Anterograde trafficking of ciliary MAP kinase–like ICK/CILK1 by the intraflagellar transport machinery is required for intraciliary retrograde protein trafficking

Kentaro Nakamura,Tatsuro Noguchi,Mariko Takahara,Yoshihiro Omori,Takahisa Furukawa,Yohei Katoh,Kazuhisa Nakayama

doi:10.1074/jbc.ra120.014142

Kentaro Nakamura, Tatsuro Noguchi + Show 5 more

Open Access

https://doi.org/10.1074/jbc.ra120.014142

Copy DOI

Abstract

ICK (also known as CILK1) is a mitogen-activated protein kinase-like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)-B complex of the IFT machinery and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of ICK knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary G protein-coupled receptors. In addition, we found that in ICK knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and released into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in ICK knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.

Highlights

Primary cilia are microtubule-based projections found on a variety of eukaryotic cells and serve as mechanosensors for extracellular stimuli, such as fluid flow, and as chemosensors for signaling molecules, such as the Hedgehog (Hh) morphogen [1]
When the intestinal cell kinase (ICK) constructs fused to mCherry were expressed in human telomerase reverse transcriptase–immortalized retinal pigment epithelial 1 cells, which are serum-starved to induce ciliogenesis, mChe-ICK(CT) and mChe-ICK(WT), but not mChe-ICK(NT), were often found localized at the ciliary tip (Fig. 1B, panels a– c), indicating that the noncatalytic region is responsible for the localization of ICK to the ciliary tip
We demonstrated the direct binding of ICK to the intraflagellar transport (IFT)-B complex via its C-terminal noncatalytic region (Fig. 1)

Summary

Results

ICK has a kinase domain in its N-terminal (NT) region and a C-terminal (CT) noncatalytic region (Fig. 1A). The exogenous expression of mChe-fused ICK(T157A) and ICK(T157E), which are a phosphorylation-defective mutant and a phosphomimetic mutant, respectively, was unable to rescue cilia elongation and IFT88 accumulation at the ciliary tip in ICK-KO cells, these mutants were able to localize at the ciliary tip (Fig. 6A, panels i and j; see Fig. 6, B and C), suggesting that the phosphorylation cycle at the TDY motif is important for the function of ICK These observations are compatible with previous studies in mouse and other organisms, demonstrating that mutations in the genes of ICK orthologs and CCRK orthologs result in cilia elongation [30, 47,48,49, 56]. These observations altogether demonstrate that the kinase activity of ICK, delivered to the ciliary tip via binding to the IFT machinery, is essential for its function, which is probably regulated by its phosphorylation within the TDY motif by CCRK/ CDK20

Discussion

Experimental procedures

VIP assay and immunoblotting analysis