Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Cells. 2020 Mar 12;9(3):694. doi: 10.3390/cells9030694.
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.
纤毛病是一组与基因突变相关的人类遗传疾病，这些突变导致初级纤毛功能障碍。纤毛发生相关激酶 1（CILK1），以前称为肠细胞激酶（ICK），是一种保守的丝氨酸和苏氨酸激酶，它限制初级（非运动）纤毛的形成和长度。CILK1 中的突变与纤毛病有关，也与青少年肌阵挛性癫痫（JME）有关。然而，JME 相关突变对 CILK1 激酶活性和 CILK1 功能的影响尚不清楚。在这里，我们报告 CILK1 N 端激酶结构域中的 JME 致病性突变会使激酶活性丧失，这表现在 Kinesin 家族成员 3A（KIF3A）的 Thr672 位点磷酸化的丧失，而 CILK1 C 端非催化结构域（CTD）中的 JME 突变对 KIF3A 磷酸化几乎没有影响。虽然 CTD 中的 CILK1 变体保留了催化活性，但它们失去了限制纤毛长度的能力，并且还获得了促进纤毛发生的功能。我们表明，野生型 CILK1 主要定位于初级纤毛的基部；相比之下，CILK1 的 JME 变体沿初级纤毛的整个轴丝分布。这些结果表明 JME 致病性突变会破坏 CILK1 的功能和细胞内定位。这些 CILK1 变体影响初级纤毛，独立于 CILK1 对 KIF3A 的磷酸化。我们的研究结果表明，与 JME 相关的 CILK1 突变导致初级纤毛形成和动态平衡的改变。
