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Degradation of Cyclin a Is Regulated by Acetylation

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Degradation of Cyclin a Is Regulated by Acetylation Oncogene (2009) 28, 2654–2666 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Degradation of cyclin A is regulated by acetylation F Mateo1, M Vidal-Laliena1, N Canela1, L Busino2, MA Martinez-Balbas3, M Pagano2,4, N Agell1 and O Bachs1 1Faculty of Medicine, Department of Cell Biology and Pathology, University of Barcelona, Barcelona, Spain; 2Department of Pathology, NYU Cancer Institute, Smilow Research Center, New York University School of Medicine, New York, NY, USA; 3Instituto de Biologı´a Molecular de Barcelona, Consejo Superior de Investigaciones Cientı´ficas, Barcelona, Spain and 4Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, USA Cyclin A accumulates at the onset of S phase,remains high activated. cdk1 together with cyclins A and B governs during G2 and early mitosis and is degraded at prometa- G2/M transition. G1 progression is under the control of phase. Here,we report that the acetyltransferase P/CAF cyclin D-cdk4/6. Cyclin E-cdk2 triggers DNA synthesis directly interacts with cyclin A that as a consequence and cyclin A-cdk2 drives S-phase progression becomes acetylated at lysines 54,68,95 and 112. Maximal (Malumbres and Barbacid, 2005). Whereas the levels acetylation occurs simultaneously to ubiquitylation at of most cdks are relatively constant during cell cycle mitosis,indicating importance of acetylation on cyclin those of cyclins fluctuate, and in that way, they bind to A stability. This was further confirmed by the observation and activate specific cdks. that the pseudoacetylated cyclin A mutant can be Cyclin A levels are low during G1 but they increase at ubiquitylated whereas the nonacetylatable mutant cannot. the onset of S phase, when it contributes to the The nonacetylatable mutant is more stable than cyclin A stimulation of DNA synthesis (Resnitzky et al., 1995; WT (cycA WT) and arrests cell cycle at mitosis. More- Rosenberg et al., 1995). The amount of cyclin A remains over,in cells treated with histone deacetylase inhibitors high after S phase and in early mitosis when, by cyclin A acetylation increases and its stability decreases, associating withcdk1, it drives theinitiation of thus supporting the function of acetylation on cyclin A chromosome condensation and possibly nuclear envel- degradation. Although the nonacetylatable mutant cannot ope breakdown (Pagano and Draetta, 1991; Furuno be ubiquitylated,it interacts with the proteins needed for et al., 1999; Gong et al., 2007). It is destroyed during its degradation (cdks,Cks,Cdc20,Cdh1 and APC/C). In prometaphase by the anaphase-promoting complex/ fact,its association with cdks is increased and its cyclosome (APC/C) by proteasome (den Elzen and complexes with these kinases display higher activity than Pines, 2001). Cyclin B levels rise during G2 and then it control cycA WT–cdk complexes. All these results indicate binds to cdk1. This complex promotes the completion of that cyclin A acetylation at specific lysines is crucial for chromosome condensation and spindle assembly, thus cyclin A stability and also has a function in the regulation driving cell-cycle progression until metaphase. Cyclin B of cycA-cdk activity. is degraded during metaphase, significantly later than Oncogene (2009) 28, 2654–2666; doi:10.1038/onc.2009.127; cyclin A (Hagting et al., 2002). Because eachcyclin is published online 1 June 2009 responsible for the phosphorylation of a specific subset of cdk substrates, it is expected that after their Keywords: acetylation; cyclin A; degradation; P/CAF; degradation their specific substrates would be depho- ubiquitylation sphorylated. Thus, the ordered destruction of the different cyclins helps to order the sequence of events in late mitosis (Bloom and Cross, 2007). In fact, on time degradation of cyclins A and B is a key event for mitosis Introduction progression and nondegradable mutants of cyclin A cause cell arrest in metaphase, whereas those of cyclin B Cell-cycle progression is governed by the family of block cells during anaphase (Parry and O’Farrell, 2001; cyclin-dependent kinases (cdks) (Morgan, 1997). Their Sullivan and Morgan, 2007). activities are regulated by binding to regulatory subunits The signals that trigger cyclin A degradation at called cyclins, phosphorylation and binding to inhibi- prometaphase are still a matter of controversy. Degra- tory proteins (Sherr and Roberts, 1999). During cell dation is induced by APC/C bound to the targeting cycle, specific pairs of cyclin-cdks are formed and subunit Cdc20 (APC/CCdc20) that is activated by phos- phorylation by cyclin B-cdk1. Cyclin A degradation is Correspondence: Professor O Bachs, Department of Cell Biology and spindle-checkpoint independent and thus, it starts as Pathology, University of Barcelona, Casanova 143, 08036 Barcelona, soon as APC/CCdc20 is activated (Geley et al., 2001; Spain. E-mail: [email protected] den Elzen and Pines, 2001). In contrast, cyclin B1 Cdc20 Received 25 November 2008; revised 11 March2009; accepted 22 April degradation by APC/C is sensitive to the spindle- 2009; published online 1 June 2009 assembly checkpoint. Therefore, at prometaphase Acetylation and cyclin A degradation F Mateo et al 2655 unattached sister chromatids generate signals that allow Figure S1). By IP withanti-cyclin A followed by WB inhibitory components of the spindle-assembly check- withanti-acetylK we observed thatendogenous cyclin A point, suchas Mad2, to bind to Cdc20 and block its was also acetylated (Figure 1a, right panel). We ability to interact withcyclin B1 (Fang et al., 1998; subsequently aimed to identify the acetyltransferases Sudakin et al., 2001). Moreover, a recent report that could be responsible for this acetylation. Thus, indicates that to maintain the cell-cycle arrest induced in vitro assays, using glutathione S-transferase (GST)- by the spindle-assembly checkpoint, Cdc20 must be cyclin A as a substrate and different acetyltransferases ubiquitylated and degraded (Nilsson et al., 2008). Only as enzymes, were performed. As shown in Figure 1b, when all chromatids are attached to the mitotic spindle cyclin A was acetylated by P/CAF but not by CBP or at metaphase, the spindle-assembly checkpoint becomes TIP60. To further determine whether P/CAF was also inactivated and then cyclin B1 can be degraded. This involved in the in vivo acetylation of cyclin A, we different behavior of cyclin A and cyclin B degradation analysed the effect of the depletion of endogenous or by the same APC/C complex indicates that distinct ectopic P/CAF on cyclin A acetylation. As shown in signals participate in targeting these cyclins for ubiqui- Figure 1c depletion of endogenous P/CAF (by a specific tylation and the subsequent degradation during mitosis siRNA) significantly decreased cyclin A acetylation. (Geley et al., 2001). Similarly, depletion of ectopic P/CAF in cells trans- The association of cyclin A to its cdk partner is fected withFlag-P/CAF resulted in a significant reduc- needed for its degradation, suggesting that Cdc20 binds tion of cyclin A acetylation (Figure 1d). Finally, to cyclin A through an extended motif that includes not decreasing the levels of ectopic GCN5 (an acetylase only its N terminus but also its cdk partner (Wolthuis homologous to P/CAF) only produced a small diminu- et al., 2008). It has recently been reported that the cyclin tion of cyclin A acetylation (Figure 1e). These results A–cdk complex must bind a Cks protein to be degraded indicate a key function of P/CAF in the in vivo in prometaphase and it has been proposed that the acetylation of cyclin A. cyclin A–cdk–Cks complex is recruited to the phos- The putative in vivo interaction between cyclin A and phorylated APC by its Cks protein. Then, its attached P/CAF was first analysed by fluorescence microscopy in Cdc20 protein causes cyclin A to be degraded regardless cyan fluorescent protein (CFP)-cyclin A-transfected and of whether the spindle checkpoint is active or not yellow fluorescent protein (YFP)-P/CAF-transfected (Wolthuis et al., 2008). cells. Results showed that both proteins colocalized in In general, cyclins have a ‘destruction box’ that is a the nucleus (Figure 2a). The interaction between both sequence recognized by the ubiquitylation machinery to proteins was further determined by IP experiments with degrade these proteins (Glotzer et al., 1991). Cyclin A anti-HA in cells co-transfected withYFP-P/CAF and also has an extended ‘destruction box’ that includes aa HA-cyclin A. Subsequent WB analysis demonstrated the 47–72 (Klotzbucher et al., 1996). However, to totally co-IP of cyclin A, P/CAF and cdk2 (Figure 2b). Finally, avoid cyclin A ubiquitylation and degradation the first surface plasmon resonance analyses demonstrated the 171 aa of cyclin A must be eliminated, revealing that in direct association between cyclin A and P/CAF addition to the extended ‘destruction box’ more (Figure 2c). We were also interested in determining the sequences from the N terminus are needed for cyclin A interaction between cyclin A and P/CAF during the cell degradation (Fung et al., 2005). cycle. For that purpose we first analysed the levels of Here we report that cyclin A can be acetylated in vivo cyclin A and P/CAF in cells synchronized at different and in vitro by the acetyltransferase P/CAF (p300/CBP- phases of the cell cycle. We observed that the levels of associated factor) at four specific lysine residues located P/CAF were high during S phase and G2/M, decreased in its N terminus. When these residues are substituted by at metaphase and remained low during G1. This arginines, cyclin A is less ubiquitylated, is muchmore behavior was similar to that observed for cyclin A stable and causes cell-cycle arrest at G2/M. Therefore, (Figure 2d, left panel). Cdk2 was detected over all the our results indicate that acetylation is a critical signal in cell cycle although the levels slightly varied depending the regulation of cyclin A degradation.
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