A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance

Abstract

Author contributions R.R.C. initiated the project, phenotyped the index proband, designed and performed all experiments except as detailed herewith, analyzed data, prepared figures, and wrote the manuscript. D.T.M assisted with designing and performing cell culture, IF, and FACS experiments, analyzed data, and edited the manuscript. H.M.L. performed μOCT experiments, analyzed data, and prepared figures. J.Y. assisted with molecular cloning, site-directed mutagenesis, and cell culture and edited the manuscript. H.E.S. performed whole exome sequencing and linkage analysis on kindreds 1-3. M.S.T. acquired clinical histopathology images and prepared figures. G.W.D. performed sequencing, molecular biology, and HSVM analysis on kindred 4. M.A.Z. led molecular analysis of kindred 5. J.C. performed and interpreted kindred 5 ciliary EM and HSVM. L.A.D. identified kindred 5 patients and provided clinical data. P.S. performed HSVM. K.E.B. and L.P.H. assisted with obtaining proband 1 clinical samples. I.A. identified bronchiectasis kindreds 2 and 3. E.M.F. assisted with analysis of phosphoproteomics data. V.V. assisted with IF experiments, analyzed data, and edited the manuscript. H.O. supervised and led kindred 4 analyses. M.R.K. supervised and led kindred 5 molecular analysis and clinical phenotyping. G.J.T. supervised μOCT experiments and analyzed data. F.S.A. supervised whole exome sequencing and linkage analysis of kindreds 1-3, analyzed genetics data, and edited the manuscript. D.M.S. supervised the project, designed experiments, and edited the manuscript.

Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

“…Glutamylation may also play an important role in ciliopathies including Joubert Syndrome [72] and hereditary retinal degeneration [24]. NEK kinases are also implicated in ciliopathies [26,73] and cancer [74][75][76]. Our work suggests that NEK kinases may represent new targets for treatment of human neurodegenerative and ciliopathic disease, and that insights into the function of NEKs might be crucial for understanding pathways by which the Tubulin Code specializes microtubule networks and ciliary integrity in human ciliopathies and neurodegenerative disorders.…”

“…NEK10 potential substrates include kinesins, IFT components, and other ciliary proteins that have homologs in C. elegans ([26,60]; Supplementary Table 4). In human bronchial epithelial cell (HBEC) culture, NEK10 mutation caused a reduction in phosphopeptides of gene products including KIF13B, KIF19, IFT140, IFT46, IFT88, and MAK [26]. These genes and their C. elegans orthologs klp-4, klp-13, che-11, dyf-6, osm-5, and dyf-5 are involved with microtubule dynamics, IFT, and ciliary structure [41,[61][62][63][64][65][66][67][68][69].…”

“…Using EMBOSS epestfind [36], we also identified a PEST domain, important for proteolytic degradation (residues 748-764) [37]. NEKL-4 is a homolog of human NEK10, a protein required for ciliogenesis and associated with ciliopathies ( [25,26]; Fig. 3c).…”

“…+ indicates any degree of suppression of the ccpp-1 phenotype, -indicates no suppression of the ccpp-1 phenotype, n/a indicates not tested. Figure 4f [26]. Original list contains genes with phosphopeptides depleted more than twofold upon NEK10 deletion in human bronchial epithelial cell (HBEC) culture.…”

“…C. elegans NEKL-4 is homologous to human NEK10, which is a kinase specific to ciliated cells and contributes to ciliogenesis in cultured human kidney cells [25]. In humans, NEK10 mutation causes bronchiectasis, a disorder of mucociliary transport in the airway due to defective motile cilia [26]. Here we show that NEKL-4 is expressed in all ciliated neurons but does not localize to cilia, suggesting that NEKL-4 indirectly influences regulation of ciliary stability by CCPP-1 and glutamylation.…”