Here we investigated the role of acetylation on p27 stability. We describe that p27 directly interacts with PCAF forming complexes in the cells, mostly located in the nucleus. This is consistent with previous reports showing that PCAF essentially displays a nuclear localization (26
). We provide evidence that p27 associates with the catalytic domain (HAT) of PCAF through a region including amino acids 91–120 that we named PID. Despite the deletion p27 mutant, lacking a region including amino acids 91–100 loses the ability to interact with PCAF (E), the fact that PCAF did no interact with the p27NT(1–110) fragment indicates that the domain amino acids 110–120 is also necessary for the interaction and thus the full PID of p27 has to considered to include amino acids 91–120.
Interestingly, this region contains a PRD (amino acids 91–95) known to interact with proteins containing a Src homology 3 (SH3) domain. The PRD of p27 has been reported to interact with other proteins that are relevant for cell-cycle control as for instance Grb2 (27
). These data are in agreement with previous observations showing that the HAT domain of GNC5, an acetyltransferase presenting high homology with PCAF, has a structure similar to an SH3 domain (28
). Due to this high homology, it can be assumed that PCAF also has this structure that would facilitate the association with the PRD of p27. However, as shown in E the interaction of PCAF with this domain of p27 is not critical because when it was deleted the association of PCAF with p27 was only slightly affected.
We further confirmed that the PID domain of p27 includes amino acids 91–120 by transfecting the deletion mutants p27(Δ91–100) or p27(Δ91–120) into the cells and analyzing their interaction with PCAF. As shown in F and G, the interaction of PCAF with these mutants was strongly reduced. The residual interaction of PCAF with these mutants can be attributed to the indirect interaction through cyclin A–cdk2 complexes. We previously reported that PCAF directly interacts with both cyclin A and cdk2 (6
). Since these p27 mutants (p27Δ91–100 and p27Δ91–120) still retain the ability to associate with cyclins and cdks, the IP with anti-Flag immunoprecipitate the cyclin A and cdk2 associated with PCAF and additionally the p27 associated with cyclin A and cdk2.
We identified K100 as the specific residue of p27 acetylated by PCAF in vitro and in the cells. K100 is very close to the PRD (amino acids 91–95) and it is included in the PID of p27. Thus, this is consistent with a scenario in which PCAF through its HAT domain associates with the PID of p27 and acetylates K100 included in this region.
Degradation of p27 is produced by ubiquitin-dependent proteolysis in a two-step process (31
). The first step takes place during G0
transition and early-mid G1
, is independent of phosphorylation at T187 of p27 and it is mediated by the ubiquitin ligase KPC (32
). However, the molecular signal triggering this degradation still remains unclear.
In contrast, the second proteolytic step is produced during G1
/S transition and S and G2
phases and it is dependent on phosphorylation at T187 by cyclin E–Cdk2 (34
). This second step is preceded by the phosphorylation of p27 at three specific tyrosine residues: Y74, Y88 and Y89. This allows to the p27-associated cyclin E–Cdk2 complexes to be partially activated. Then, these complexes can phosphorylate the T187 of p27 inducing its ubiquitin-dependent degradation (36
). Interestingly, these tyrosine residues are very close to the PRD of p27. Moreover, the tyrosine kinases, members of the Src family (i.e. c-Src, Bcr-Abl, Lyn) that phosphorylate these p27 tyrosine residues have an SH3 domain that allows the interaction with the PRD of p27 (38–41
). Collectively, these results indicate a key participation of the PID (a region including amino acids 91–120) of p27 in the regulation of its stability: by associating with PCAF and acetylating K100 and by interacting with tyrosine kinases that phosphorylate Y74, Y88 and/or Y89 thus allowing the further phosphorylation of T187.
We propose that degradation of p27 mediated by acetylation could operate during G1. We observed that the knockdown of PCAF increases the levels of p27 and conversely overexpression of PCAF decreases its amount. Moreover, we also observed that the half-life of p27 is decreased when PCAF is overexpressed and than that of the non-acetylatable mutant p27K100R is higher than that of p27WT. Moreover, as shown in , we observed that differently to p27WT, the non-acetylatable mutants p27K100R and p27Δ91–120 were not degraded during G1.
We also report here several evidences indicating that the acetylation induced p27 degradation could be performed via proteasome. Specifically, we describe that proteasome inhibitors as ALLN and MG132 block the p27 degradation induced by PCAF overexpression and that p27 acetylation by PCAF induced its K100 dependent ubiquitylation, because the non-acetylatable p27K100R mutant was not ubiquitylated under the same experimental conditions (E). The observation that the levels of acetylated p27 correlates with the levels of p27 during the first 6
h after mitogenic stimulation of the cells (C and F) also supports the relationship between acetylation and degradation during G1. These results indicate that there is a basal acetylation of p27 in quiescent cells. A possibility is that this basal acetylation could be related to a basal turn over of the protein in the nucleus. At 2
h an increase of p27 acetylation was observed by WB. Interestingly, at this point the levels of p27 are still high (C). This indicates that at this point degradation still has not been initiated and suggests that p27 is already targeted for its subsequent degradation that will immediately start. This is supported by the fact that at times 4–6
h the total levels of p27 are progressive decreasing concomitantly with the decrease of acetylated p27. This indicates that specifically is the acetylated form of p27 the one that is degraded at that time. Finally, at times 8–10
h the subsequent degradation of p27 is probably performed via phosphorylation of T187 via Skp2 that is produced later on at the late G1 and G1/S transition. At this point acetylation of p27 returns to its basal levels.
Interestingly, we also report here that the acetylation dependent degradation of p27 is independent of Skp2 (F). These data also support the role of acetylation induced degradation of p27 during G1. Finally, it has been reported that a domain of p27 including amino acids 42–102 is necessary for the ubiquitylation and degradation of p27 mediated by the E-ubiquitin ligase KPC at G0
). Our results indicating that K100 is included in this domain are compatible with the possibility that acetylation could signal p27 for recognition by KPC at G0
. Another mechanism involved in the degradation of p27 during G1
related to the activation of the Wnt–Cul4A pathway has been reported (42
). However, the possibility that acetylation of p27 could participate in this mechanism remains to be explored.
The functional relevance of p27 acetylation on its role on transcription still remains to be explored. However, because p27 is a transcriptional repressor of its target genes, our data showing that acetylation at K100 by PCAF induces its degradation allows us to speculate that it would facilitate the expression of the p27-TGs. This speculation is compatible with the well-known role of PCAF as a transcriptional coactivator. Experiments to clarify this point are currently underway in our laboratory.
We also speculate that this new mechanism regulating p27 stability could be also relevant during oncogenesis. It is known that p27 is decreased in many human tumors correlating with a bad prognosis (43
). In a number of cases, this decrease has been correlated with increased levels of Skp2 that mediates p27 degradation at S/G2
. However, in other cases, this correlation could not be established. Results reported here allow us to speculate that the decrease of p27 levels in tumor cells could be also produced by alterations in its acetylation status.
In conclusion, we describe here a new mechanism regulating p27 stability that involves acetylation of K100 of p27. We postulate that this new mechanism could be relevant for the transcriptional regulatory role of p27.