Dataset Information

LF2 phosphorylation of the CDKL5 activation loop controls CDKL5’s activity

ABSTRACT: Cdkl5 Deficiency Disorder (CDD) is caused by variants in the protein kinase CDKL5, leading to symptoms such as seizures, developmental delay, and severe intellectual disability. The Chlamydomonas homologue of human CDKL5 is the flagellar protein LF5, whose absence results in a long flagella phenotype. We found that mouse CDKL5 similarly localizes within cilia, and its loss causes long cilia. Chlamydomonas cells lacking CDKL5 exhibit altered flagellar waveforms and reduced motility. CDKL5 kinase activity is essential for flagella length control and proper localization as a kinase-dead mutant, CDKL5(K33R), fails to rescue the long flagella phenotype and does not properly localize to the proximal end of flagella. In wild-type cells, CDKL5 is highly phosphorylated at residues S162, T164, and Y166 within its activation loop and can undergo autophosphorylation in vitro. Interestingly, CDKL5 lacking kinase activity maintains similar phosphorylation at these residues. However, CDKL5 isolated from cells that lack the protein kinase LF2 has reduced activation loop phosphorylation, diminished autophosphorylation capacity, and altered ciliary localization, suggesting that LF2 phosphorylates CDKL5’s activation loop to regulate its kinase activity. Disruption of Cdk20, the mouse ortholog of LF2, likewise alters the ciliary localization of Cdkl5 in primary cilia. CDKL5 likely regulates intraflagellar transport (IFT) as the loss CDKL5 increased the ciliary abundance of IFT proteins while decreasing the phosphorylation of IFT74.

INSTRUMENT(S):

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Gregory Pazour

LAB HEAD: Gregory J.

PROVIDER: PXD066796 | Pride | 2025-11-15

REPOSITORIES: Pride

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Activation of the ciliary kinase CDKL5 is mediated by the cyclin-dependent kinase CDK20/LF2 to control flagellar length.

Hou Yuqing Y Omi Oranti Ahmed OA Stuck Michael W MW Cheng Xi X Walker Bethany B Lam Ying-Wai YW Schmoker Anna M AM Nguyen Son N SN Gonzalez-Perez Maria Paz MP Ballif Bryan A BA Lechtreck Karl F KF Witman George B GB Pazour Gregory J GJ

PLoS biology 20251212 12

Variants in the protein kinase CDKL5 cause CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental condition characterized by seizures, developmental delay, and intellectual disability. The Chlamydomonas homolog of CDKL5, LF5, is a flagellar protein whose loss leads to elongated flagella. Here, we combine live-cell imaging, immunofluorescence, and biochemical approaches including mass spectrometry to define how CDKL5 activity is regulated and how its loss alters ciliary function. We find th ...[more]

PMID: 41385589

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Project description:Study Smoking and COPD are associated with decreased mucociliary clearance and healthy smokers have shorter cilia in the large airway than nonsmokers. Intraflagellar transport (IFT) is the process by which cilia are produced and maintained. We assessed expression of IFT-related genes in smokers and nonsmokers and evaluated cilia length in the large and small airway of nonsmokers, healthy smokers, and smokers with COPD. Methods Airway epithelium was obtained via bronchoscopic brushing. Affymetrix microarrays were used to evaluate IFT gene expression in 2 independent data sets from large and small airway. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject. Results All 40 IFT genes were expressed in the human large and small airway epithelium. In the large airway, 10 IFT genes were down-regulated and 1 up-regulated in smokers. In the small airway, 11 genes were down-regulated and 3 up-regulated in smokers. A set of 8 IFT genes was down-regulated in both data sets. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. Answer to the Question These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies. Gene expression was assessed for 40 intraflagellar transport related genes in the LAE of nonsmokers (n=21) and healthy smokers (n=31) and the SAE of an independent group of nonsmokers (n=28) and healthy smokers (n=69). Cilia length was assessed in a total of 228 airway epithelium samples, including 120 LAE samples and 108 SAE samples; a subset of the 228 samples is represented among the 149 samples in this Series.

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