Intestinal Cell Kinase Research Articles

Isc10, an inhibitor of the Smk1 MAPK, prevents activation loop autophosphorylation and substrate phosphorylation through separate mechanisms

Many eukaryotic protein kinases are activated by the intramolecular autophosphorylation of activation loop residues. Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in yeast that autophosphorylates its activation loop tyrosine and thereby upregulates catalytic output. This reaction is controlled by an inhibitor, Isc10, that binds the MAPK during meiosis I and an activator, Ssp2, that binds Smk1/Isc10 during meiosis II. Upon completion of the meiotic divisions, Isc10 is degraded, and Smk1 undergoes autophosphorylation to generate the high activity form of the MAPK that controls spore formation. How Isc10 inhibits Smk1 is not clear. Here, we use a bacterial coexpression/reconstitution system to define a domain in the carboxy-terminal half of Isc10 that specifically inhibits Smk1 autophosphorylation. Nevertheless, Smk1 bound by this domain is able to phosphorylate other substrates, and it phosphorylates the amino-terminal half of Isc10 on serine 97. In turn, the phosphorylated motif in Isc10 inhibits the Smk1 active site. These data show that Isc10 inhibits autophosphorylation and the phosphorylation of substrates by separate mechanisms. Furthermore, we demonstrate Isc10 can inhibit the autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. These findings define a novel class of developmentally regulated molecules that prevent the self-activation of MAPKs and MAPK-like enzymes.

BROMI/TBC1D32 together with CCRK/CDK20 and FAM149B1/JBTS36 contributes to intraflagellar transport turnaround involving ICK/CILK1.

Primary cilia are antenna-like organelles that contain specific proteins, and are crucial for tissue morphogenesis. Anterograde and retrograde trafficking of ciliary proteins are mediated by the intraflagellar transport (IFT) machinery. BROMI/TBC1D32 interacts with CCRK/CDK20, which phosphorylates and activates the intestinal cell kinase (ICK)/CILK1 kinase, to regulate the change in direction of the IFT machinery at the ciliary tip. Mutations in BROMI, CCRK, and ICK in humans cause ciliopathies, and mice defective in these genes are also known to demonstrate ciliopathy phenotypes. We show here that BROMI interacts not only with CCRK but also with CFAP20, an evolutionarily conserved ciliary protein, and with FAM149B1/ Joubert syndrome (JBTS)36, a protein in which mutations cause JBTS. In addition, we show that FAM149B1 interacts directly with CCRK as well as with BROMI. Ciliary defects observed in CCRK-knockout (KO), BROMI-KO, and FAM149B1-KO cells, including abnormally long cilia and accumulation of the IFT machinery and ICK at the ciliary tip, resembled one another, and BROMI mutants that are defective in binding to CCRK and CFAP20 were unable to rescue the ciliary defects of BROMI-KO cells. These data indicate that CCRK, BROMI, FAM149B1, and probably CFAP20 altogether regulate the IFT turnaround process under the control of ICK.

Isc10 is a Dual Mechanism Inhibitor of CMGC Kinases

Autophosphorylation of the activation loop is a common step that converts many protein kinases from an inactive to an active state. Despite the physiological importance of these reactions, the mechanisms that control activation loop autophosphorylation are poorly understood. Smk1 is a meiosis‐specific MAPK in yeast that autophosphorylates its activation loop on tyrosine (Y), thereby upregulating its own catalytic activity. Previously, we identified Isc10 as an inhibitor of Smk1. Here, we show that Isc10 inhibits Smk1 by two mechanisms. First, the C‐terminal segment of Isc10 binds to Smk1 and specifically inhibits the cis‐autophosphorylation of Y209 in its activation‐loop. Second, prior to Smk1 autophosphorylation, the low activity state Smk1 that is bound to Isc10 phosphorylates it on serine 97 (S97). The phosphorylated S97, in turn, becomes trapped in the Smk1 active site. This prevents phosphorylation of downstream targets before activating signals promote Isc10 degradation and the release of active Smk1. Further, Isc10 inhibits autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. Taken together, these findings define a novel class of developmentally‐regulated molecules that inhibit CMGC‐group kinases.

Mice Harboring a Non-Functional CILK1/ICK Allele Fail to Model the Epileptic Phenotype in Patients Carrying Variant CILK1/ICK.

CILK1 (ciliogenesis associated kinase 1)/ICK (intestinal cell kinase) is a highly conserved protein kinase that regulates primary cilia structure and function. CILK1 mutations cause a wide spectrum of human diseases collectively called ciliopathies. While several CILK1 heterozygous variants have been recently linked to juvenile myoclonic epilepsy (JME), it remains unclear whether these mutations cause seizures. Herein, we investigated whether mice harboring either a heterozygous null Cilk1 (Cilk1+/−) mutation or a heterozygous loss-of-function Cilk1 mutation (Cilk1R272Q/+) have epilepsy. We first evaluated the spontaneous seizure phenotype of Cilk1+/− and Cilk1R272Q/+ mice relative to wildtype littermates. We observed no electrographic differences among the three mouse genotypes during prolonged recordings. We also evaluated electrographic and behavioral responses of mice recovering from isoflurane anesthesia, an approach recently used to measure seizure-like activity. Again, we observed no electrographic or behavioral differences in control versus Cilk1+/− and Cilk1R272Q/+ mice upon isoflurane recovery. These results indicate that mice bearing a non-functional copy of Cilk1 fail to produce electrographic patterns resembling those of JME patients with a variant CILK1 copy. Our findings argue against CILK1 haploinsufficiency being the mechanism that links CILK1 variants to JME.

Elucidating CCRK’s function in ciliary assembly and cilium‐dependent signaling

Our studies are focused on the questions of how ciliary length and ciliary import of cell signaling regulators are controlled. Despite many advances, these questions have yet to be fully answered. One clue as to the mechanistic basis of these processes comes from studies on Cell Cycle‐Related Kinase (CCRK). Work on the Clamydomonas homolog, LF2, indicates that this kinase ensures that flagella are neither too long nor too short, likely mediated by regulated transport of ciliary tubulin ( Craft et al., 2015). We previously showed that mammalian CCRK is similarly required for ciliary length control and that it is needed for cells to achieve appropriate responses to Hedgehog (Hh) signals ( Snouffer et al., 2017). Additional work indicated that CCRK promotes the efficient transport of Hh pathway regulatory factors, such as Smoothened and Gli2, into cilia. However, the manner by which the kinase directs these processes is unknown. One target of CCRK phosphorylation is the ciliary length regulator Intestinal Cell Kinase (ICK); CCRK‐mediated phosphorylation in ICK’s TDY motif promotes ICK activity. Our analysis of Ccrk/Ick double mutants indicates that CCRK must also function via ICK‐independent mechanisms and that ICK promotes functions in the absence of CCRK‐mediated phosphorylation. To gain insight into the ICK‐independent CCRK functions in ciliary length control and ciliary cargo import, we performed phosphoproteomic screens for potential mediators of CCRK function. This screen has pointed to candidate mediators that regulate microfilament and microtubule dynamics. Consistent with a role for CCRK’s action via microfilament dynamics, Ccrk mutant, but not wild‐type, cells showed greatly reduced import of ciliary cargo upon treatment with sub‐threshold doses of the actin polymerization inhibitor Cytochalasin D. Our ongoing work on this mechanism will be presented.Support or Funding InformationThis work was supported by National Institute of Child Health and Human Development grant number R01HD050761 and by the Office of the Vice President of Research at the University of Georgia.

Functional Alterations in Ciliogenesis-Associated Kinase 1 (CILK1) that Result from Mutations Linked to Juvenile Myoclonic Epilepsy.

Ciliopathies are a group of human genetic disorders associated with mutations that give rise to the dysfunction of primary cilia. Ciliogenesis-associated kinase 1 (CILK1), formerly known as intestinal cell kinase (ICK), is a conserved serine and threonine kinase that restricts primary (non-motile) cilia formation and length. Mutations in CILK1 are associated with ciliopathies and are also linked to juvenile myoclonic epilepsy (JME). However, the effects of the JME-related mutations in CILK1 on kinase activity and CILK1 function are unknown. Here, we report that JME pathogenic mutations in the CILK1 N-terminal kinase domain abolish kinase activity, evidenced by the loss of phosphorylation of kinesin family member 3A (KIF3A) at Thr672, while JME mutations in the C-terminal non-catalytic domain (CTD) have little effect on KIF3A phosphorylation. Although CILK1 variants in the CTD retain catalytic activity, they nonetheless lose the ability to restrict cilia length and also gain function in promoting ciliogenesis. We show that wild type CILK1 predominantly localizes to the base of the primary cilium; in contrast, JME variants of CILK1 are distributed along the entire axoneme of the primary cilium. These results demonstrate that JME pathogenic mutations perturb CILK1 function and intracellular localization. These CILK1 variants affect the primary cilium, independent of CILK1 phosphorylation of KIF3A. Our findings suggest that CILK1 mutations linked to JME result in alterations of primary cilia formation and homeostasis.

Activation of sonic hedgehog signaling by a Smoothened agonist restores congenital defects in mouse models of endocrine-cerebro-osteodysplasia syndrome.

BackgroundEndocrine-cerebro-osteodysplasia (ECO) syndrome is a genetic disorder associated with congenital defects of the endocrine, cerebral, and skeletal systems in humans. ECO syndrome is caused by mutations of the intestinal cell kinase (ICK) gene, which encodes a mitogen-activated protein (MAP) kinase-related kinase that plays a critical role in controlling the length of primary cilia. Lack of ICK function disrupts transduction of sonic hedgehog (SHH) signaling, which is important for development and homeostasis in humans and mice. Craniofacial structure abnormalities, such as cleft palate, are one of the most common defects observed in ECO syndrome patients, but the role of ICK in palatal development has not been studied.MethodsUsing Ick-mutant mice, we investigated the mechanisms by which ICK function loss causes cleft palate and examined pharmacological rescue of the congenital defects.FindingsSHH signaling was compromised with abnormally elongated primary cilia in the developing palate of Ick-mutant mice. Cell proliferation was significantly decreased, resulting in failure of palatal outgrowth, although palatal adhesion and fusion occurred normally. We thus attempted to rescue the congenital palatal defects of Ick mutants by pharmacological activation of SHH signaling. Treatment of Ick-mutant mice with an agonist for Smoothened (SAG) rescued several congenital defects, including cleft palate.InterpretationsThe recovery of congenital defects by pharmacological intervention in the mouse models for ECO syndrome highlights prenatal SHH signaling modulation as a potential therapeutic measure to overcome congenital defects of ciliopathies.

Subcellular Targeting of the Euplotes raikovi Kinase Er-MAPK1, as Revealed by Expression in Different Cell Systems.

In the ciliate Euplotes raikovi, a 631-amino acid Er-MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key component of an autocrine, cell-growth promoting self-signaling mechanism. While its 283-amino acid N-terminal domain includes all the structural specificities of the mitogen-activated protein kinases required for a catalytic function, the 348-amino acid C-terminal domain is structurally unique with undetermined functions. By expressing the two Er-MAPK1 domains tagged with the green fluorescent protein in mammalian fibroblasts, the yeast Schizosaccharomyces pombe and the ciliate Tetrahymena thermophila, evidence was obtained that the C-terminal domain contains all the sequence information responsible for the Er-MAPK1 subcellular localization. However, in fibroblasts and S. pombe this information determined a nucleolar localization of the GFP-tagged C-terminal domain, and a ciliary localization in T. thermophila. In the light of these findings, the Er-MAPK1 localization in E. raikovi was re-examined via immunoreactions and shown to be ciliary besides that nuclear, as is the case for the mammalian intestinal cell kinase with which the Er-MAPK1 N-terminal domain shares a strong sequence identity and a catalytic function.

Ciliogenesis associated kinase 1: targets and functions in various organ systems.

Ciliogenesis associated kinase 1 (CILK1) was previously known as intestinal cell kinase because it was cloned from that origin. However, CILK1 is now recognized as a widely expressed and highly conserved serine/threonine protein kinase. Mutations in the human CILK1 gene have been associated with ciliopathies, a group of human genetic disorders with defects in the primary cilium. In mice, both Cilk1 knock-out and Cilk1 knock-in mutations have recapitulated human ciliopathies. Thus, CILK1 has a fundamental role in the function of the cilium. Several candidate substrates have been proposed for CILK1 and the challenge is to relate these to the mutant phenotypes. In this review, we summarize what is known about CILK1 functions and targets, and discuss gaps in current knowledge that motivate further experimentation to fully understand the role of CILK1 in organ development in humans.

Ciliopathy-Associated Protein Kinase ICK Requires Its Non-Catalytic Carboxyl-Terminal Domain for Regulation of Ciliogenesis.

Loss-of-function mutations in the human ICK (intestinal cell kinase) gene cause dysfunctional primary cilia and perinatal lethality which are associated with human ciliopathies. The enzyme that we herein call CAPK (ciliopathy-associated protein kinase) is a serine/threonine protein kinase that has a highly conserved MAPK-like N-terminal catalytic domain and an unstructured C-terminal domain (CTD) whose functions are completely unknown. In this study, we demonstrate that truncation of the CTD impairs the ability of CAPK to interact with and phosphorylate its substrate, kinesin family member 3A (KIF3A). We also find that deletion of the CTD of CAPK compromises both localization to the primary cilium and negative regulation of ciliogenesis. Thus, CAPK substrate recognition, ciliary targeting, and ciliary function depend on the non-catalytic CTD of the protein which is predicted to be intrinsically disordered.

Fibroblast growth factor receptor influences primary cilium length through an interaction with intestinal cell kinase

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

Enriched expression of the ciliopathy gene Ick in cell proliferating regions of adult mice

Cilia are essential for sensory and motile functions across species. In humans, ciliary dysfunction causes “ciliopathies”, which show severe developmental abnormalities in various tissues. Several missense mutations in intestinal cell kinase (ICK) gene lead to endocrine-cerebro-osteodysplasia syndrome or short rib-polydactyly syndrome, lethal recessive developmental ciliopathies. We and others previously reported that Ick-deficient mice exhibit neonatal lethality with developmental defects. Mechanistically, Ick regulates intraflagellar transport and cilia length at ciliary tips. Although Ick plays important roles during mammalian development, roles of Ick at the adult stage are poorly understood. In the current study, we investigated the Ick gene expression in adult mouse tissues. RT-PCR analysis showed that Ick is ubiquitously expressed, with enrichment in the retina, brain, lung, intestine, and reproductive system. In the adult brain, we found that Ick expression is enriched in the walls of the lateral ventricle, in the rostral migratory stream of the olfactory bulb, and in the subgranular zone of the hippocampal dentate gyrus by in situ hybridization analysis. We also observed that Ick staining pattern is similar to pachytene spermatocyte to spermatid markers in the mature testis and to an intestinal stem cell marker in the adult small intestine. These results suggest that Ick is expressed in proliferating regions in the adult mouse brain, testis, and intestine.

Variant Intestinal-Cell Kinase in Juvenile Myoclonic Epilepsy

BackgroundIn juvenile myoclonic epilepsy, data are limited on the genetic basis of networks promoting convulsions with diffuse polyspikes on electroencephalography (EEG) and the subtle microscopic brain dysplasia called microdysgenesis.MethodsUsing Sanger sequencing, we sequenced the exomes of six members of a large family affected with juvenile myoclonic epilepsy and confirmed cosegregation in all 37 family members. We screened an additional 310 patients with this disorder for variants on DNA melting-curve analysis and targeted real-time DNA sequencing of the gene encoding intestinal-cell kinase (ICK). We calculated Bayesian logarithm of the odds (LOD) scores for cosegregating variants, odds ratios in case–control associations, and allele frequencies in the Genome Aggregation Database. We performed functional tests of the effects of variants on mitosis, apoptosis, and radial neuroblast migration in vitro and conducted video-EEG studies in mice lacking a copy of Ick.ResultsA variant, K305T (c.914A→C), cosegregated with epilepsy or polyspikes on EEG in 12 members of the family affected with juvenile myoclonic epilepsy. We identified 21 pathogenic ICK variants in 22 of 310 additional patients (7%). Four strongly linked variants (K220E, K305T, A615T, and R632X) impaired mitosis, cell-cycle exit, and radial neuroblast migration while promoting apoptosis. Tonic–clonic convulsions and polyspikes on EEG resembling seizures in human juvenile myoclonic epilepsy occurred more often in knockout heterozygous mice than in wild-type mice (P=0.02) during light sleep with isoflurane anesthesia.ConclusionsOur data provide evidence that heterozygous variants in ICK caused juvenile myoclonic epilepsy in 7% of the patients included in our analysis. Variant ICK affects cell processes that help explain microdysgenesis and polyspike networks observed on EEG in juvenile myoclonic epilepsy. (Funded by the National Institutes of Health and others.)

Modulation of GSK3β autoinhibition by Thr‐7 and Thr‐8

Glycogen synthase kinase 3β (GSK-3β) is a pivotal signaling node that regulates a myriad of cellular functions and is deregulated in many pathological conditions, making it an attractive therapeutic target. Inhibitory Ser-9 phosphorylation of GSK3β by AKT is an important mechanism for negative regulation of GSK3β activity upon insulin stimulation. Here, we report that Thr-7 and Thr-8 residues located in the AKT/PKB substrate consensus sequence on GSK3β are essential for insulin-stimulated Ser-9 phosphorylation invivo and for GSK3β inactivation. Intestinal cell kinase (ICK) phosphorylates GSK3β Thr-7 invitro and invivo. Thr-8 phosphorylation partially inhibits GSK3β, but Thr-7 phosphorylation promotes GSK3β activity and blocks phospho-Ser-9-dependent GSK3β autoinhibition. Our findings uncover novel mechanistic and signaling inputs involved in the autoinhibition of GSK3β.

The role of intestinal cell kinase in interneurons, a protein mutated in Juvenile Myoclonic Epilepsy

The role of intestinal cell kinase in interneurons, a protein mutated in Juvenile Myoclonic Epilepsy

A Murine Model for Human ECO Syndrome Reveals a Critical Role of Intestinal Cell Kinase in Skeletal Development.

An autosomal-recessive inactivating mutation R272Q in the human intestinal cell kinase (ICK) gene caused profound multiplex developmental defects in human endocrine-cerebro-osteodysplasia (ECO) syndrome. ECO patients exhibited a wide variety of skeletal abnormalities, yet the underlying mechanisms by which ICK regulates skeletal development remained largely unknown. The goal of this study was to understand the structural and mechanistic basis underlying skeletal anomalies caused by ICK dysfunction. Ick R272Q knock-in transgenic mouse model not only recapitulated major ECO skeletal defects such as short limbs and polydactyly but also revealed a deformed spine with defective intervertebral disk. Loss of ICK function markedly reduced mineralization in the spinal column, ribs, and long bones. Ick mutants showed a significant decrease in the proliferation zone of long bones and the number of type X collagen-expressing hypertrophic chondrocytes in the spinal column and the growth plate of long bones. These results implicate that ICK plays an important role in bone and cartilage development by promoting chondrocyte proliferation and maturation. Our findings provided new mechanistic insights into the skeletal phenotype of human ECO and ECO-like syndromes.

An essential role of intestinal cell kinase in lung development is linked to the perinatal lethality of human ECO syndrome

Human endocrine-cerebro-osteodysplasia (ECO) syndrome, caused by the loss-of-function mutation R272Q in the intestinal cell kinase (ICK) gene, is a neonatal-lethal developmental disorder. To elucidate the molecular basis of ECO syndrome, we constructed an Ick R272Q knock-in mouse model that recapitulates ECO pathological phenotypes. Newborns bearing Ick R272Q homozygous mutations die at birth due to respiratory distress. Ick mutant lungs exhibit not only impaired branching morphogenesis associated with reduced mesenchymal proliferation but also significant airspace deficiency in primitive alveoli concomitant with abnormal interstitial mesenchymal differentiation. ICK dysfunction induces elongated primary cilia and perturbs ciliary Hedgehog signaling and autophagy during lung sacculation. Our study identifies an essential role for ICK in lung development and advances the mechanistic understanding of ECO syndrome.

Ick Ciliary Kinase Is Essential for Planar Cell Polarity Formation in Inner Ear Hair Cells and Hearing Function.

Cellular asymmetries play crucial roles in development and organ function. The planar cell polarity (PCP) signaling pathway is involved in the establishment of cellular asymmetry within the plane of a cell sheet. Inner ear sensory hair cells (HCs), which have several rows of staircase-like stereocilia and one kinocilium located at the vertex of the stereocilia protruding from the apical surface of each HC, exhibit a typical form of PCP. Although connections between cilia and PCP signaling in vertebrate development have been reported, their precise nature is not well understood. During inner ear development, several ciliary proteins are known to play a role in PCP formation. In the current study, we investigated a functional role for intestinal cell kinase (Ick), which regulates intraflagellar transport (IFT) at the tip of cilia, in the mouse inner ear. A lack of Ick in the developing inner ear resulted in PCP defects in the cochlea, including misorientation or misshaping of stereocilia and aberrant localization of the kinocilium and basal body in the apical and middle turns, leading to auditory dysfunction. We also observed abnormal ciliary localization of Ift88 in both HCs and supporting cells. Together, our results show that Ick ciliary kinase is essential for PCP formation in inner ear HCs, suggesting that ciliary transport regulation is important for PCP signaling.SIGNIFICANCE STATEMENT The cochlea in the inner ear is the hearing organ. Planar cell polarity (PCP) in hair cells (HCs) in the cochlea is essential for mechanotransduction and refers to the asymmetric structure consisting of stereociliary bundles and the kinocilium on the apical surface of the cell body. We reported previously that a ciliary kinase, Ick, regulates intraflagellar transport (IFT). Here, we found that loss of Ick leads to abnormal localization of the IFT component in kinocilia, PCP defects in HCs, and hearing dysfunction. Our study defines the association of ciliary transport regulation with PCP formation in HCs and hearing function.

An inactivating mutation in intestinal cell kinase, ICK, impairs hedgehog signalling and causes short rib-polydactyly syndrome.

The short rib polydactyly syndromes (SRPS) are a group of recessively inherited, perinatal-lethal skeletal disorders primarily characterized by short ribs, shortened long bones, varying types of polydactyly and concomitant visceral abnormalities. Mutations in several genes affecting cilia function cause SRPS, revealing a role for cilia function in skeletal development. To identify additional SRPS genes and discover novel ciliary molecules required for normal skeletogenesis, we performed exome sequencing in a cohort of patients and identified homozygosity for a missense mutation, p.E80K, in Intestinal Cell Kinase, ICK, in one SRPS family. The p.E80K mutation abolished serine/threonine kinase activity, resulting in altered ICK subcellular and ciliary localization, increased cilia length, aberrant cartilage growth plate structure, defective Hedgehog and altered ERK signalling. These data identify ICK as an SRPS-associated gene and reveal that abnormalities in signalling pathways contribute to defective skeletogenesis.

A novel ICK mutation causes ciliary disruption and lethal endocrine-cerebro-osteodysplasia syndrome.

BackgroundEndocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology.ResultsThrough homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G > T; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells.ConclusionsThrough identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13630-016-0029-1) contains supplementary material, which is available to authorized users.