Ciliopathy-Associated Protein Kinase ICK Requires Its Non-Catalytic Carboxyl-Terminal Domain for Regulation of Ciliogenesis

Abstract

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.

Functional Alterations in Ciliogenesis-Associated Kinase 1 (CILK1) that Result from Mutations Linked to Juvenile Myoclonic Epilepsy

“…Previously, we reported that the unstructured CTD is required for CILK1 targeting to the cilium base [12]. Here, our data indicate that JME variants in the kinase domain that inactivate CILK1 mislocalize from the cilium base to the axoneme.…”

“…Previous studies have shown CILK1 phosphorylation of KIF3A at Thr-672 in vitro and in vivo and suggested that KIF3A may be the CILK1 substrate that mediates its downstream effect on anterograde IFT, which is critical for cilia formation and growth [12,13]. An intriguing observation we made in this study is that CILK1 variants in the CTD that retain the ability to phosphorylate KIF3A-Thr672 produce defects in both cilia formation and cilia length.…”

“…This study explored how mutations found in JME affect the functions of CILK1. Recently, we showed that both kinase activity and ciliary targeting involving the non-catalytic C terminal domain are essential for CILK1 to suppress ciliogenesis [12]. In the current study, we found that some CILK1 mutations in the non-catalytic CTD do not compromise catalytic activity but eliminate inhibition of ciliogenesis.…”

Ciliogenesis associated kinase 1: targets and functions in various organ systems

“…It is worth pointing out that the current CILK1 substrate consensus sequence was determined using only the N‐terminal kinase domain. Since the CTD of CILK1 has been subsequently shown as indispensable for substrate recognition and phosphorylation , we speculate that CILK1 substrate specificity may be influenced by the MAPK‐like ‘docking’ interactions through its CTD. This would probably not affect interactions at the CILK1 active site that contribute to specificity with peptide substrates.…”

“…For example, we have shown that mutation of the conserved R272 within the PKKRP motif in the CTD is sufficient to exclude CILK1 from the nucleus [7]. Furthermore, we have shown the non-catalytic CTD of CILK1 is required for ciliary localization [15]. It has been shown that CILK1 with different ciliopathy mutations exhibits distinct patterns of subcellular localization [31,32], which predicts that these ciliopathy mutations may cause their disease phenotypes through different molecular mechanisms.…”