APC/CFZR-1 regulates centrosomal ZYG-1 to limit centrosome number

Abstract

Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegans, the anaphase-promoting complex/cyclosome and co-activator FZR-1 (APC/CFZR-1) ubiquitin ligase negatively regulates centrosome assembly through SAS-5 degradation. In this study, we report the C. elegans ZYG-1 (Plk4 in human) as a potential substrate of APC/CFZR-1. Inhibiting APC/CFZR-1 or mutating a ZYG-1 destruction (D)-box leads to elevated ZYG-1 levels at centrosomes, restoring bipolar spindles and embryonic viability to zyg-1 mutants, suggesting that APC/CFZR-1 influences centrosomal ZYG-1 via D-box motif. We also show the Slimb/βTrCP-binding (SB) motif is critical for ZYG-1 degradation, substantiating a conserved mechanism by which ZYG-1/Plk4 stability is regulated by SCFSlimb/βTrCP-dependent proteolysis via the conserved SB motif in C. elegans. Furthermore, we show that co-mutating ZYG-1 SB and D-box motifs stabilizes ZYG-1 in an additive manner, suggesting that APC/CFZR-1 and SCFSlimb/βTrCP ubiquitin ligases function cooperatively for timely ZYG-1 destruction in C. elegans embryos where ZYG-1 activity remains at threshold level to ensure normal centrosome number.

Site-specific phosphorylation of ZYG-1 regulates ZYG-1 stability and centrosome number

“…Autoradiograph illustrates that CK2 phosphorylates KD-ZYG-1 K41M , and this phosphorylation was blocked by a CK2 kinase inhibitor (TBB). Furthermore, our immunoprecipitation (IP) assays using embryonic lysates suggest a physical interaction between 2xMyc::ZYG-1 and KIN-3::V5 in vivo 20 , 50 ( Figure 1 C). These results support our hypothesis that CK2 directly phosphorylates ZYG-1.…”

“…A compelling candidate is the kinase ZYG-1, a master regulator of centrosome duplication. 12 In C. elegans , increased centrosomal ZYG-1 or overexpressing ZYG-1 leads to extra centrosomes in early embryos 20 , 29 and seam cells. 49 Because CK2 kinase activity negatively regulates centrosome duplication by controlling ZYG-1 levels at centrosomes, 25 we hypothesized CK2 directly phosphorylates ZYG-1, and that the phosphorylation state of ZYG-1 influences ZYG-1 activity in regulating centrosome assembly.…”

“…Considering sequence conservation, we focused on S279 and neighboring serine residues within the L1 domain that is critical for ZYG-1 function in centrosome assembly. 17 , 20 S279 is part of a serine cluster (aa260-285: Figure 2 A) conforming to the minimal CK2 consensus motif and highly conserved throughout the Caenorhabditis genus ( Figure S2 A). Given that the C. elegans ZYG-1 is evolutionarily divergent from Plk4 orthologs, S279 and adjacent serine sites do not appear to be conserved in the human Plk4.…”

“…To facilitate this assay, we introduced an N-terminal 2xMyc tag at the endogenous zyg-1 locus by CRISPR/Cas9 editing. 20 By immunostaining embryos with anti-Myc, we quantified the fluorescence intensity of centrosome-associated 2xMyc::ZYG-1 during the first anaphase ( Figure 2 C). The NP-ZYG-1 4A still localizes to centrosomes, indicating alanine replacements at these serine sites did not impair ZYG-1 loading to centrosomes.…”

“… 18 , 19 This regulatory mechanism appears to be evolutionarily conserved, involving the SCF-Slimb/βTrCP E3 ubiquitin ligase and the Slimb/βTrCP-binding motif (SBM) in C. elegans , Drosophila , and human cells. 19 , 20 , 21 , 22 In Drosophila and human cells, autophosphorylation of the SBM triggers SCF Slimb/βTrCP binding to Plk4, promoting Plk4 degradation. 18 , 23 , 24 However, it remains elusive whether autophosphorylation of ZYG-1 in C. elegans has a similar role in SCF Slimb/βTrCP -mediated degradation of ZYG-1.…”