Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension

abstract: ...Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.

... ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis. Biogenesis of the primary cilium begins after cell cycle exit, but the regulatory steps for its formation are poorly defined. Here the authors show that proteasome-mediated removal of the ciliogenesis inhibitor, trichoplein, from mother centrioles initiates the first step of ciliogenesis. The primary cilium is a membrane-bound, microtubule-based sensory organelle that is composed of nine doublet

... ciliogenesis by removing trichoplein from the mother centrioles. Our global E3 screening has identified KCTD17 as a substrate-adaptor for the Cul3-RING ubiquitin ligases (CRL3s) that polyubiquitylates trichoplein at Lys-50 and Lys-57. The CRL3-KCTD17-meidated trichoplein polyubiquitylation and degradation plays a pivotal role in the initial step of axonemal extension during ciliogenesis through the inactivation of centriolar Aurora-A. Results UPS targets trichoplein to proteolysis during ciliogenesis When

...-binding protein KCTD17 controls ciliogenesis To identify a ubiquitin E3 ligase that controls ciliogenesis through trichoplein degradation, we carried out the two-stepped global E3 screen ( Fig. 3a ). In the primary screen, 1,172 E3 ligase proteins (including putative E3s; listed in Supplementary Table 1 ) were purified from the human proteome expression resource library (HuPEX) using wheat germ cell-free expression system 38 39 . We evaluated their binding to bacterially purified MBP-trichoplein by protein array, and identified ten potential E3 ligases ( Supplementary Fig. 3 ). We then performed the secondary screen using four distinct small interfering RNAs (siRNAs) per potential E3 ligase; depletion of KCTD17 (K + channel tetramerization domain-containing 17) protein by four independent siRNAs considerably interfered with the serum starvation-induced ciliogenesis both in RPE1 cells and IMR-90 fibroblasts ( Fig. 3b and Supplementary Fig. 4 ). Immunofluoresence analyses of cell cycle markers, including cyclin A and BrdU incorporation, exclude the possibility that the defective ciliogenesis is due to the inappropriate cell cycle re-entry ( Supplementary Fig. 5 ). In addition, KCTD17 silencing attenuated the serum starvation-induced polyubiquitylation of trichoplein ( Fig. 3c ). Thus, KCTD17 is a novel regulator of ciliogenesis that involves in trichoplein polyubiquitylation. CRL3-KCTD17 and UbcH5a/b polyubiquitylate trichoplein KCTD17 interacted with trichoplein (residues 3965aa) through its coiled-coil-containing carboxyl-terminal region (residues 193297aa; Fig. 3d and Supplementary Fig. 6 ). To examine the mechanism by which KCTD17 modulates polyubiquitylation of trichoplein, we explored the other components of the KCTD17-containing E3 ligase complex. Pull-down and co-immunoprecipitation assays revealed that KCTD17 bound to Cul3, which serves as a scaffold for CRL3s 40 41 42 43 , and that this interaction was mediated via its BTB (broad complex, tramtrack, bric-a-brac) domain (residues

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Gene: KCTD17 - potassium channel tetramerization domain containing 17 - (NCBI)

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