RESUMO
BACKGROUND: Kinesin family member 3A (KIF3A) is a molecular motor protein in the heterotrimeric kinesin-2 complex that drives anterograde intraflagellar transport. This process plays a pivotal role in both biogenesis and maintenance of the primary cilium that supports tissue development. Ciliogenesis associated kinase 1 (CILK1) phosphorylates human KIF3A at Thr672. CILK1 loss of function causes ciliopathies that manifest profound and multiplex developmental defects, including hydrocephalus, polydactyly, shortened and hypoplastic bones and alveoli airspace deficiency, leading to perinatal lethality. Prior studies have raised the hypothesis that CILK1 phosphorylation of KIF3A is critical for its regulation of organ development. RESULTS: We produced a mouse model with phosphorylation site Thr674 in mouse Kif3a mutated to Ala. Kif3a T674A homozygotes are viable and exhibit no skeletal and brain abnormalities, and only mildly reduced airspace in alveoli. Mouse embryonic fibroblasts carrying Kif3a T674A mutation show a normal rate of ciliation and a moderate increase in cilia length. CONCLUSION: These results indicate that eliminating Kif3a Thr674 phosphorylation by Cilk1 is insufficient to reproduce the severe developmental defects in ciliopathies caused by Cilk1 loss of function. This suggests KIF3A-Thr672 phosphorylation by CILK1 is not essential for tissue development and other substrates are involved in CILK1 ciliopathies.
Assuntos
Cílios , Ciliopatias/genética , Cinesinas/fisiologia , Mutação com Perda de Função , Proteínas Serina-Treonina Quinases/genética , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos Transgênicos , FenótipoRESUMO
Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEFs) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia, and upregulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild-type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. The CILK1 A615T mutant protein was not increased to the same level as the wild-type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single-residue variant of CILK1 is compromised, and this impairs the maintenance of primary cilia and Hedgehog signaling.
Assuntos
Cílios , Epilepsia , Proteínas Hedgehog , Transdução de Sinais , Cílios/metabolismo , Animais , Proteínas Hedgehog/metabolismo , Camundongos , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/patologia , Humanos , Fibroblastos/metabolismo , Mutação/genética , Proteínas Serina-Treonina QuinasesRESUMO
Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent of the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEF) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia and up-regulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. CILK1 A615T mutant protein was not increased to the same level as the wild type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single residue variant of CILK1 is compromised and this impairs the maintenance of primary cilia and Hedgehog signaling.