Cyclin A must be degraded at prometaphase in order to allow mitosis progression. Nevertheless, the signals that trigger cyclin A degradation at mitosis have been largely elusive. In the present paper, we review the status of cyclin A degradation in the light of recent evidence indicating that acetylation plays a role in cyclin A stability. The emerging model proposes that the acetyltransferase PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] [perhaps also its homologue GCN5 (general control non-derepressible 5)] acetylates cyclin A at Lys54, Lys68, Lys95 and Lys112 during mitosis, leading to its ubiquitylation by the anaphase-promoting factor/cyclosome and its subsequent degradation via proteasome. Interestingly, these four lysine residues in cyclin A also participate in the regulation of cyclin A–Cdk (cyclin-dependent kinase) activity by modulating its interaction with Cdks.
- cAMP-response-element-binding protein (CREB)
- p300/CREB-binding protein-associated factor (PCAF)
- cyclin A
Degradation of cyclins A and B is needed for mitosis progression
Cell cycle progression is governed by the family of Cdks (cyclin-dependent kinases) . Their activities are regulated by binding to regulatory subunits called cyclins, by phosphorylation at specific sites and by binding to inhibitory proteins . 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 . Whereas the levels of most Cdks are relatively constant during the cell cycle, those of cyclins fluctuate, and in this 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 [4,5]. 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 nuclear envelope breakdown [6–8]. It is destroyed during prometaphase by the APC/C (anaphase-promoting complex/cyclosome) via proteasome . Cyclin B levels rise during G2 and then cyclin B 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 . On time degradation of cyclins A and B is a key event for mitosis progression, and non-degradable mutants of cyclin A cause cell arrest in metaphase, whereas those of cyclin B block cells during anaphase [11,12].
The signals that trigger cyclin A degradation at prometaphase have been largely elusive. Degradation is induced by APC/C bound to the targeting subunit Cdc20 (cell division cycle 20) (APC/CCdc20) that is activated by phosphorylation by cyclin B–Cdk1. Cyclin A degradation is spindle checkpoint independent [9,13]. In contrast, cyclin B1 degradation by APC/CCdc20 is sensitive to this checkpoint. Therefore, at prometaphase, unattached sister chromatids generate signals that allow components of the checkpoint, such as Mad2, to bind to Cdc20 and block its ability to interact with cyclin B1 [14,15]. Under these conditions Cdc20 is ubiquitylated and subsequently degraded . The different behaviour 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 degradation during mitosis .
The association of cyclin A with its Cdk partner is needed for its degradation. Moreover, the cyclin A–Cdk complex must bind a Cks (Cdk subunit) protein to be degraded in prometaphase. Thus it has been proposed that the cyclin A–Cdk–Cks complex is recruited to the phosphorylated APC/C 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 .
In general, cyclins have a ‘destruction box’, which is a sequence that is recognized by the ubiquitylation machinery in order to degrade these proteins . Cyclin A also has an extended ‘destruction box’ that includes amino acids 57–72 . However, in order to totally avoid cyclin A ubiquitylation and degradation, the first 171 amino acids 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 .
Cyclin A is acetylated at specific lysine residues by the PCAF (p300/CREB-binding protein-associated factor)
It has recently been shown that the acetyltransferase PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] interacts with cyclin A, both in vivo and in vitro . Using fluorescence microscopy and immunoprecipitation experiments, the interaction between both proteins was clearly demonstrated and surface plasmon resonance analysis revealed a direct interaction between both proteins. The interaction between both proteins is high during S-phase and G2/M and low at metaphase and G1 phase .
By different experimental approaches it was observed that both endogenous and ectopic cyclin A are acetylated in the cells. In vitro acetylation analysis indicated that cyclin A was acetylated by PCAF but not by CBP or Tip60. Moreover, depletion of endogenous or ectopic PCAF by using specific siRNAs (small interfering RNAs) significantly decreased cyclin A acetylation. Finally, decreasing the levels of ectopic GCN5 (general control non-derepressible 5; an acetylase homologous with PCAF) also produced a slight diminution of cyclin A acetylation . These results indicate a key role of PCAF (and perhaps GCN5) in the in vivo acetylation of cyclin A. Interestingly, maximal acetylation of cyclin A was observed during G2/M although the protein was also acetylated during S-phase.
PCAF is an acetyltransferase principally involved in the acetylation of histones. However, PCAF also participates in the reversible acetylation of various transcriptional regulators such as the general transcription factors TFIIEβ and TFIIF  and the sequence-specific transcription factors E2F1 , c-Myc , myoD  and p53 [25,26]. It has been implicated in many important cellular processes such as transcription, differentiation, proliferation and apoptosis . In the cell, PCAF is a subunit of multiprotein complexes that possess global histone acetylation activity and locus-specific co-activator functions together with acetyltransferase activity on non-histone substrates . Recently, it has been described that PCAF possesses an intrinsic ubiquitylation activity . In that work it was demonstrated that, in addition to acetylating p53, PCAF controls the stability of the oncoprotein Hdm2 (human double minute 2), indicating an important role of this acetylase in the DNA damage checkpoint.
The putative acetylation sites of cyclin A were first identified by spot mapping analysis using short cyclin A peptides as substrates and PCAF as the acetylase. These experiments revealed that Lys54, Lys68, Lys95 and Lys112 from cyclin A were acetylated. The evidence that these four lysine residues were the in vivo acetylation sites was obtained from cycA 4R (a cyclin A in which Lys54, Lys68, Lys95 and Lys112 were replaced by arginine residues). In vitro acetylation assays revealed that cycA 4R was not acetylated. It was also observed that this mutant was not acetylated when transfected into the cells .
Cyclin A acetylation leads to its ubiquitylation and degradation
The non-acetylatable mutant cycA 4R is much more stable than cycA WT (wild-type). Specifically, cycA WT has a half-life of approx. 6 h, whereas that of cycA 4R is much longer. Interestingly, acetylation of cyclin A, achieved by treatment of cells with HDACs (histone deacetylase) inhibitors such as sodium butyrate or trichostatin A, reduced its half-life to 2 h . All these results clearly indicate a role of acetylation in cyclin A stability. The observation of simultaneous peaks of cyclin A acetylation and ubiquitylation at mitosis strongly suggested that cyclin A acetylation can be related to its ubiquitylation. This possibility was confirmed by the evidence that the non-acetylatable mutant cycA 4R cannot be ubiquitylated. In contrast, the pseudoacetylated mutant, cycA 4Q (cyclin A in which Lys54, Lys68, Lys95 and Lys112 were replaced by glutamine residues), was ubiquitylated similarly to cycA WT .
It was also observed that 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. Therefore all these results indicate that Lys54, Lys68, Lys95 and Lys112 can be acetylation sites needed for cyclin A ubiquitylation. These results are in agreement with a number of reports revealing that lysine acetylation might act as a direct signal, enhancing protein degradation for proteins such as E2F1 , HIF-1α (hypoxia-inducible factor-1α) , SV40 (simian virus 40) T-antigen  and pRB .
These four specific lysine residues are located in the N-terminal domain of cyclin A that has been observed to be involved in the stability of the protein . In fact, two of these lysine residues, Lys54 and Lys68, were already described as important residues for the ubiquitylation and degradation of cyclin A. Specifically, it was reported that replacement of Lys37, Lys54 and Lys68 by arginine residues generates a more stable cyclin A, but this mutant was still ubiquitylated . The observation that Lys54, Lys68, Lys95 and Lys112 are critical residues for acetylation indicates that at least Lys54 and Lys68 can be both acetylated and ubiquitylated. Thus it is likely that when these lysine residues are acetylated, alternative ubiquitylation sites could be used.
Overexpression of the non-acetylatable mutant cycA 4R blocks cell cycle progression at metaphase
As overexpression of non-degradable mutants of cyclin A, lacking significant portions of the N-terminus, induced cell arrest in metaphase [13,20], the effect of the non-acetylatable mutant cycA 4R on cell cycle progression was analysed. Interestingly, it was shown that cycA 4R overexpression induced a substantial block in metaphase. To study the mechanism by which cycA 4R blocks cell cycle progression, its interaction with some components of the ubiquitylation machinery (Cdc20, Cdh1, APC3, Cks1/2, Cdk1 and Cdk2) was analysed. It was observed that both cycA WT and cycA 4R interacted with Cdc20 and APC3 in a similar manner. However, cycA 4R showed an increased interaction with Cdks, Cks and Cdh1 .
To better understand the reason for the increased in vivo association of cycA 4R with Cks, Cdks and Cdh1, an analysis of these interactions using purified proteins was performed. As cyclin A interacts with Cks indirectly through Cdks, only the interaction with Cdks and Cdh1 was analysed. Under these experimental conditions, the cyclin A association with Cdh1 and Cdks was similar in the two cyclin A forms . Thus these results indicate that the in vivo increased association of cycA 4R with both Cdh1 and Cdks is produced not by a higher affinity for these proteins but by an unknown mechanism related to the in vivo complexes. Moreover, 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 Lys54, Lys68, Lys95 and Lys112 is needed for the correct incorporation of ubiquitin molecules into specific sites of cyclin A.
Lys54, Lys68, Lys95 and Lys112 of cyclin A are also involved in the regulation of cyclin A–Cdk2 activity.
The increased in vivo interaction of cycA 4R and cycA 4Q with Cdks is of particular interest because it might affect their kinase activity. In fact, it has been observed that cycA 4R–Cdk and cycA 4Q complexes display higher kinase activity than that of cycA WT–Cdk complexes . So, in addition to the role in cyclin A stability, Lys54, Lys68, Lys95 and Lys112 also play a role in the regulation of cyclin A interaction with Cdk and its associated kinase activity. The fact that cycA 4Q can be ubiquitylated, whereas cycA 4R cannot indicate that the elevated kinase activity of these complexes does not play a role in cyclin A ubiquitylation.
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 . The present review summarizes recent results indicating that cyclin A acetylation at specific sites in the N-terminus by the acetyltransferase PCAF participates in the signalling pathway that targets cyclin A for degradation at early mitosis.
Cyclin A associates with PCAF during S-phase and this interaction is maintained until early mitosis; then, before metaphase, this complex is disrupted. Concomitant with its association with PCAF, cyclin A becomes acetylated at four specific sites, namely Lys54, Lys68, Lys95 and Lys112. A more detailed time-course analysis indicates that cyclin A acetylation increases at early mitosis simultaneously with cyclin A ubiquitylation (Figure 1). All these results support that acetylation by PCAF targets cyclin A for its ubiquitylation.
This work was supported by the Ministerio de Educación y Ciencia of Spain [grant number SAF2006-05212] and the Instituto de Salud Carlos III [grant number RETICS RD06/0020/0010].
Ubiquitin–Proteasome System, Dynamics and Targeting: 4th Intracellular Proteolysis Meeting, a Biochemical Society Focused Meeting held at Institut d'Estudis Catalans, Casa de Convalescència, Barcelona, Spain, 27–29 May 2009. Organized and Edited by Bernat Crosas (Institute of Molecular Biology of Barcelona, Spain), Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (University of Barcelona, Spain), Manuel Rodríguez (CIC bioGUNE, Derio, Spain) and Timothy Thomson (Institute of Molecular Biology of Barcelona, Spain)
Abbreviations: APC/C, anaphase-promoting complex/cyclosome; Cdc20, cell division cycle 20; Cdk, cyclin-dependent kinase; Cks, Cdk subunit; CREB, cAMP-response-element-binding protein; cycA 4R, cyclin A in which Lys54, Lys68, Lys95 and Lys112 were replaced by arginine residues; cycA 4Q, cyclin A in which Lys54, Lys68, Lys95 and Lys112 were replaced by glutamine residues; GCN5, general control non-derepressible 5; HDAC, histone deacetylase; PCAF, p300/CREB-binding protein-associated factor; WT, wild-type
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