Ciliopathies are a group of human genetic disorders associated with mutations that give rise to the dysfunction of primary cilia. Ciliogenesis-associated kinase 1 (CILK1), formerly known as intestinal cell kinase (ICK), is a conserved serine and threonine kinase that restricts primary (non-motile) cilia formation and length. Mutations in CILK1 are associated with ciliopathies and are also linked to juvenile myoclonic epilepsy (JME). However, the effects of the JME-related mutations in CILK1 on kinase activity and CILK1 function are unknown. Here, we report that JME pathogenic mutations in the CILK1 N-terminal kinase domain abolish kinase activity, evidenced by the loss of phosphorylation of kinesin family member 3A (KIF3A) at Thr672, while JME mutations in the C-terminal non-catalytic domain (CTD) have little effect on KIF3A phosphorylation. Although CILK1 variants in the CTD retain catalytic activity, they nonetheless lose the ability to restrict cilia length and also gain function in promoting ciliogenesis. We show that wild type CILK1 predominantly localizes to the base of the primary cilium; in contrast, JME variants of CILK1 are distributed along the entire axoneme of the primary cilium. These results demonstrate that JME pathogenic mutations perturb CILK1 function and intracellular localization. These CILK1 variants affect the primary cilium, independent of CILK1 phosphorylation of KIF3A. Our findings suggest that CILK1 mutations linked to JME result in alterations of primary cilia formation and homeostasis.

Loss-of-function mutations in the human ICK (intestinal cell kinase) gene cause dysfunctional primary cilia and perinatal lethality which are associated with human ciliopathies. The enzyme that we herein call CAPK (ciliopathy-associated protein kinase) is a serine/threonine protein kinase that has a highly conserved MAPK-like N-terminal catalytic domain and an unstructured C-terminal domain (CTD) whose functions are completely unknown. In this study, we demonstrate that truncation of the CTD impairs the ability of CAPK to interact with and phosphorylate its substrate, kinesin family member 3A (KIF3A). We also find that deletion of the CTD of CAPK compromises both localization to the primary cilium and negative regulation of ciliogenesis. Thus, CAPK substrate recognition, ciliary targeting, and ciliary function depend on the non-catalytic CTD of the protein which is predicted to be intrinsically disordered.