Protein Phosphatase 2A-SUR-6/B55 Regulates Centriole Duplication in C. elegans by Controlling the Levels of Centriole Assembly Factors

Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Developmental Cell (Impact Factor: 9.71). 04/2011; 20(4):563-71. DOI: 10.1016/j.devcel.2011.03.007
Source: PubMed


Centrioles play a crucial role in mitotic spindle assembly and duplicate precisely once per cell cycle. In worms, flies, and humans, centriole assembly is dependent upon a key regulatory kinase (ZYG-1/Sak/Plk4) and its downstream effectors SAS-5 and SAS-6. Here we report a role for protein phosphatase 2A (PP2A) in centriole duplication. We find that the PP2A catalytic subunit LET-92, the scaffolding subunit PAA-1, and the B55 regulatory subunit SUR-6 function together to positively regulate centriole assembly. In PP2A-SUR-6-depleted embryos, the levels of ZYG-1 and SAS-5 are reduced and the ZYG-1- and SAS-5-dependent recruitment of SAS-6 to the nascent centriole fails. We show that PP2A physically associates with SAS-5 in vivo and that inhibiting proteolysis can rescue SAS-5 levels and the centriole duplication defect of PP2A-depleted embryos. Together, our findings indicate that PP2A-SUR-6 promotes centriole assembly by protecting ZYG-1 and SAS-5 from degradation.

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Available from: mi hye Song, May 09, 2014
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    • "In this way, PLK4 can control its own availability (Brownlee and Rogers, 2012; Cunha-Ferreira et al., 2009; Guderian et al., 2010; Holland et al., 2010; Rogers et al., 2009) or that of other components of the centriole duplication machinery (such as SAS6) (Puklowski et al., 2011), to restrict centriole number. By contrast, protein phosphatase 2 (PP2A) was reported to promote centriole formation by facilitating SAS6 accumulation at the nascent centriole (Brownlee et al., 2011; Kitagawa et al., 2011; Song et al., 2011). These results support the existence of an intrinsic important balance between kinase and phosphatase activities during centriole duplication and highlight the importance of PLK4 to the centriole assembly machinery. "
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    ABSTRACT: Centriole biogenesis depends on the Polo-like kinase PLK4 and a small group of structural proteins. The spatiotemporal regulation of these proteins at pre-existing centrioles is critical to ensure that centriole duplication occurs once per cell cycle. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) plays an important role in centriole fidelity. Depending upon an association with CEP152, PIPKIγ localized in a ring-like pattern in the intermediate pericentriolar materials around the proximal end of the centriole in G1, S, and G2 phases, but not in M phase. Without detaining cells in S or M phase, depletion of PIPKIγ led to centriole amplification in a PLK4/SAS-6 dependent manner. Expression of exogenous PIPKIγ reduced centriole amplification resulted from endogenous PIPKIγ depletion, hydroxyurea treatment, or PLK4 overexpression, suggesting that PIPKIγ likely functions at the PLK4 level to restrain centriole duplication. Importantly, we found that PIPKIγ bound to the cryptic Polo-Box domain of PLK4 and this binding reduced PLK4 kinase activity. Together, our findings suggest that PIPKIγ is a novel negative regulator of centriole duplication by modulating the homeostasis of PLK4 activity.
    Preview · Article · Jan 2014 · Journal of Cell Science
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    • "The B subunit variants are encoded by seven genes belonging to three families (B55, B56, and B72) in C. elegans, and sur-6, identified in our screen, is the only representative of the C. elegans B55 family. sur-6/B55 is involved in the regulation of several cellular processes in different model systems, such as oncogenesis by the AKT pathway (Kuo et al. 2008), vulva development in C. elegans, where it inhibits PAR-1 (Hurd and Kemphues 2003), embryonic development (Kao et al. 2004), centriole duplication (Song et al. 2011), and axon guidance (Ogura et al. 2010). The specificity of the genetic interaction between sur-6, par-1, and pkc-3 in C. elegans is supported by the fact that down-regulation of the other PP2A regulatory subunit family members did not result in reduced viability of exoc mutant animals (Figure 2). "
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    ABSTRACT: The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNAi screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified six kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic SNARE proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the PP2A phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular GFP signal for the same proteins. Double RNAi experiments for par-1, pkc-3 and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in post-embryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that aPKC kinase and phosphatase PP2A regulate exocyst-dependent insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process.
    Full-text · Article · Nov 2013 · G3-Genes Genomes Genetics
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    • "The C. elegans PP2A holoenzyme has many overlapping functions during the first mitotic division. Loss of either the catalytic or structural subunit results in a severe pleiotropic phenotype with defects in chromosome alignment, progression of the cell cycle, and exit from mitosis (Fig. 1A) (Schlaitz et al., 2007; Song et al., 2011). The separate tasks attributable to PP2A are revealed when any one regulatory subunit is depleted. "
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    ABSTRACT: Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A) enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B', B″) but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly) is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts), and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo.
    Full-text · Article · Jan 2013 · Biology Open
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