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

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 that Chlamydomonas CDKL5 is activated by LF2, a cyclin-dependent kinase, through phosphorylation of its activation loop. This activation controls CDKL5 localization in steady-state cilia, down-regulates its IFT-mediated transport as flagella reach steady-state, controls ciliary abundance of IFT proteins, and controls phosphorylation of the tubulin-binding domain of IFT74, thereby influencing flagellar length. Mouse Cdkl5 shows similar properties: it localizes within cilia, its loss leads to ciliary elongation, and its localization depends on both its kinase activity and Cdk20, the mammalian ortholog of LF2. These results extend our understanding of ciliary length control, challenge the prevailing model that CDKL5 is activated by autophosphorylation, and suggest that CDD pathogenesis arises, at least in part, from disruption of this conserved ciliary regulatory pathway.