Thus, the mark Lys residue(s) of ubiquitination for Plk2 degradation within these six Lys residues, aswell simply because the acetylation of these residues, blocks ubiquitination. Open in a separate window Figure 3. SIRT1 Destabilizes Plk2(A) Lysates prepared from HEK293T cells transfected with the indicated expression plasmids were blotted with antibodies to GFP and -actin. (B) Cells transfected with indicated plasmids were exposed to CHX (100 M) for 8 hr. Plk2 is decreased, resulting in a rapid accumulation of centrosomal Plk2, which contributes to the timely initiation of centriole duplication. Collectively, our findings uncover a critical role of SIRT1 in centriole duplication and provide a mechanistic insight into SIRT1-mediated centrosome-associated functions. Graphical Abstract In Brief Ling et al. demonstrate that SIRT1 deacetylates and thereby destabilizes Plk2, which in turn results in the suppression of centriole duplication. INTRODUCTION The centrosome is the major microtubule-organizing center in animal cells. A mature centrosome contains a pair of centrioles and surrounding amorphous pericentriolar material, which direct the formation of bipolar spindles in mitosis and ensure the equal distribution of chromosomes to daughter cells. Centrosome (as well as centriole) duplication occurs once in each cell cycle. After mitosis, a daughter cell inherits one centrosome (two centrioles) from the mother cell. The centrosome initiates duplication in late G1 and early S phases, in which each centriole inherited from the mother cell serves as a template for procentriole production (Doxsey, 2001; Hinchcliffe and Sluder, 2001, 2002). Defects in the regulatory mechanism for centrosome/centriole duplication may lead to multiple rounds of duplication (reduplication) in a single cell cycle, resulting in the generation of centrosome (>2) or centriole (>4) amplification and, in turn, aberrant mitoses and chromosomal segregation errors. Centrosome amplification is common in various types of cancer and is one of the major causes of chromosomal instability (Winey, 1996; DAssoro et al., 2002; Fukasawa, 2005). Many proteins participate in the regulation of centrosome duplication, and their activities are mainly controlled by posttranslational modifications. Among various modifications, past research efforts have primarily focused on phosphorylation. Indeed, many kinases and phosphatases have been identified as regulators of centrosome duplication (Fukasawa, 2007). Acetylation that occurs on the s-amino group of lysine (Lys) residues is another common modification, which is catalyzed by histone acetyltransferases (HATs) and reversed by histone deacetylases (HDACs). Acetylation eliminates positive charges and potentially affects the protein interactions, activity, and localization of the target substrates. The acetylation and deacetylation reactions are also known to cross-talk with other Cor-nuside modifications. For instance, acetylation often antagonizes ubiquitination, hence stabilizing the target protein, either directly by competing for the same Lys residues or indirectly by altering the overall structure of the target proteins. Reversible (de)acetylation and its modifiers, HDACs and HATs, have been thus implicated in many biological processes (Caron et al., 2005; Yang and Seto, 2008). However, their roles in centrosome duplication have not been closely studied. Rabbit Polyclonal to MOK Previously, in an effort to systematically examine the effects of all identified human HDACs, including 11 classical HDACs (HDAC1C11) and 7 Sirtuins (SIRT1C7), on centrosome duplication, we found that SIRT1, the founding member of class III HDACs (Sirtuins), can localize to centrosomes and suppress centrosome duplication in cycling cells and centrosome reduplication in arrested cells (Ling et al., 2012). Here, we further report that SIRT1 deacetylates polo-like kinase 2 (Plk2) to control centriole duplication. Plk2 is an important centrosome duplication regulator (Warnke et al., 2004; Cizmecioglu et al., 2008; Ling et al., 2015), which drives centrosome duplication through phosphorylating several centrosome targets (Krause and Hoffmann, 2010; Chang et al., 2010; Cizmecioglu et al., 2012). We found that Plk2 is acetylated primarily by p300, which protects Plk2 from being ubiquitinated, whereas SIRT1-mediated deacetylation promotes ubiquitination, and hence degradation, of Plk2. Moreover, phosphorylation of SIRT1 by aurora kinase A (AURKA), which occurs during mitosis of the mother cell, upregulates the binding affinity of SIRT1 to Plk2, and is critical for the timely accumulation of centrosomal Plk2 and the initiation of centrosome duplication in daughter cells. We thus delineated a series of events involving SIRT1, p300, Plk2, AURKA, and ubiquitin (Ub)dependent proteolytic machineries, which contribute to the timely initiation of centrosome duplication. RESULTS Plk2 Is an Acetylated Centrosomal Protein U2OS human osteosarcoma cells were co-immunostained for acetyl-lysine (AcK) and -tubulin, as well as SAS-6. The anti-AcK antibody-reactive signals were readily detected at both unduplicated and duplicated centrosomes (Figure 1A). Immuno- blotting of the centrosome-enriched fraction with the anti-AcK antibody (Figure 1B, fraction 3) showed that multiple centrosomal proteins are acetylated in human cells. Mass spectrometry (MS) analysis was performed to identify potential acetylated centrosomal Cor-nuside proteins (Figure S1), of which we focused on Plk2, a protein that has been Cor-nuside implicated in the regulation of centriole and centrosome duplication and.
Thus, the mark Lys residue(s) of ubiquitination for Plk2 degradation within these six Lys residues, aswell simply because the acetylation of these residues, blocks ubiquitination