CCRK/CDK20 regulates ciliary retrograde protein trafficking via interacting with BROMI/TBC1D32

Abstract

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.

BROMI/TBC1D32 together with CCRK/CDK20 and FAM149B1/JBTS36 contributes to intraflagellar transport turnaround involving ICK/CILK1

“…Consistent with the role of ICK downstream of CCRK, mutations of ICK and CCRK homologues in various organisms cause a long cilia/flagella phenotype and often increase the variation in the ciliary length of individual cells (Asleson and Lefebvre, 1998;Berman et al, 2003;Bengs et al, 2005;Burghoorn et al, 2007;Tam et al, 2007;Omori et al, 2010;Yang et al, 2013;Moon et al, 2014;Okamoto et al, 2017;Yi et al, 2018;Jiang et al, 2019;Maurya et al, 2019;Wang et al, 2019;Nakamura et al, 2020). Furthermore, mutations of CCRK and BROMI (also known as TBC1D32), which interact with CCRK (Ko et al, 2010;Noguchi et al, 2021), in humans cause ciliopathies, and CCRK and BROMI mutant mice demonstrate defects in embryonic development caused by aberrant Hh signaling (Ko et al, 2010;Adly et al, 2014;Snouffer et al, 2017;Wang et al, 2018;Alsahan and Alkuraya, 2020;Hietamäki et al, 2020). We have recently shown that not only in ICKknockout (KO) cells but also in CCRK-KO cells derived from human telomerase reverse transcriptase-immortalized retinal pigment epithelial 1 (hTERT-RPE1) cells, the average ciliary length is longer and the variation in ciliary length is larger than in control RPE1 cells, and that excessively accumulated proteins at the bulged ciliary tips of these KO cells are eliminated via extracellular vesicles (ECVs) (Noguchi et al, 2021).…”

Mechanisms of Regulation in Intraflagellar Transport

“…CDK20) [ 159 ]. This kinase was shown to be involved in cancer, cancer metastatic suppression, hedgehog signaling and IFT regulation [ 183 , 184 , 185 , 186 ]. Here we focus on the effect of CCRK and its homologs (LF2 in Chlamydomonas , DYF-18 in C. elegans , Table 1 ) have on IFT.…”

“…Here we focus on the effect of CCRK and its homologs (LF2 in Chlamydomonas , DYF-18 in C. elegans , Table 1 ) have on IFT. Knockout mutants of CCRK in mammals, Chlamydomonas and C. elegans have longer cilia with more length variability [ 160 , 161 , 175 , 186 ]. Furthermore, in mammalian cilia, accumulations of IFT components and shedding of vesicles from the tip have been observed [ 185 , 186 ].…”

“…Knockout mutants of CCRK in mammals, Chlamydomonas and C. elegans have longer cilia with more length variability [ 160 , 161 , 175 , 186 ]. Furthermore, in mammalian cilia, accumulations of IFT components and shedding of vesicles from the tip have been observed [ 185 , 186 ]. Overall, the phenotype is similar to loss of RCK function (see above).…”

“…IFT regulation by mammalian CCRK is dependent on another protein, Bromi (Broad-minded). Bromi has been suggested to form a complex with CCRK, promoting its stability [ 185 , 186 , 187 ]. Homologs of Bromi have not been reported in Chlamydomonas or in C. elegans .…”