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1.
Genet Med ; 23(6): 1050-1057, 2021 06.
Article in English | MEDLINE | ID: mdl-33495529

ABSTRACT

PURPOSE: To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. METHODS: Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19G28R and CDK19Y32H. RESULTS: We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). CONCLUSION: CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.


Subject(s)
Intellectual Disability , Neurodevelopmental Disorders , Animals , Cyclin-Dependent Kinases/genetics , Gain of Function Mutation , Humans , Infant , Mutation, Missense , Zebrafish/genetics
2.
J Hum Genet ; 66(10): 1021-1027, 2021 Oct.
Article in English | MEDLINE | ID: mdl-33640901

ABSTRACT

CDK9 has been considered a candidate gene involved in the CHARGE-like syndrome in a pair of cousins. We report an 8-year-old boy with a strikingly similar phenotype including facial asymmetry, microtia with preauricular tags and bilateral hearing loss, cleft lip and palate, cardiac dysrhythmia, and undescended testes. Joint contracture, no finger flexion creases, and large halluces were the same as those of a previously reported patient with homozygous CDK9 variants. The ocular phenotype included blepharophimosis, lacrimal duct obstruction, eyelid dermoids, Duane syndrome-like abduction deficit, and congenital cataracts. Optical coherence tomography and electroretinography evaluations revealed severe retinal dystrophy had developed at an early age. Trio-based whole-exome sequencing identified compound heterozygous variants in CDK9 [p.(A288T) of maternal origin and p.(R303C) of paternal origin] in the patient. Variants' kinase activities were reduced compared with wild type. We concluded that CDK9 biallelic variants cause a CHARGE-like malformation syndrome with retinal dystrophy as a distinguishing feature.


Subject(s)
Blepharophimosis/genetics , CHARGE Syndrome/genetics , Cyclin-Dependent Kinase 9/genetics , Retinal Dystrophies/genetics , Alleles , Blepharophimosis/diagnosis , Blepharophimosis/pathology , CHARGE Syndrome/diagnosis , CHARGE Syndrome/diagnostic imaging , CHARGE Syndrome/pathology , Child , Cleft Lip/diagnostic imaging , Cleft Lip/genetics , Cleft Lip/pathology , Cleft Palate/diagnostic imaging , Cleft Palate/genetics , Cleft Palate/pathology , Electroretinography , Homozygote , Humans , Lacrimal Duct Obstruction/diagnosis , Lacrimal Duct Obstruction/genetics , Lacrimal Duct Obstruction/pathology , Male , Mutation/genetics , Pedigree , Phenotype , Retinal Dystrophies/diagnosis , Retinal Dystrophies/diagnostic imaging , Retinal Dystrophies/pathology , Tomography, Optical Coherence , Exome Sequencing
3.
EMBO J ; 31(8): 1904-15, 2012 Apr 18.
Article in English | MEDLINE | ID: mdl-22373574

ABSTRACT

Nemo-like kinase (NLK/Nlk) is an evolutionarily conserved protein kinase involved in Wnt/ß-catenin signalling. However, the roles of NLK in Wnt/ß-catenin signalling in vertebrates remain unclear. Here, we show that inhibition of Nlk2 function in zebrafish results in decreased Lymphoid enhancer factor-1 (Lef1)-mediated gene expression and cell proliferation in the presumptive midbrain, resulting in a reduction of midbrain tectum size. These defects are related to phosphorylation of Lef1 by Nlk2. Thus, Nlk2 is essential for the phosphorylation and activation of Lef1 transcriptional activity in neural progenitor cells (NPCs). In NPC-like mammalian cells, NLK is also required for the phosphorylation and activation of LEF1 transcriptional activity. Phosphorylation of LEF1 induces its dissociation from histone deacetylase, thereby allowing transcription activation. Furthermore, we demonstrate that NLK functions downstream of Dishevelled (Dvl) in the Wnt/ß-catenin signalling pathway. Our findings reveal a novel role of NLK in the activation of the Wnt/ß-catenin signalling pathway.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Stem Cells/physiology , Transcription Factors/metabolism , Wnt1 Protein/metabolism , Zebrafish Proteins/metabolism , beta Catenin/metabolism , Animals , Brain/embryology , Brain/physiology , Cells, Cultured , Humans , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phosphorylation , Zebrafish
4.
Nat Commun ; 14(1): 7194, 2023 11 08.
Article in English | MEDLINE | ID: mdl-37938219

ABSTRACT

In vertebrate embryos, the canonical Wnt ligand primes the formation of dorsal organizers that govern dorsal-ventral patterns by secreting BMP antagonists. In contrast, in Drosophila embryos, Toll-like receptor (Tlr)-mediated NFκB activation initiates dorsal-ventral patterning, wherein Wnt-mediated negative feedback regulation of Tlr/NFκB generates a BMP antagonist-secreting signalling centre to control the dorsal-ventral pattern. Although both Wnt and BMP antagonist are conserved among species, the involvement of Tlr/NFκB and feedback regulation in vertebrate organizer formation remains unclear. By imaging and genetic modification, we reveal that a negative feedback loop between canonical and non-canonical Wnts and Tlr4/NFκB determines the size of zebrafish organizer, and that Tlr/NFκB and Wnts switch initial cue and feedback mediator roles between Drosophila and zebrafish. Here, we show that canonical Wnt signalling stimulates the expression of the non-canonical Wnt5b ligand, activating the Tlr4 receptor to stimulate NFκB-mediated transcription of the Wnt antagonist frzb, restricting Wnt-dependent dorsal organizer formation.


Subject(s)
NF-kappa B , Zebrafish , Animals , Feedback , Ligands , Drosophila , Wnt Signaling Pathway
5.
Sci Rep ; 10(1): 17575, 2020 10 16.
Article in English | MEDLINE | ID: mdl-33067521

ABSTRACT

Cyclin-dependent kinase 8 (CDK8) is a member of the CDK/Cyclin module of the mediator complex. A recent study reported that heterozygous missense CDK8 mutations cause a neurodevelopmental disorder in humans. The mechanistic basis of CDK8-related disorder has yet to be delineated. Here, we report 2 patients with de novo missense mutations within the kinase domain of CDK8 along with the results of in vitro and in vivo functional analyses using a zebrafish model. Patient 1 and Patient 2 had intellectual disabilities and congenital anomalies. Exome analyses showed that patient 1 had a heterozygous de novo missense p.G28A variant in the CDK8 (NM_001260.3) gene and patient 2 had a heterozygous de novo missense p.N156S variant in the CDK8 gene. We assessed the pathogenicity of these two variants using cultured-cells and zebrafish model. An in vitro kinase assay of human CDK8 showed that enzymes with a p.G28A or p.N156S substitution showed decreased kinase activity. An in vivo assays of zebrafish overexpression analyses also showed that the p.G28A and p.N156S alleles were hypomorphic alleles. Importantly, the inhibition of CDK8 kinase activity in zebrafish embryos using a specific chemical inhibitor induced craniofacial and heart defects similar to the patients' phenotype. Taken together, zebrafish studies showed that non-synonymous variants in the kinase domain of CDK8 act as hypomorphic alleles causing human congenital disorder.


Subject(s)
Cyclin-Dependent Kinase 8/genetics , Mutation, Missense , Neurodevelopmental Disorders/genetics , Point Mutation , Abnormalities, Multiple/genetics , Animals , Child , Craniofacial Abnormalities/genetics , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinase 8/deficiency , Cyclin-Dependent Kinase 8/physiology , Cyclin-Dependent Kinases/physiology , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/enzymology , Female , Heart Defects, Congenital/genetics , Heterozygote , High-Throughput Nucleotide Sequencing , Humans , Infant , Intellectual Disability/genetics , Loss of Function Mutation , Male , Protein Domains , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Zebrafish/embryology , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/physiology
6.
J Neurochem ; 111(5): 1104-18, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19840224

ABSTRACT

Nerve growth factor (NGF) promotes neurite outgrowth through regulating cytoskeletal organization and cell adhesion. These activities are modulated by protein phosphorylation. Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinase-like kinase that phosphorylates several transcription factors. Although NLK is known to be expressed at relatively high levels in the nervous system, its function is not well understood. We found that NGF promotes the translocation of NLK to PC12 cells' leading edges, and triggers NLK kinase activity in them. Activated NLK directly phosphorylates microtubule-associated protein-1B (MAP1B) and the focal adhesion adaptor protein, paxillin. Knockdown of NLK attenuates the phosphorylation of both paxillin and MAP1B and inhibits both the NGF-induced re-distribution of F-actin and neurite outgrowth. We also discovered that NLK is a LiCl-sensitive kinase. LiCl is known to block NGF-induced neurite outgrowth and the phosphorylation of MAP1B and paxillin in PC12 cells. Therefore, the effects of LiCl are mediated in part by blocking NLK activity. These results suggest that NLK controls the dynamics of the cytoskeleton downstream of NGF signaling.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Nerve Growth Factor/pharmacology , Neurites/drug effects , Paxillin/metabolism , Protein Serine-Threonine Kinases/metabolism , Adenosine Triphosphate/pharmacology , Animals , Cell Line/cytology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Green Fluorescent Proteins/genetics , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Immunoprecipitation/methods , Intracellular Signaling Peptides and Proteins/genetics , Models, Biological , Neurons/cytology , Neurons/drug effects , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/genetics , Protein Transport/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Serine/metabolism , Signal Transduction/drug effects , Time Factors , Transfection/methods
7.
Nat Commun ; 10(1): 4710, 2019 10 17.
Article in English | MEDLINE | ID: mdl-31624259

ABSTRACT

Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength-dependent manner to pattern developing tissues. However, developing tissues also undergo dynamic morphogenesis, which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradients. Here we show that a cell competition-related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/ß-catenin signalling gradient, which acts as a morphogen to establish embryonic anterior-posterior patterning, identify that unfit cells with abnormal Wnt/ß-catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/ß-catenin gradient formation and consequent anterior-posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues, this system may support tissue robustness and disease prevention.


Subject(s)
Body Patterning/genetics , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Morphogenesis/genetics , Wnt Signaling Pathway/genetics , Zebrafish Proteins/genetics , beta Catenin/genetics , Animals , Animals, Genetically Modified , Apoptosis/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/embryology , Microscopy, Fluorescence , Time-Lapse Imaging/methods , Zebrafish , Zebrafish Proteins/metabolism , beta Catenin/metabolism
8.
Cell Rep ; 8(5): 1391-404, 2014 Sep 11.
Article in English | MEDLINE | ID: mdl-25159144

ABSTRACT

The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/ß-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/ß-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/ß-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/ß-catenin signaling.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Phosphoproteins/metabolism , Protein Phosphatase 1/metabolism , Protein Serine-Threonine Kinases/metabolism , Wnt Signaling Pathway , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Animals , Base Sequence , Carrier Proteins/genetics , Dishevelled Proteins , HeLa Cells , Humans , Molecular Sequence Data , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Phosphatase 1/genetics , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Proteolysis , Wnt3A Protein/genetics , Wnt3A Protein/metabolism , Zebrafish
9.
Cell Signal ; 25(1): 190-7, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23000342

ABSTRACT

Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinase-related kinase. Although NLK was originally identified as a Drosophila gene affecting cell movement during eye development, recent studies show that NLK also contributes to cell proliferation, differentiation, and morphological changes during early embryogenesis and nervous system development in vertebrates. In addition, NLK has been reported to be involved in the development of several human cancers. NLK is able to play a role in multiple processes due to its capacity to regulate a diverse array of signaling pathways, including the Wnt/ß-catenin, Activin, IL-6, and Notch signaling pathways. Although the molecular mechanisms that regulate NLK activity remain unclear, our recent research has presented a new model for NLK activation. Here, we summarize the current understanding of the function and regulation of NLK and discuss the aspects of NLK regulation that remain to be resolved.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Animals , Catenins/metabolism , Humans , Lymphoid Enhancer-Binding Factor 1/metabolism , MicroRNAs/metabolism , Mitogen-Activated Protein Kinases/classification , Mitogen-Activated Protein Kinases/genetics , Nervous System/growth & development , Signal Transduction , Wnt Proteins/metabolism
10.
Mol Biol Cell ; 22(2): 266-77, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-21118996

ABSTRACT

Nemo-like kinase (NLK) is an evolutionarily conserved protein kinase that phosphorylates several transcription factors. However, the molecular mechanisms that regulate NLK activity have been poorly understood. Here we show that homodimerization of NLK is required for its activation and nuclear localization. Biochemical analysis revealed that NLK is activated through intermolecular autophosphorylation of NLK dimers at Thr-286. Mutation of NLK at Cys-425, which corresponds to the defect in the Caenorhabditis elegans NLK homologue lit-1, prevented NLK dimerization, rendering NLK defective in both nuclear localization and kinase activity. By contrast, the external addition of nerve growth factor, which has been previously identified as an NLK activator, induced dimerization and Thr-286 autophosphorylation of endogenous NLK proteins. In addition, both dimerization and Thr-286 phosphorylation of NLK were found to be essential for induction of neurite-like cellular processes by NLK. The present findings suggest that dimerization is an initial key event required for the functional activation of NLK.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Animals , Cysteine/genetics , Enzyme Activation , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Nerve Growth Factor/pharmacology , Neurites/metabolism , PC12 Cells , Phosphorylation , Protein Multimerization , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Protein Transport , Rats , Sequence Alignment , Threonine/metabolism
11.
Nat Cell Biol ; 12(3): 278-85, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20118921

ABSTRACT

The Notch signalling pathway has a crucial function in determining cell fates in multiple tissues within metazoan organisms. On binding to ligands, the Notch receptor is cleaved proteolytically and releases its intracellular domain (NotchICD). The NotchICD enters the nucleus and acts cooperatively with other factors to stimulate the transcription of target genes. High levels of Notch-mediated transcriptional activation require the formation of a ternary complex consisting of NotchICD, CSL (CBF-1, suppressor of hairless, LAG-1) and a Mastermind family member. However, it is still not clear how the formation of the ternary complex is regulated. Here we show that Nemo-like kinase (NLK) negatively regulates Notch-dependent transcriptional activation by decreasing the formation of this ternary complex. Using a biochemical screen, we identified Notch as a new substrate of NLK. NLK-phosphorylated Notch1ICD is impaired in its ability to form a transcriptionally active ternary complex. Furthermore, knockdown of NLK leads to hyperactivation of Notch signalling and consequently decreases neurogenesis in zebrafish. Our results both define a new function for NLK and reveal a previously unidentified mode of regulation in the Notch signalling pathway.


Subject(s)
Gene Expression Regulation/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Notch/metabolism , Signal Transduction/physiology , Amino Acid Substitution/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line, Tumor , DNA/metabolism , ELAV Proteins/metabolism , ELAV-Like Protein 3 , Embryo, Nonmammalian/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mitogen-Activated Protein Kinases/genetics , Models, Biological , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurogenesis/physiology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotides, Antisense/genetics , Phosphorylation/physiology , Protein Binding/physiology , Protein Interaction Domains and Motifs/physiology , Protein Serine-Threonine Kinases/genetics , RNA, Small Interfering/genetics , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Receptors, Notch/genetics , Transcription Factor HES-1 , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , Xenopus , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
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