coordinate initiation of ciliogenesis. Nat Commun , 10777. PubMed PMC

Yang TT, et al (2018). Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components. Nat Commun , 10.1038/s41467-018-04469-1. PubMed DOI PMC

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