APC/C^FZR-1Controls ZYG-1 Levels to Regulate Centrosome Assembly

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 identify the C. elegans ZYG-1 (Plk4 in human) as a new 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 targets ZYG-1 for proteasomal degradation 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, inhibiting both APC/CFZR-1 and SCFSlimb/βTrCP, by co-mutating ZYG-1 SB and D-box motifs, stabilizes ZYG-1 in an additive manner, conveying 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, but the CK2 kinase inhibitor (TBB) blocks phosphorylation of KD-ZYG-1 K41M by CK2. Furthermore, our immunoprecipitation (IP) assays using embryonic lysates suggests a physical interaction between 2xMyc::ZYG-1 and KIN-3::V5 in vivo 20,50 (Fig 1C). Collectively, our data support a 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, it has been shown that increased centrosomal ZYG-1 or hyperactive 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.…”

“…For prediction of potential phosphorylation residues, we used in silico approaches (KinasePhos3.0 53 ; and GPS 5.0 54 ) and identified several ZYG-1 residues at S279, S280, S620 and T621 showing the highest likelihood of CK2 consensus motifs (Table S1). These potential CK2 target sites are located in two distinct regions: S279/S280 in the Based on the sequence conservation, we focused on S279 and neighboring serine residues within the L1 domain critical for ZYG-1 function in centrosome assembly 17,20 . S279 is located within a cluster of serine residues (aa260-285: Fig 2A ).…”

“…Given that inhibiting CK2 kinase activity leads to elevated centrosomal ZYG-1 25 , we examined how the NP-and PM-ZYG-1 mutations affected ZYG-1 levels at centrosomes. To facilitate this assay, we introduced a 2xMyc tag at the endogenous N-terminal zyg-1 locus by CRISPR/Cas9 editing 20 . By immunostaining embryos with anti-Myc, we quantified centrosome-associated 2xMyc::ZYG-1 fluorescence intensity during the first mitosis (Fig 2C).…”

“…The regulatory mechanism of Plk4/ZYG-1 abundance appears to be evolutionarily conserved. In C. elegans, Drosophila, and human cells, the SCF Slimb/bTrCP E3 ubiquitin ligase controls levels of Plk4/ZYG-1 through the conserved Slimb/bTrCP-binding motif (SBM) [19][20][21][22] . In Drosophila and human cells, autophosphorylation of the SBM triggers SCF Slimb/bTrCP binding to Plk4, promoting Plk4 degradation 18,23,24 .…”