Rootletin prevents Cep68 from VHL-mediated proteasomal degradation to maintain centrosome cohesion

Abstract

Centrosome cohesion, mostly regarded as a proteinaceous linker between parental centrioles, ensures the interphase centrosome(s) to function as a single microtubule-organizing center. Maintenance of centrosome cohesion counts on a number of centrosomal linker proteins because depletion of any of those leads to premature centrosome separation in interphase, termed centrosome splitting. However, the underlying mechanisms of the dependence are unknown. Here, we show that absence of Rootletin triggers the von Hippel-Lindau tumour suppressor protein (VHL)-mediated proteasomal degradation of Cep68 and, in turn, results in centrosome splitting. The VHL E3 ligase complex ubiquitinates Cep68 in vitro and in vivo. Co-silencing of Rootletin and VHL reverts Cep68 loss and centrosome splitting. Expression of a stable mutant of Cep68, either diminishing its polyubiquitylation or eliminating binding to β-domain of VHL, also suppresses centrosome splitting provoked by Rootletin depletion. We propose that the archetypal linker protein Rootletin maintains centrosome cohesion in part through inhibition of VHL-mediated Cep68 degradation.

Centrosome Linker–induced Tetraploid Segregation Errors Link Rhabdoid Phenotypes and Lethal Colorectal Cancers

“…S7C). Thus, BRAF-mutant/MSI colorectal cancer cell lines, in which centrosome and microtubule stability is damaged by genetic hypermutation, CROCC depletion may determine a major impact in the progression of mitotic errors (36)(37)(38). Consistently, an increased frequency of micronuclei (median 11% CROCC KD vs. 1% ShCon cells P ¼ 0.0003) and gH2AX nuclear foci (median 43% CROCC KD vs. 18% ShCon cells P ¼ 0.011) were observed ( Fig.…”

“…Besides (Rootletin), multiple proteins including C-NAP1 (CEP250), CEP68, and LRRC45 have been implicated in centrosome linker formation and function. CROCC is able to maintain centrosome cohesion in part through inhibition of VHL-mediated Cep68 degradation (36). It has recently been proposed that a vast network of repeating CROCC units with C-Nap1 as ring organizer and CEP68 as filament modulator forms the centrosome linker structure (37).…”

Cep44 functions in centrosome cohesion by stabilizing rootletin

“…Because the centrosome linker is a highly dynamic structure, we speculate that linker proteins might turn over quickly. One linker component Cep68 has been shown to be ubiquitylated by two ubiquitin ligases, βTrCP and VHL (Pagan et al, 2015;Yin et al, 2017). Although it is known that VHL-mediated ubiquitylation and degradation of Cep68 is inhibited by rootletin and enhanced by MCM7 (Kong et al, 2017;Yin et al, 2017), it remains unclear whether the level of rootletin itself is regulated.…”

“…One linker component Cep68 has been shown to be ubiquitylated by two ubiquitin ligases, βTrCP and VHL (Pagan et al, 2015;Yin et al, 2017). Although it is known that VHL-mediated ubiquitylation and degradation of Cep68 is inhibited by rootletin and enhanced by MCM7 (Kong et al, 2017;Yin et al, 2017), it remains unclear whether the level of rootletin itself is regulated. Based on our findings, it appears that none of the previously known linker proteins, namely C-Nap1, LRRC45, Cep215 and Cep68, is able to impinge on rootletin protein level.…”

“…Since Cep44 controls the stability of rootletin, which influences the stability of Cep68 (Yin et al, 2017), ablation of Cep44 might compromise Cep68 localization and protein level. We indeed found that Cep68 is delocalized from the centrosome upon Cep44 loss; however, the protein level of Cep68 remained unchanged.…”