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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, CDKL5K33R, 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 long flagella 2 cells, which lack the protein kinase LF2 shows 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: Ying Wai Lam

LAB HEAD: Gregory J

PROVIDER: PXD066877 | Pride | 2025-11-18

REPOSITORIES: Pride

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			Action	DRS
	C12-LF5GFP_K33R_Y166F_CC-1690_CC-4560_lf2-5.tif	Other
	CC-1690_CC-4560.tif	Other
	Clarissa_QE_1129.raw	Raw
	Clarissa_QE_1130.raw	Raw
	Clarissa_QE_1244.raw	Raw

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Project description:Zebrafish has been recognized as a robust model organism in biomedical research for the investigation of human genetic disorders. Recently, we proposed the homozygous cdkl5sa21938 mutant zebrafish as a model of CDKL5 deficiency disorder (CDD), a rare developmental epileptic encephalopathy associated with broad range of symptoms. Since neurological phenotypes are the primary focus of investigations, the understanding of CDKL5 function beyond the brain remains limited. Therefore, the aim of this study was to expand the knowledge about Cdkl5-associated molecular mechanisms using the proposed cdkl5 zebrafish model and to evaluate their similarity to findings in mammalian systems. For this purpose, we conducted RNA-sequencing of whole cdkl5-/- mutant zebrafish and compare it to their wild-type siblings at two different stages of development (5 and 35 dpf). Most significant DEGs were related to muscle, neuronal and visual systems which are affected in CDD. Gene Ontology (GO) analysis revealed that at both stages the downregulated DEGs were mainly enriched in muscle development, extracellular matrix and actin cytoskeleton functions while at 35 dpf the upregulated DEGs were mainly enriched in eye development functions. KEGG pathway analysis showed the enrichment of downregulated DEGs in focal adhesion and ECM-receptor interaction pathways at both stages. Additionally, we found that DEGs related to neuronal development were mainly downregulated at both stages while several upregulated DEGs were enriched in synaptic signaling at 35dpf. Moreover, we identified many downregulated key DEGs of cartilage development at both stages and bone development at 35 dpf, potential explaining the impaired cdkl5-/- mutant craniofacial cartilage and bone mineralization defects and the CDD skeletal phenotypes. In conclusion, this study shows that Cdkl5 loss in the cdkl5-/- mutant zebrafish lead to the dysregulation of several genes involved in functions known to be associated with CDKL5 in mammalian systems and provide novel insights into the less studied CDKL5 functions and phenotypes.

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

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