Degradation of cyclin A is regulated by acetylation

Introduction

Cell-cycle progression is governed by the family of cyclin-dependent kinases (cdks) (Morgan, 1997). Their activities are regulated by binding to regulatory subunits called cyclins, phosphorylation and binding to inhibitory proteins (Sherr and Roberts, 1999). During cell cycle, specific pairs of cyclin-cdks are formed and activated. cdk1 together with cyclins A and B governs G₂/M transition. G₁ progression is under the control of cyclin D-cdk4/6. Cyclin E-cdk2 triggers DNA synthesis and cyclin A-cdk2 drives S-phase progression (Malumbres and Barbacid, 2005). Whereas the levels of most cdks are relatively constant during cell cycle those of cyclins fluctuate, and in that way, they bind to and activate specific cdks.

Cyclin A levels are low during G₁ but they increase at the onset of S phase, when it contributes to the stimulation of DNA synthesis (Resnitzky et al., 1995; Rosenberg et al., 1995). The amount of cyclin A remains high after S phase and in early mitosis when, by associating with cdk1, it drives the initiation of chromosome condensation and possibly nuclear envelope breakdown (Pagano and Draetta, 1991; Furuno et al., 1999; Gong et al., 2007). It is destroyed during prometaphase by the anaphase-promoting complex/cyclosome (APC/C) by proteasome (den Elzen and Pines, 2001). Cyclin B levels rise during G₂ and then it binds to cdk1. This complex promotes the completion of chromosome condensation and spindle assembly, thus driving cell-cycle progression until metaphase. Cyclin B is degraded during metaphase, significantly later than cyclin A (Hagting et al., 2002). Because eachcycli n is responsible for the phosphorylation of a specific subset of cdk substrates, it is expected that after their degradation their specific substrates would be dephosphorylated. 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 progression and nondegradable mutants of cyclin A cause cell arrest in metaphase, whereas those of cyclin B block cells during anaphase (Parry and O’Farrell, 2001; Sullivan and Morgan, 2007).

The signals that trigger cyclin A degradation at prometaphase are still a matter of controversy. Degradation is induced by APC/C bound to the targeting subunit Cdc20 (APC/C^{) that is activated by phosphorylation by cyclin B-cdk1. Cyclin A degradation is spindle-checkpoint independent and thus, it starts as soon as APC/C is activated (}Geley et al., 2001; den Elzen and Pines, 2001). In contrast, cyclin B1 degradation by APC/C ^{is sensitive to the spindle-assembly checkpoint. Therefore, at prometaphase unattached sister chromatids generate signals that allow inhibitory components of the spindle-assembly checkpoint, such as Mad2, to bind to Cdc20 and block its ability to interact with cyclin B1 (}Fang et al., 1998; Sudakin et al., 2001). Moreover, a recent report indicates that to maintain the cell-cycle arrest induced by the spindle-assembly checkpoint, Cdc20 must be ubiquitylated and degraded (Nilsson et al., 2008). Only when all chromatids are attached to the mitotic spindle at metaphase, the spindle-assembly checkpoint becomes inactivated and then cyclin B1 can be degraded. This different behavior of cyclin A and cyclin B degradation by the same APC/C complex indicates that distinct signals participate in targeting these cyclins for ubiquitylation and the subsequent degradation during mitosis (Geley et al., 2001).

The association of cyclin A to its cdk partner is needed for its degradation, suggesting that Cdc20 binds to cyclin A through an extended motif that includes not only its N terminus but also its cdk partner (Wolthuis et al., 2008). It has recently been reported that the cyclin A–cdk complex must bind a Cks protein to be degraded in prometaphase and it has been proposed that the cyclin A–cdk–Cks complex is recruited to the phosphorylated APC by its Cks protein. Then, its attached Cdc20 protein causes cyclin A to be degraded regardless of whether the spindle checkpoint is active or not (Wolthuis et al., 2008).

In general, cyclins have a ‘destruction box’ that is a sequence recognized by the ubiquitylation machinery to degrade these proteins (Glotzer et al., 1991). Cyclin A also has an extended ‘destruction box’ that includes aa 47–72 (Klotzbucher et al., 1996). However, to totally avoid cyclin A ubiquitylation and degradation the first 171 aa of cyclin A must be eliminated, revealing that in addition to the extended ‘destruction box’ more sequences from the N terminus are needed for cyclin A degradation (Fung et al., 2005).

Here we report that cyclin A can be acetylated in vivo and in vitro by the acetyltransferase P/CAF (p300/CBP-associated factor) at four specific lysine residues located in its N terminus. When these residues are substituted by arginines, cyclin A is less ubiquitylated, is much more stable and causes cell-cycle arrest at G₂/M. Therefore, our results indicate that acetylation is a critical signal in the regulation of cyclin A degradation.

Results

The putative in vivo acetylation of cyclin A was analysed in HCT116 cells transfected with HA-cy clin A. By immunoprecipitation (IP) with anti-HA antibodies followed by western blotting (WB) with anti-acetylated lysines (acetylK), we observed that cyclin A was acetylated in the cells (Figure 1a, left panel). In vivo labeling of ectopic HA-cyclin A was also observed in cells incubated with [^{H]Na acetate. After labeling, cells were subjected to IP with anti-HA and acetylated cyclin A was visualized by autoradiography (}Supplementary Figure S1). By IP with anti-cyclin A followed by WB with anti-acetylK we observed that endogenous cyclin A was also acetylated (Figure 1a, right panel). We subsequently aimed to identify the acetyltransferases that could be responsible for this acetylation. Thus, in vitro assays, using glutathione S-transferase (GST)-cyclin A as a substrate and different acetyltransferases as enzymes, were performed. As shown in Figure 1b, cyclin A was acetylated by P/CAF but not by CBP or TIP60. To further determine whether P/CAF was also involved in the in vivo acetylation of cyclin A, we analysed the effect of the depletion of endogenous or ectopic P/CAF on cyclin A acetylation. As shown in Figure 1c depletion of endogenous P/CAF (by a specific siRNA) significantly decreased cyclin A acetylation. Similarly, depletion of ectopic P/CAF in cells transfected with Flag-P/CA F resulted in a significant reduction of cyclin A acetylation (Figure 1d). Finally, decreasing the levels of ectopic GCN5 (an acetylase homologous to P/CAF) only produced a small diminution of cyclin A acetylation (Figure 1e). These results indicate a key function of P/CAF in the in vivo acetylation of cyclin A.

Figure 1

Cyclin A is acetylated by P/CAF (p300/CBP-associated factor) in vivo and in vitro. (a) HCT116 cells transfected with HA-cyclin A were subjected to immunoprecipitation (IP) with anti-HA or IgG as a control, followed by western blotting (WB) with anti-HA or anti-acetylK (left panel). To analyse the acetylation of the endogenous protein, HCT116 cells were synchronized in G₂/M (when levels of cyclin A are highest) and endogenous cyclin A was immunoprecipitated from cell extracts using anti-cyclin A or IgG as a control, followed by WB with anti-cyclin A or anti-acetylK (right panel). (b) Purified glutathione S-transferase (GST)-cyclin A was subjected to in vitro acetylation assays using the catalytic domain of P/CAF (GST-HAT_P/CAF), GST-CBP or GST-TIP60 in the presence of [^{C]acetylCoA. Purified GST was used as a negative control substrate. In the assays with P/CAF or TIP60, their autoacetylation was used as a positive control, whereas in the case of CBP, histones were used as a positive control substrate. Acetylated proteins were visualized by autoradiography (top panel). A loading control gel was stained with Coomassie blue (bottom panel). (}c) HCT116 cells were transfected with HA-cyclin A and control or P/CAF siRNA. Extracts were prepared and expression of endogenous P/CAF was analysed by WB (top panel). Part of the extracts was subjected to IP against HA or IgG as a control, followed by WB with anti-HA or anti-acetylK (bottom panel). (d) The same as in (c), but coexpressing HA-cyclin A and Flag-P/CAF together with control or P/CAF siRNA. (e) HCT116 cells were transfected with HA-cyclin A, Flag-GCN5 and control or GCN5 siRNA. Expression of GCN5 was analysed by WB (top panel) and IP was performed with the cell extracts using antibodies against HA or IgG as a control, followed by WB with anti-HA or anti-acetylK (bottom panel).

The putative in vivo interaction between cyclin A and P/CAF was first analysed by fluorescence microscopy in cyan fluorescent protein (CFP)-cyclin A-transfected and yellow fluorescent protein (YFP)-P/CAF-transfected cells. Results showed that both proteins colocalized in the nucleus (Figure 2a). The interaction between both proteins was further determined by IP experiments with anti-HA in cells co-transfected with YFP-P/C AF and HA-cyclin A. Subsequent WB analysis demonstrated the co-IP of cyclin A, P/CAF and cdk2 (Figure 2b). Finally, surface plasmon resonance analyses demonstrated the direct association between cyclin A and P/CAF (Figure 2c). We were also interested in determining the interaction between cyclin A and P/CAF during the cell cycle. For that purpose we first analysed the levels of cyclin A and P/CAF in cells synchronized at different phases of the cell cycle. We observed that the levels of P/CAF were high during S phase and G₂/M, decreased at metaphase and remained low during G₁. This behavior was similar to that observed for cyclin A (Figure 2d, left panel). Cdk2 was detected over all the cell cycle although the levels slightly varied depending on the cell-cycle phase. The interaction between cyclin A and P/CAF was analysed by IP experiments followed by WB. Results indicated that cyclin A and P/CAF mostly interacted during S phase and G₂/M, when both proteins were most abundant. At these specific points of the cell cycle, cyclin A also interacted with cdk2. Interestingly, maximal acetylation of cyclin A was observed during G₂/M although the protein was also acetylated during S phase (Figure 2d, right panel).

Figure 2

Cyclin A interacts with P/CAF (p300/CBP-associated factor) and is acetylated at S and G₂/M phases of the cell cycle. (a) COS cells were transfected with cyan fluorescent protein (CFP)-cyclin A and yellow fluorescent protein (YFP)-P/CAF and colocalization of both proteins was studied by fluorescence microscopy. (b) HeLa cells were transfected with HA-cyclin A and YFP-P/CAF. Cell extracts were subjected to immunoprecipitation (IP) using HA or IgG as a control followed by western blotting (WB) with antibodies against HA, P/CAF and cdk2. A sample of cell lysate (input) was used as a control. (c) The putative direct interaction between P/CAF and cyclin A was studied by surface plasmon resonance as described in Materials and methods section. P/CAF was fixed on the matrix and cyclin A was left to circulate on the chip. The interaction was represented in the sensorgram. (d) HeLa cells were transfected with HA-cyclin A and YFP-P/CAF and synchronized as described in Materials and methods section. Then, the levels of P/CAF, cyclin A and cdk2 were determined by WB (left panel). To confirm the time of mitosis, a WB with anti-H3P was performed (bottom, left panel). Cell extracts were subjected to IP with anti-HA and the amount of P/CAF, cyclin A, acetylated cyclin A and cdk2 was analysed by WB (right panel).

Among other regulatory functions, acetylation of lysine residues might affect protein stability in different ways. Thus, we decided to investigate whether acetylation could participate in the regulation of cyclin A stability. Cyclin A degradation largely depends on its N-terminal region and the lysine residues involved in cyclin A ubiquitylation and degradation are located in the first 171 aa of its sequence (Figure 3a) (Geley et al., 2001; den Elzen and Pines, 2001). To study whether the acetylation sites were located in this cyclin A region that contains 12 lysines, we performed in vitro spot mapping assays. Thus, 13 peptides (each one containing one or two consecutive lysines) belonging to this N-terminal region of cyclin A were synthesized and spotted on a membrane that was subjected to acetylation assays with P/CAF (Supplementary Figure S2). An acetylatable peptide from histone H3 was used as a control. Results indicated that peptides containing K54, K68, K95 and K112 were acetylated (Figure 3b). To analyse whether these lysines were the major acetylation sites in the full-length protein, we generated a cyclin A mutant in which lysines 54, 68, 95 and 112 were substituted by arginines (cycA 4R). This mutant was used for in vitro acetylation assays with P/CAF. Results indicated that differently from cyclin A WT (cycA WT) that was clearly acetylated by P/CAF, cycA 4R was not (Figure 3c). Finally, to further determine whether these four lysines were the major in vivo acetylation sites, we transfected cells with Flag-cycA WT or Flag-cycA 4R, subsequently they were subjected to IP with anti-Flag and then cyclin A acetylation was determined by WB. Results revealed that cycA WT was acetylated whereas the mutant cycA 4R was not, indicating that these four lysines are the major in vivo acetylation sites (Figure 3d).

Figure 3

Cyclin A is acetylated at lysines 54, 68, 95 and 112. (a) Schematic representation of cyclin A lysine residues and domains. (b) Thirteen peptides containing one or two consecutive lysines from the cyclin A fragment including aa 1–171 were spotted on a membrane (see also Supplementary Figure S2). As a positive control, a peptide from histone H3 was added. The membrane was subjected to in vitro acetylation assays with P/CAF (p300/CBP-associated factor) and [^{C]acetylCoA. Acetylation was visualized by autoradiography. (}c) Purified glutathione S-transferase (GST)-cycA WT and GST-cycA 4R were subjected to in vitro acetylation assays with P/CAF. Acetylation was visualized by autoradiography (left panel). A loading control gel was stained with Coomassie blue (right panel). (d) HeLa cells were transfected with Flag-cycA WT or Flag-cycA 4R. Cell extracts were subjected to immunoprecipitation (IP) with anti-Flag followed by western blotting (WB) with anti-Flag and anti-acetylK.

To further study the function of acetylation on cyclin A stability, we determined the half-life of cycA WT and cycA 4R in transfected cells. Figure 4a shows that the half-life of cycA WT is of around 6 h whereas that of cycA 4R is much longer. These results suggested that acetylation participates in the degradation of cyclin A. To further confirm this possibility, we aimed to determine the stability of cyclin A under experimental conditions that increase cyclin A acetylation. For this purpose, cells were treated with histone deacetylase inhibitors such as sodium butyrate (NaB) or trichostatin A (TSA) (Abramova et al., 2006; Baek et al., 2006). After these treatments, the acetylation levels of histones (that were used as a control) and those of cyclin A were clearly increased (Figure 4b). It can also be observed that treated cells show reduced levels of cyclin A. Interestingly, the half-life of cyclin A in NaB- or TSA-treated cells was strongly reduced (2 h) respect to that observed in nontreated cells (6 h) (Figure 4c).

Figure 4

Acetylation affects cyclin A stability. (a) HeLa cells were transfected with Flag-cycA WT or Flag-cycA 4R, treated with cycloheximide (CHX), collected at different times of treatment and cyclin A levels analysed by WB. Cdk2 levels were used as a loading control. The amount of Flag-cyclin A was quantitated and represented in the graph. Results are the mean ± s.e. of eight independent experiments. (b) HCT116 cells were treated with histone deacetylase inhibitors sodium butyrate (NaB, 4mM) or trichostatin A (TSA, 500 nM) for 16 h. Then extracts were prepared and levels of cyclin A, cdk2, acetylated histones and actin were determined by western blotting (WB; left panel). Part of the extracts was used to perform immunoprecipitation (IP) with anti-cyclin A or IgG as a control followed by WB with anti-cyclin A and anti-acetylK (right panel). (c) HCT116 cells were treated with 4 mM of NaB or 500 nM TSA for 16 h. Then CHX was added to the cells and samples were collected at different times of CHX treatment. Levels of endogenous cyclin A were analysed by WB and cdk2 was used as a loading control. The amount of cyclin A was quantitated and represented in a graph. Results are the mean±s.e. of six independent experiments. (d) Flag-cyclin A-transfected cells were treated with the proteasome inhibitor ALLN, collected at different times after the release of a double-thymidine block and subjected to IP with anti-Flag. Cyclin A ubiquitylation was determined by WB with anti-Flag (top panel) and cyclin A acetylation with anti-acetylK (bottom panel). A shorter exposure of WB with anti-Flag is shown in the middle panel.

Next we analysed the acetylation and ubiquitylation of cyclin A during cell cycle. Thus, Flag-cycA WT-transfected cells were synchronized by a double-thymidine block, treated with the proteasome inhibitor ALLN and subjected to IP with anti-Flag at different times after the release of the blockade. When WB with anti-acetylK was performed, a peak of cyclin A acetylation was observed at 4–6 h after the release (Figure 4d, bottom panel). Interestingly, during the same period of time a peak of cyclin A ubiquitylation was also observed (Figure 4d, upper panel). The simultaneous acetylation and ubiquitylation of cyclin A also supports that acetylation is involved in cyclin A ubiquitylation and degradation. Fluorescence-activated cell sorting (FACS) analysis revealed that under our experimental conditions at 4–6 h after the release of the double-thymidine block cells are in mitosis (Supplementary Figure S3).

As it has been reported that nondegradable forms of cyclin A (lacking a part of the N terminus) cause arrest of cells in metaphase (Geley et al., 2001; Fung et al., 2005), we aimed to study the effect of the overexpression of the nonacetylatable and more stable cycA 4R mutant on cell-cycle progression. FACS analysis revealed a substantial block in G₂/M in cells transfected with the cycA 4R mutant, whereas only a slight effect was observed in cells transfected with cycA WT (Figure 5a). To further analyse whether cycA 4R arrested cells in mitosis, cycA WT- or 4R-transfected cells were analysed by immunocytochemistry using anti-phosphorylated histone H3. Figure 5b shows that the number of mitosis was increased in cycA 4R-transfected cells and quantification analysis revealed a significant increase in the percentage of metaphases in these cells (Table 1).

Figure 5

Cyclin A 4R blocks cell-cycle progression at G₂/M. (a) HeLa cells were mock-transfected as a control or transfected with cyan fluorescent protein (CFP)-cycA WT or CFP-cycA 4R. Fluorescence-activated cell sorting (FACS) analysis of the population of transfected cells was performed and represented in a graph. Control of expression of CFP-cycA WT and CFP-cycA 4R is shown in Supplementary Figure S5. (b) HeLa cells were transfected with pEFHA empty vector or HA-cycA WT or HA-cycA 4R. 24h after transfection, cells were fixed and immunofluorescence against HA and phosphorylated histone H3 was performed.

We further explored the possibility that the increased stability of cycA 4R could be due to defects in its ubiquitylation. Thus, in vivo ubiquitylation assays were performed. Cells were transfected with HA-ubiquitin plus Flag-cycA WT or Flag-cycA 4R, then subjected to IP with anti-Flag and finally the ubiquitylation levels were analysed by WB with anti-Flag and anti-HA. Results indicated that cycA WT was clearly ubiquitylated whereas ubiquitylation of cycA 4R was strongly reduced (Figure 6a). To study the function of acetylation in cyclin A ubiquitylation, we analysed the in vivo ubiquitylation of Flag-cycA 4Q, a mutant in which lysines 54, 68, 95 and 112 of cyclin A were substituted by glutamines. This form is considered to be a pseudoacetylated mutant because of the structure similarity between glutamine (Q) and the acetylated-lysine residue (Hecht et al., 1995; Li et al., 2002). Results indicated that differently from cycA 4R, the mutant cycA 4Q was ubiquitylated similarly to cycA WT (Figure 6a). Interestingly, the half-life of cycA 4Q is shorter than that of cycA 4R but not as short as that of cycA WT, indicating that cycA 4Q can be degraded although not so efficiently as cycA WT (Supplementary Figure S4). Therefore, these results indicate that K54, K68, K95 and K112 are acetylation sites needed for cyclin A ubiquitylation.

Figure 6

Cyclin A WT, 4R and 4Q present differential ubiquitylation and interaction with components of the ubiquitylation machinery and cdk1 and cdk2. (a) HeLa cells were transfected with HA-ubi quitin plus Flag-cycA WT, Flag-cycA 4R or Flag-cycA 4Q and treated with the proteasome inhibitor ALLN. Then, they were lysed and subjected to immunoprecipitation (IP) with anti-Flag or IgG as a control. The levels of ubiquitylated cyclin A were determined by western blotting (WB) with anti-Flag and anti-HA. (b) 293T cells were transfected with Flag-cycA WT, 4R or 4Q, lysed and immunoprecipitated with anti-Flag. The presence of Flag-cyclin A, APC3, Cdh1, Cdc20, Cks1/2, cdk1 and cdk2 in the immunoprecipitates was determined by WB. (c) As described in panel b, but co-transfecting Flag-Cdc20 with CFP-c ycA WT, 4R or 4Q, or co-transfecting HA-Cdh1 with Flag-cycA WT, 4R or 4Q. (d) Pull-down experiments using purified recombinant glutathione S-transferase (GST)-cycA WT, 4R and 4Q or GST as a control bound to glutathione Sepharose beads and in vitro translated, radiolabeled, Cdc20, Cdh1, cdk2, Cks1 and Cks2. Interacting proteins were visualized by autoradiography.

We subsequently aimed to analyse the mechanisms underlying the differential ubiquitylation of cyclin A mutants. It is known that to be degraded cyclin A has to form a cyclin A–cdk–Cks complex that is recruited to the phosphorylated APC/C by its Cks protein. Cdc20 attached to this complex causes cyclin A to be degraded (Wolthuis et al., 2008). Thus, we analysed the interactions of Flag-cycA WT, Flag-cycA 4R and Flag-cycA 4Q with Cdc20, Cdh1, APC3, Cks1/2, cdk1 and cdk2 in asynchronously growing cells. Results showed that the three forms of cyclin A interacted with Cdc20 and APC3 in a similar manner. However, cycA 4R and cycA 4Q showed an increased interaction with cdk1, cdk2, Cks and Cdh1 (Figure 6b). The specific interactions of the three cyclin A forms with Cdc20 and Cdh1 were further confirmed by IP experiments in cells transfected with Flag-Cdc20 or HA-Cdh1 plus each one of cyclin A forms (Figure 6c). Experiments carried out in cells synchronized in S phase or G₂/M revealed that the interactions of the different cycA forms with all these proteins at these specific points are similar to those observed in asynchronously growing cells (data not shown).

Finally, we studied the direct interaction of cycA WT, cycA 4R and cycA 4Q with Cdc20, Cdh1, cdk2 and Cks1/2 by pull down using purified proteins. As shown in Figure 6d none of these cyclin A forms directly interacted with Cks 1/2 and the interaction with Cdc20, Cdh1 and cdk2 was similar in all the three cyclin A forms. These results indicate that the in vivo increased association of both cycA mutants with Cdh1 and cdks is not produced by a higher affinity for these proteins but by an unknown mechanism related to the in vivo complexes.

The increased in vivo interaction of cycA 4R and cycA 4Q with cdk1 and cdk2 is of particular interest because it might affect their kinase activity. To analyse this possibility, we subjected cells transfected with Flag-cycA WT or Flag-cycA 4R to IP with anti-Flag and the associated cdk activity was determined in the immunoprecipitates. We observed that cycA 4R–cdk complexes display higher kinase activity than that of cycA WT–cdk complexes (Figure 7a). When cells were subjected to IP with anti-cdk2 we also observed an increased association of cycA 4R with cdk2 respect to that shown by cycA WT, and also a higher kinase activity in the cycA 4R– cdk2 complexes (Figure 7b). Similar results were observed with the cycA 4Q mutant (data not shown). So, in addition to the function in cyclin A stability, lysines 54, 68, 95 and 112 also have a function in the regulation of cyclin A interaction with cdk and its associated kinase activity.

Figure 7

Increased interaction of cycA 4R with cdk2 correlates with an increase in cyclin A-associated kinase activity. 293T cells were transfected with Flag-cy cA WT or Flag-cycA 4R, lysed and immunoprecipitated with anti-Flag (a) or anti-cdk2 (b). Kinase assays of the immunoprecipitates were performed and phosphorylation of histone H1 was detected by PhosphorImager. Kinase activity was normalized to the amount of immunoprecipitated cdk2 and represented in the graphs. Results shown are the mean±s.e. of three independent experiments.

Discussion

The different timing of cyclin A and cyclin B degradation at mitosis and the diverse sensitivity of these cyclins to the spindle-assembly checkpoint indicates that specific mechanisms target each one of these cyclins for degradation (van Leuken et al., 2008). We report here that cyclin A acetylation by the acetyltransferase P/CAF participates in the signaling pathway that targets cyclin A for degradation at early mitosis. Acetylation is a posttranslational modification occurring at the Nε-amino group of lysines that might regulate protein functions in many different ways as for instance, protein–protein and protein–DNA interactions and protein stability. Lately, a number of reports have revealed that lysine acetylation might act as a direct signal enhancing protein degradation for proteins such as E2F1 (Galbiati et al., 2005), HIF-1α (Jeong et al., 2002), SV40T antigen (Shimazu et al., 2006) and pRB (Leduc et al., 2006). Moreover, the interplay between lysine acetylation and ubiquitylation has been reinforced by the evidence that at least four acetyltransferases (p300, CBP, P/CAF and TAF1) possess intrinsic ubiquitin activating/conjugating or ligase activities (Sadoul et al., 2008).

We report here that P/CAF acetylates cyclin A at lysines K54, K68, K95 and K112 and that these lysines are the major acetylation sites both in vivo and in vitro. These specific residues are located in the N-terminal domain of cyclin A that has been involved in the stability of the protein (Wolthuis et al., 2008). In fact, two of these lysines, K54 and K68, were already described as important residues for the ubiquitylation and degradation of cyclin A. Specifically, the authors reported that substitution of K37, K54 and K68 by arginines generates a more stable cyclin A but this mutant was still ubiquitylated (Fung et al., 2005). As our observations indicate that lysines K54, K68, K95 and K112 are critical residues for acetylation, this means that at least K54 and K68 can be both acetylated and ubiquitylated. Thus, it is likely that when these lysines are acetylated, alternative ubiquitylation sites could be used. It has also been reported that in Drosophila the substitution of eight lysines in the N-terminal domain of cyclin A by arginines generates an unusual enrichment of overcondensed metaphase chromosomes in injected embryos, similar to what has been observed with stable cyclin A (Ramachandran et al., 2007). These data are compatible with our results indicating that these lysines are critical for cyclin A stability. Thus, our results suggest that likely cyclin A acetylation at these specific lysines signals this protein for the subsequent ubiquitylation and degradation.

According to our observations, P/CAF is the principal histone acetyltransferase involved in cyclin A acetylation. In addition to acetylating histones, P/CAF also participates in the reversible acetylation of various transcriptional regulators such as the general transcription factors TF_IIEβ and TF_IIF (Imhof et al., 1997) and the sequence-specific transcription factors E2F1 (Martinez- Balbas et al., 2000), c-myc (Patel et al., 2004), myo D (Patel et al., 2004) and p53 (Gu and Roeder, 1997; Sakaguchi et al., 1998). It has been implicated in many important cellular processes such as transcription, differentiation, proliferation and apoptosis (Schiltz and Nakatani, 2000). In the cell P/CAF is a subunit of multiprotein complexes that possess global histone acetylation activity and locus-specific coactivator functions together with acetyl transferase activity on non-histone substrates (Nagy and Tora, 2007). Recently, it has been described that in addition to acetylate p53, the intrinsic ubiquitylation activity of P/CAF controls the stability of the oncoprotein Hdm2, indicating an important function of this acetylase in the DNA damage checkpoint (Linares et al., 2007).

Interestingly, the levels of P/CAF oscillate during cell cycle similarly to those of cyclin A. They are low at G₁, increase during S phase and remain high during G₂ and early mitosis to finally decrease before metaphase. As the decrease of cyclin A is produced by degradation by the APC/C ^{complex, the similar behavior of P/CAF suggests that it could also be an APC/C substrate. However, this is something that needs to be investigated.}

Cyclin A associates with P/CAF during S phase and this interaction is maintained until early mitosis, then before metaphase this complex is disrupted. Concomitant to its association with P/CAF, cyclin A becomes acetylated. A more detailed time-course analysis indicates that cyclin A acetylation increases at early mitosis simultaneously to cyclin A ubiquitylation. All these data support that acetylation by P/CAF targets cyclin A for its ubiquitylation.

To be degraded cyclin A has to be bound to a cdk subunit that in turn has to be associated with a Cks protein. This cyclin A–cdk–Cks complex is then recruited to the phosphorylated APC/C by its Cks subunit. The Cdc20 attached to cyclin A causes cyclin A to be degraded regardless of the spindle checkpoint being active or not (Wolthuis et al., 2008). Thus, a possibility was that cyclin A acetylation at these specific lysines might be required for the interaction with some of these proteins of the ubiquitylation machinery. However, the analysis of the interactions of cycA 4R and cycA 4Q with the proteins of the APC/C ^{ubiquitylation complex ruled out this possibility because these mutants retain the ability to interact withal l the proteins of the complex. Thus, the lack of ubiquitylation of cycA 4R is not due to a reduced ability to form ubiquitylation complexes. A possible interpretation of these results is that acetylation of lysines 54, 68, 95 and 112 is needed for the correct incorporation of ubiquitin molecules on specific sites of cyclin A.}

An unexpected result was the observation that the levels of cdk1, cdk2 and Cks associated with cycA4R and cycA 4Q are much higher than those bound to cycA WT. These results could be interpreted in the sense that both cycA mutants display a much higher affinity for these proteins than cycA WT. However, this possibility was ruled out by results showing that the direct interaction between the cycA mutants and these proteins was similar to that shown by cycA WT. Thus, the in vivo increased interaction between cyclin A mutants and cdks is due to still unknown mechanisms. The increased interaction of cycA 4R and cycA 4Q with cdks is accompanied by a higher activity of these complexes. The fact that cycA 4Q can be ubiquitylated whereas cycA 4R cannot indicates that the elevated kinase activity of these complexes does not have a function in cyclin A ubiquitylation.

As a summary, results presented here reveal that acetylation at specific lysines is a new mechanism that targets cyclin A for degradation at early mitosis. In addition to that, our results also revealed an unexpected new mechanism for the regulation of cdk activity that depends on the integrity of four specific lysines of cyclin A.

Materials and methods

Plasmids

cDNA of wild-type cyclin A was cloned into pGEX6P1, pEFHA, pcDNA3-Flag and pECFP-C1 vectors. cycA 4R and cycA 4Q mutants were generated by site-directed mutagenesis. pCX-Flag-P/CAF, pGEX2TKP-HAT_P/CAF (352–658), pGEX4T2-P/CAF (full length), pGEX2T-CBP and pGEX2T-TIP60 were a kind gift from MA Martínez-Balbás. pcDNA3.1-HA-Cdh1 and Flag-Cdc20 were a kind gift from M Pagano. pcDNA3-HA-Cks 1 and 2 were a kind gift from R Wolthuis. P/CAF shRNA and control shRNA were purchased from Sigma (St Louis, MO, USA).

Antibodies and reagents

Antibodies against cyclin A (H-432), cyclin A (BF-683)-AC (agarose conjugated), cdk2 (M-2), Cdc20 (H175) and Cks1/2 (FL-79) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Cdh1 (34–2000) was purchased from Zymed (South San Francisco, CA, USA). Anti-acetylated lysines (9441) and anti-phospho-histone H3 (Ser28) (9713) were from Cell Signaling (Danvers, MA, USA). Antibodies against Flag (F7425), HA (H6908) and P/CAF (P7493) were purchased from Sigma, and APC3/Cdc27 (ab10538) from Abcam (Cambridge, UK) Anti-HA 12C5A for immunofluorescence was purchased from Roche (Basel, Switzerland). For IP we used monoclonal anti-HA agarose and monoclonal anti-Flag M2 affinity gel from Sigma. [^{C]acetylCoA was purchased from PerkinElmer (Waltham, MA, USA). Thymidine, nocodazol, cycloheximide, sodium butyrate and TSA were from Sigma. ALLN was from Calbiochem (Merck Chemicals Ltd., Nottingham, UK). For transfection assays we used Lipofectamine 2000 from Invitrogen (Paisley, UK).}

Protein purification and in vitro acetylation

Protein expression and purification was performed as described (Canela et al., 2006). Acetylase assays were performed as described (Martinez-Balbas et al., 2000). For cyclin A acetylation assays, 1–10 μl of the different acetylases (5000–10 000 c.p.m. activity on histones) were incubated with 6 μM of purified GST or GST-cyclin A and 0.02 μCi [^{C]acetylCoA. For the spot-mapping experiment, the membrane was incubated in 3ml of HAT buffer (50mM Tris-HCl (pH 8), 500mM NaCl, 0.1mM EDTA, 5% glycerol, 0.1% Nonidet P-40 (NP-40)) in the presence of GST-HAT}_P/CAF and [^{C]acetylCoA, for 30 min at 30 °C. Then the membrane was washed, dried and subjected to autoradiography.}

Immunoprecipitation

Cells were lysed in RIPA buffer (50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1mM EDTA, 1mM DTT, 1mM PMSF, 0.1mM Na₃VO₄, 0.5 μg/μl aprotinin, 10 μg/μl leupeptin) for 30 min on ice. Lysates (0.2–2 mg of protein) were incubated with Flag or HA or cyclin A agarose beads for 2 hat 4 °C. After three washes with RIPA buffer, Laemmli buffer was added to the samples and they were electrophoresed.

Surface plasmon resonance experiments

The surface plasmon resonance analysis was performed at room temperature using a Biacore T100 (Biacore International AB, GE Healthcare, New Jersey, NJ, USA). P/CAF purified protein was immobilized on a carboxymethylated dextran sensor chip (CM5) using the amine coupling method as described by the manufacturer. A blank immobilization was performed using the same method and was used as the reference surface. Purified full-length cyclin A was diluted in HBS-EP buffer (Biacore International AB) and was injected over the flow cells at a flow rate of 30 μl/min for 60 s. Following a dissociation time of 120 s, final regeneration of the surface was performed with a short pulse of 0.05% (w/v) SDS. The interaction between P/CAF and cyclin A was detected and presented as a sensorgram by plotting resonance units against time. Cells were fixed with 70% cold ethanol for 2h at 4 °C, washed with phosphate-buffered saline (PBS), and finally incubated with 50μg/ml of propidium iodide (Sigma) and 200 μg/ml RNase for 30 min at room temperature. Analysis of DNA content was carried out in a BD Biosciences FACS Canto II (BD Biosciences, Erembodegen, Belgium). Data were analysed with WinMDI 2.9 software (http://facs.scripps.edu/software.html).

Immunofluorescence

To detect HA-cyclin A and H3P by immunocytochemistry, cells were grown in coverslips, fixed in 4% paraformaldehyde/PBS for 15 min at room temperature, washed in PBS and blocked with 1% BSA, 0.1% Triton X-100 in PBS for 1h. Then, coverslips were incubated with anti-HA (Roche; 12C5A, mouse monoclonal, 1:200 dilution) and anti-H3P (Cell Signaling; 9713, rabbit polyclonal, 1:200 dilution) for 1 hat 37 °C in a humidified atmosphere. They were then washed in PBS and incubated for 45 min at 37 °C with Alexa-Fluor 594 (Invitrogen, A11005, goat anti-mouse, dilution 1:500) and Alexa-Fluor 488 (Invitrogen, Molecular Probes; A11008, goat anti-rabbit, dilution 1:250). After that coverslips were washed, mounted on glass slides with Mowiol (Calbiochem) and analysed by fluorescence microscopy.

In vivo ubiquitylation assays

Cells were transfected with indicated plasmids. At 24 h after transfection, cells were replated and treated with 100 μM ALLN for 16 h. Then, cells were harvested and subjected to IP as described (Fung et al., 2005).

In vitro protein-binding assay

The GST and GST-cycA WT, 4R and 4Q were expressed in Escherichia coli. These fusion proteins were bound to glutathione Sepharose beads (Glutathione Sepharose 4B; GE Healthcare, Chalfont St Giles, UK) and washed with NETN (20mM Tris-HCl (pH 8), 1mM EDTA (pH 8), 0.5% NP-40, 100mM NaCl). Beads were then incubated for 1 h at room temperature with ^{S-labeled Flag-CDK2, Flag-Cdc20, HA-Cdh1, HA-Cks1 or HA-Cks2 proteins,} in vitro transcribed and translated using the Promega TnT kit (Promega, Madison, WI, USA). Beads were extensively washed with NETN 150mM NaCl, and the bound material was analysed by SDS– polyacrylamide gel followed by autoradiography.

Immunoprecipitation and kinase assays

After three washes in IP buffer and two in kinase buffer (50mM HEPES (pH 7.4), 2.5mM EGTA, 10mM MgCl₂) immunoprecipitates were resuspended in a final volume of 30 μl of kinase buffer containing 15μM ATP, 10 μCi of [^{P]ATP, 2mM dithiothreitol and 2 μg of histone H1 for 30 min at 30 °C. Reactions were stopped by the addition of Laemmli buffer. Samples were then electrophoresed on 12% SDS–PAGE and then stained with Coomassie blue and dried. The radioactivity associated to the gels was detected with a PhosphorImager (BIO-RAD laboratories, Hercules, CA, USA).}

Supplementary Material

Supplementary Data

Acknowledgments