P53 Nuclear Export Research Articles

P53-Ubl fusions as models of ubiquitination, sumoylation and neddylation of p53

The tumour suppressor p53 has been shown to be modified at its C-terminus with ubiquitin and the ubiquitin-like proteins SUMO and NEDD8. Whereas monoubiquitination of p53 is strongly associated with nuclear export, the effects of sumoylation and neddylation remain unclear. In this study we have generated p53-Ub, p53-SUMO-1 and p53-NEDD8 fusion proteins as models for the effect of these modifications on the localization and function of p53. As shown before, the ubiquitin fusion clearly drives nuclear export of p53 and we now find that this is also partially the case for a SUMO-1 fusion, which does not localise to PML bodies. In contrast a NEDD8 fusion has little effect on nuclear export, and mutating NEDD8 to more closely resemble ubiquitin did not restore nuclear export. Despite their differing subcellular localization, we find that both p53-ubiquitin and p53-NEDD8 retain similar transcriptional activity and both induce apoptosis at a similar level to unfused p53. The p53-ubiquitin fusion protein is potentially a good model for studying the role of p53 outside the nucleus. However, in our experiments we find that the export of p53 from the nucleus is not sufficient to activate its cytoplasmic apoptotic function which may depend on the ability to deubiquitinate cytoplasmic p53.

Cooperative Roles of c-Abl and Cdk5 in Regulation of p53 in Response to Oxidative Stress

The p53 tumor suppressor protein, a critical modulator of cellular stress responses, is activated through diverse mechanisms that result in its stabilization and transcriptional activation. p53 activity is controlled by transcriptional, translational, and post-translational regulation. The major mechanisms of p53 regulation occur primarily through interactions with HDM2, an E3 ubiquitin ligase that leads to p53 nuclear export and degradation. Here, we demonstrate that hydrogen peroxide-induced oxidative stress elicits down-regulation of HDM2. c-Abl mediates down-regulation of HDM2, leading to an increase of p53 level. Moreover, Cdk5 (cyclin-dependent kinase 5), a proline-directed Ser/Thr kinase, additionally increases p53 stability via post-translational modification of p53 in response to hydrogen peroxide. The p53 protein stabilized by c-Abl and Cdk5 is transcriptionally active; however, transcription of its target gene is differentially regulated with selective binding of p53 on promoter regions of its target genes by c-Abl. In addition, c-Abl modulates Cdk5 activity via phosphorylation of tyrosine 15 in cooperation with cleavage of p35 to p25. Our results show that c-Abl and Cdk5 cooperatively regulate maximal activation of p53, resulting in neuronal death in response to oxidative stress by hydrogen peroxide. These findings aid in clarifying the mechanism underlying the occurrence of neuronal apoptosis as a result of c-Abl and Cdk5-mediated p53 stabilization and transcriptional activation.

Regulation of p53 tetramerization and nuclear export by ARC

Inactivation of the transcription factor p53 is central to carcinogenesis. Yet only approximately one-half of cancers have p53 loss-of-function mutations. Here, we demonstrate a mechanism for p53 inactivation by apoptosis repressor with caspase recruitment domain (ARC), a protein induced in multiple cancer cells. The direct binding in the nucleus of ARC to the p53 tetramerization domain inhibits p53 tetramerization. This exposes a nuclear export signal in p53, triggering Crm1-dependent relocation of p53 to the cytoplasm. Knockdown of endogenous ARC in breast cancer cells results in spontaneous tetramerization of endogenous p53, accumulation of p53 in the nucleus, and activation of endogenous p53 target genes. In primary human breast cancers with nuclear ARC, p53 is almost always WT. Conversely, nearly all breast cancers with mutant p53 lack nuclear ARC. We conclude that nuclear ARC is induced in cancer cells and negatively regulates p53.

Expression of Androgen Receptor Is Negatively Regulated By p53

Increased expression of androgen receptor (AR) in prostate cancer (PC) is associated with transition to androgen independence. Because the progression of PC to advanced stages is often associated with the loss of p53 function, we tested whether the p53 could regulate the expression of AR gene. Here we report that p53 negatively regulates the expression of AR in prostate epithelial cells (PrECs). We found that in LNCaP human prostate cancer cells that express the wild-type p53 and AR and in human normal PrECs, the activation of p53 by genotoxic stress or by inhibition of p53 nuclear export downregulated the expression of AR. Furthermore, forced expression of p53 in LNCaP cells decreased the expression of AR. Conversely, knockdown of p53 expression in LNCaP cells increased the AR expression. Consistent with the negative regulation of AR expression by p53, the p53-null HCT116 cells expressed higher levels of AR compared with the isogenic HCT116 cells that express the wildtype p53. Moreover, we noted that in etoposide treated LNCaP cells p53 bound to the promoter region of the AR gene, which contains a potential p53 DNA-binding consensus sequence, in chromatin immunoprecipitation assays. Together, our observations provide support for the idea that the loss of p53 function in prostate cancer cells contributes to increased expression of AR.

Keeping p53 in the Nucleus

One of the functions of the tumor suppressor p53 is as a transcriptional regulator, and this activity is stimulated in response to DNA damage. Among the many types of posttranslational modifications that control p53’s various functions is the addition of poly(ADP-ribose), mediated by poly(ADP-ribose) polymerase 1 (PARP-1), the activity of which is stimulated by DNA damage. Kanai et al . identified the residues in mouse p53 that were the major sites of poly(ADP-ribosyl)ation as Glu 255 , Asp 256 , and Glu 268 and then studied how expression of a p53 fusion protein in which these residues were changed to alanine (GFP-p53EDE/A) influenced the localization and transcriptional activity of the protein compared with expression of a wild-type GFP p53 fusion protein (GFP-WT p53) in mouse embryo fibroblasts (MEFs) deficient for p53. In p53 –/– MEFs, GFP-WT p53 was predominantly nuclear, whereas GFP-p53EDE/A was predominantly cytosolic, even when the cells were subjected to the DNA-damaging agent doxorubicin, which suggests that poly(ADP-ribosyl)ation is important for nuclear accumulation of p53. In response to DNA damage, cells expressing GFP-WT p53 showed increased abundance of p21, which is encoded by a p53-target gene, whereas cells expressing GFP-p53EDE/A did not. Consistent with PARP-1 as the enzyme responsible for the poly(ADP-ribosyl)ation of p53, MEFs deficient for PARP-1 showed decreased stability of p53 and failed to up-regulate p21 in response to DNA damage. In cells in which nuclear export was pharmacologically inhibited, the GFP-p53EDE/A mutant increased the abundance of p21 as effectively as WT p53, thus showing that poly(ADP-ribosyl)ation was not required for transactivation activity but that its effect on transactivation activity of p53 was due to decreased accumulation of p53 in the nucleus. The interaction of tagged Crm1, a nuclear export receptor, with tagged p53 or p53EDE/A was enhanced for the p53EDE/A mutant, suggesting that poly(ADP-ribosyl)ation interferes with the binding of p53 with Crm1. In vitro binding assays with poly(ADP-ribosyl)ated p53 and nonmodified p53 and Crm1 supported this interpretation. Thus, the authors propose a model whereby DNA damage promotes the hyperactivation of PARP-1, which targets p53, and poly(ADP-ribosyl)ated p53 then accumulates in the nucleus because it fails to interact with the nuclear export receptor Crm1. M. Kanai, K. Hanashiro, S.-H. Kim, S. Hanai, A. H. Boulares, M. Miwa, K. Fukasawa, Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation. Nat. Cell Biol. 9 , 1175-1183 (2007). [PubMed]

Inhibition of Crm1–p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.

Roles for CSN5 in control of p53/MDM2 activities

The 5th subunit of COP9 signalosome (CSN5, also known as Jab1 or COPS5) is implicated in regulating p53 activity and is overexpressed in various tumors. However, the precise roles of CSN5 in p53 network and tumorigenesis are not well characterized. Here we show that CSN5 is a critical regulator of both p53 and MDM2. We show that curcumin, an important inhibitor of CSN-associated kinases, can downregulate not only CSN5 but also MDM2, which results in p53 stabilization. Importantly, CSN5 interacts with p53. CSN5 expression leads to p53 degradation, facilitating MDM2-mediated p53 ubiquitination, and promoting p53 nuclear export. Additionally, CSN5 expression results in stabilization of MDM2 through reducing MDM2 self-ubiquitination and decelerating turnover rate of MDM2. Significantly, we further show that CSN5 antagonizes the transcriptional activity of p53. These results demonstrate that CSN5 is a pivotal regulator for both p53 and MDM2. Our studies may pave the way for targeting CSN5 for anti-cancer drug development.

Functional implications of caspase-mediated RhoGDI2 processing during apoptosis of HL60 and K562 leukemia cells

RhoGDI2, a cytosolic regulator of Rho GTPase, is cleaved during apoptosis in a caspase-3 dependent fashion. By using 2D-gel electrophoresis, mass spectrometry and Western blotting we investigate in this paper the functional consequences of RhoGDI2 processing. We can show that loss of the N-terminal 19 amino acids results in a shift of the isoelectric point of the truncated RhoGDI2 (NDelta19) to a more basic value due to the removal of 9 acidic amino acids from the N-terminus, which may be responsible for enhanced retention of the N-terminally truncated protein within the nuclear compartment. Fusion of the p53 nuclear export signaling sequence MFRELNEALELK to NDelta19 (NDelta19NES) abolished its apoptosis promoting properties, while overexpression of NDelta19 significantly increased the susceptibility to apoptosis induction by the proteasome inhibitor PSI and by staurosporine. These results suggest that cleavage of RhoGDI2 by caspase-3 is not a functionally irrelevant bystander effect of caspase activation during apoptosis, but rather expedites progression of the apoptotic process.

Mechanistic Studies of MDM2-mediated Ubiquitination in p53 Regulation

As a central regulator for cell cycle arrest, apoptosis, and cellular senescence, p53 requires multiple layers of regulatory control to ensure correct temporal and spatial functions. It is well accepted that Mdm2-mediated ubiquitination plays a crucial role in p53 regulation. In addition to proteasome-mediated degradation, ubiquitination of p53 by Mdm2 acts a key signal for its nuclear export. Nuclear export has previously been thought to require the disassociation of the p53 tetramer and exposure of the intrinsic nuclear export signal. To elucidate the molecular mechanism of degradation-independent repression on p53 by Mdm2, we have developed a two-step approach to purify ubiquitinated forms of p53 induced by Mdm2 from human cells. Surprisingly, however, we found that ubiquitination has no effect on the tetramerization/oligomerization of p53, arguing against this seemingly well accepted model. Moreover, nuclear export of p53 alone is not sufficient to completely abolish p53 activity. Ubiquitination-mediated repression of p53 by Mdm2 acts at least, in part, through inhibiting the sequence-specific DNA binding activity. Thus, our results have important implications regarding the mechanisms by which Mdm2 acts on p53.

Regulation of p53 Nuclear Export through Sequential Changes in Conformation and Ubiquitination

Wild-type p53 is a conformationally labile protein that undergoes nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 nuclear export by exposing or activating a nuclear export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation. The cytoplasmic localization of mutant p53s required the C-terminal NES and an intact ubiquitination pathway. Mutant p53 ubiquitination occurred at lysines in both the DNA-binding domain (DBD) and C terminus. Interestingly, Lys to Arg mutations that inhibited ubiquitination restored nuclear localization to mutant p53 but had no apparent effect on p53 conformation. Further studies revealed that wild-type p53, like mutant p53, is ubiquitinated by MDM2 in both the DBD and C terminus and that ubiquitination in both regions contributes to its nuclear export. MDM2 binding can induce a conformational change in wild-type p53, but this conformational change is insufficient to promote p53 nuclear export in the absence of MDM2 ubiquitination activity. Taken together, these results support a stepwise model for mutant and wild-type p53 nuclear export. In this model, the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination. The addition of ubiquitin to DBD and C-terminal lysines then promotes nuclear export via the C-terminal NES.

C-terminal modifications regulate MDM2 dissociation and nuclear export of p53

p53 functions to prevent malignant progression, in part by inhibiting proliferation or inducing the death of potential tumour cells. One of the most important regulators of p53 is MDM2, a RING domain E3 ligase that ubiquitinates p53, leading to both proteasomal degradation and relocation of p53 from the nucleus to the cytoplasm. Previous studies have suggested that although polyubiquitination is required for degradation, monoubiquitination of p53 is sufficient for nuclear export. Using a p53-ubiquitin fusion protein we show that ubiquitination contributes to two steps before export: exposure of a carboxy-terminal nuclear export sequence (NES), and dissociation of MDM2. Monoubiquitination can directly promote further modifications of p53 with ubiquitin-like proteins and MDM2 promotes the interaction of the SUMO E3 ligase PIASy with p53, enhancing both sumoylation and nuclear export. Our results suggest that modifications such as sumoylation can regulate the strength of the p53-MDM2 interaction and participate in driving the export of p53.

Two Populations of p27 Use Differential Kinetics to Phosphorylate Ser-10 and Thr-187 via Phosphatidylinositol 3-Kinase in Response to Fibroblast Growth Factor-2 Stimulation

The cyclin-dependent kinase inhibitor p27 regulates cell cycle progression. We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 on serine 10 (Ser-10) and threonine 187 (Thr-187) and whether the two phosphorylation sites were differentially regulated. FGF-2 stimulation dramatically increased p27 phosphorylation at Ser-10 and Thr-187 using differential kinetics, and the FGF-2-induced p27 phosphorylation was completely blocked at both sites by LY294002. We determined the physical and biochemical interaction of p27 with the Cdk2-cyclin E complex in response to FGF-2 stimulation. Maximal p27 binding to Cdk2-cyclin E occurred at 12 h; the maximal level of p27 phosphorylation at Thr-187 in the ternary complex was observed at 16 h; ubiquitination of the Thr-187-phosphorylated p27 (pp27Thr-187) was observed starting at 12 h and continuing up to 24 h. However, maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 h after FGF-2 stimulation; maximal export of Ser-10-phosphorylated p27 (pp27Ser-10) occurred 8 h after FGF-2 treatment, and pp27Ser-10 was simultaneously ubiquitinated. We further investigated which of the two phosphorylated p27 was involved in G(1)/S progression. LY294002 blocked 64% of the cell proliferation stimulated by FGF-2. Use of leptomycin B to block nuclear export of pp27Ser-10 greatly decreased the FGF-2-stimulated cell proliferation (44%), suggesting that phosphorylation of p27 at Ser-10 is the major mechanism for G(1)/S transition. Our results suggest that differential kinetics are observed in p27 phosphorylation at Ser-10 and Thr-187 and that pp27Thr-187 and pp27Ser-10 may represent two populations of p27 observed in the G(1) phase of the cell cycle.

Regulation of p53 Tetramerization and Nuclear Export by ARC

Regulation of p53 Tetramerization and Nuclear Export by ARC

Jab1 Induces the Cytoplasmic Localization and Degradation of p53 in Coordination with Hdm2

The biological mechanisms for maintaining the basal level of p53 in normal cells require nuclear exclusion and cytoplasmic degradation. Here, we showed that Jab1 facilitates p53 nuclear exclusion and its subsequent degradation in coordination with Hdm2. p53 was excluded from the nucleus in the presence of Jab1; this exclusion was prevented by leptomycin B treatment. Nuclear export of p53 was accompanied by a decrease in the levels of p53, as well as of its target proteins, which include p21 and Bax. Domain analyses of Jab1 showed that the N-terminal domain, 1-110, was capable of inducing cytoplasmic translocation of p53. Furthermore, 110-191 was required to facilitate the degradation of p53. Neither of these mutants incorporated into the CSN complex, indicating that Jab1 could affect the levels of p53 independent of intact CSN complex. Conversely, Jab1 was incapable of translocating and degrading two p53 mutants, W23S and 6KR, neither of which could be modified by Hdm2. Moreover, Jab1 did not affect the cellular localization or protein levels of p53 in p53 and Hdm2 double-null mouse embryo fibroblasts. We further observed that the ablation of endogenous Jab1 by small interfering RNA prevented Hdm2-mediated p53 nuclear exclusion. Under stressed conditions, which could sequester Hdm2 in its inactive state, Jab1 did not affect p53. Our studies implicate that Jab1 is required to remove post-translationally modified p53 and provide a novel target for p53-related cancer therapies.

P53 Stabilization and Transactivation by a von Hippel-Lindau Protein

von Hippel-Lindau (VHL) disease is a rare autosomal dominant cancer syndrome. Although hypoxia-inducible factor-alpha (HIFalpha) is a well-documented substrate of von Hippel-Lindau tumor suppressor protein (pVHL), it remains unclear whether the dysregulation of HIF is sufficient to account for de novo tumorigenesis in VHL-deleted cells. Here we found that pVHL directly associates with and stabilizes p53 by suppressing Mdm2-mediated ubiquitination and nuclear export of p53. Moreover, upon genotoxic stress, pVHL invoked an interaction between p53 and p300 and the acetylation of p53, which ultimately led to an increase in p53 transcriptional activity and p53-mediated cell cycle arrest and apoptosis. These results suggest that the tumor suppressor pVHL has an unexpected function to upregulate the tumor suppressor p53.

Nuclear Accumulations of p53 and Mdm2 are Accompanied by Reductions in c-Abl and p300 in Zinc-Depleted Human Hepatoblastoma Cells

The influence of zinc status on the expression of proteins known to be involved in the stability of p53, the human tumor suppressor gene product, was examined in hepatoblastoma (HepG2) cells. Cells were cultured in zinc-deficient (ZD0.2, ZD0.4), zinc normal (ZN), zinc adequate (ZA), or zinc-supplemented (ZS) medium, which contained 0.2, 0.4, 4, 16, or 32 microM zinc, respectively. Nuclear p53 levels were almost 100% and 40% higher in ZD0.2 and ZD0.4 cells, respectively, than in ZN cells. Mdm2 protein, which mediates p53 degradation, was 174% and 148% higher in the nucleus of ZD0.2 and ZD0.4 cells, respectively, than in ZN cells. In addition, the observed reductions of nuclear c-Abl in ZD0.2 and ZD0.4 cells to 50% and 60% of ZN cells, respectively, may be a cellular response attempting to normalize nuclear p53 accumulation because nuclear c-Abl is known to down-regulate ubiquitination and nuclear export of p53. Moreover, no changes in total cellular p53, Mdm2, and c-Abl or nuclear Mdmx were observed among the treatment groups. Furthermore, in ZD0.2 and ZD0.4 cells, the reduction in total and nuclear p300, which is known to complex with CREB-binding protein and Mdm2 in the nucleus for the generation of degradable polyubiquitinated form of p53, may have depressed the degradation pathway for p53 and Mdm2, and contributed to the nuclear accumulation of these proteins in ZD cells.

Activation of PKR promotes the nucleocytoplasmic export of p53

Wild type p53 accumulates in the nucleus upon different types of stress. We previously reported that ER stress downregulates p53. ER stress inhibits protein synthesis by inducing the phosphorylation of eIF2α. The eIF2α kinase PKR plays a major role in the antiviral response. We have shown that PKR activation can also be induced upon ER stress. Our goal was to understand the molecular mechanisms of p53 downregulation by PKR by means that induce eIF2α phosphorylation such as virus infection and pharmacological inducers of ER stress. We report that PKR activation upon ER stress or dsRNA promotes p53 downregulation. This decrease in p53 protein levels was independent on the eIF2α-mediated translational inhibition properties of PKR. We observe that PKR mediates the nuclear export of p53 while promoting its degradation through the 26S proteasome pathway. This downregulation of p53 can be rescued by inhibiting the protein degradation pathway with MG132, as well as the prevention of p53 nuclear export with leptomycin B (LMB) despite the robust activation of PKR and eIF2α phosphorylation. This study provides evidence that PKR plays a role other than the inhibition of protein synthesis. This could explain how some cancer cells, bearing wild type p53, that overexpress PKR may overcome the tumor suppressor effects of p53 by inducing its nuclear export and degradation. This work was supported by NCIC.

Charge Modification at Multiple C-terminal Lysine Residues Regulates p53 Oligomerization and Its Nucleus-Cytoplasm Trafficking

The basal level of the tumor suppressor p53 is regulated by MDM2-mediated ubiquitination at specific lysines, which leads to p53 nuclear export and degradation. Upon p53 activation, however, these lysines become acetylated by p300/CREB-binding protein. Here we have reported an unexpected finding that p300-mediated acetylation also regulates p53 subcellular localization and can promote cytoplasmic localization of p53. This activity is independent of MDM2 but requires a p53 nuclear export signal and acetylation of multiple lysines by p300. Mechanistically, we showed that conversion of a minimal four of these lysines to alanines but not arginines mimics p300-mediated p53 nuclear export, and these lysine-neutralizing mutations effectively prevent p53 tetramerization, thus exposing the oligomerization-regulated nuclear export signal. Our study suggested a threshold mechanism whereby the degree of acetylation regulates p53 nucleus-cytoplasm trafficking by neutralizing a lysine-dependent charge patch, which in turn, controls oligomerization-dependent p53 nuclear export.

A herbal medicine for the treatment of lung cancer

Lung cancer is the leading cause of cancer-related deaths throughout the world. Extracts of medicinal plants are believed to contain different chemopreventive or chemotherapeutic compounds. In this study, we determined the anti-cancer property of one of the traditional Indian medicine Rasagenthi Lehyam (RL) for the treatment of lung cancer. Two lung cancer cell lines (A-549 and H-460) and one normal bronchial epithelial (BEAS-2B) cell line were used to test the chemotherapeutic effect of RL. Out of five fractions of RL, chloroform fraction of RL (cRL) demonstrated a significant inhibition of cell proliferation and induction of apoptosis in A-549 and H-460 cells but not in normal BEAS-2B cells. The cRL fraction up-regulated the pro-apoptotic genes p53 and Bax and induced caspase-3 activation, and down-regulated the pro-survival gene Bcl-2 in both the lung cancer cell lines. Also, nuclear export of p53 was seen in cRL-treated lung cancer cells. In addition, cRL induced G2/M arrest of cell cycle and enhanced the radio-sensitivity of both the lung cancer cell lines. This study suggests that cRL may prove to be a potent anti-cancer agent that may be used for the treatment of lung cancer. However, further studies are required to bring cRL into the mainstream of medicine in the treatment of lung cancer.

Role of Serine 10 Phosphorylation in p27 Stabilization Revealed by Analysis of p27 Knock-in Mice Harboring a Serine 10 Mutation

The inhibition of cyclin-dependent kinase activity by p27 contributes to regulation of cell cycle progression. Serine 10 is the major phosphorylation site of p27, and its phosphorylation has been shown to affect the stability and nuclear export of p27 at the G0-G1 transition in transfected cultured cells. To investigate the physiological relevance of p27 phosphorylation on Ser10, we generated p27 "knock-in" mice that harbor an S10A mutation in this protein. Mice homozygous for the mutation (p27(S10A/S10A) mice) were normal in body size, but the abundance of p27 was decreased in many organs, including brain, thymus, spleen, and testis. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27(S10A/S10A) mice compared with that in wild-type cells, whereas p27 stability in S phase was similar in cells of the two genotypes. The degradation of p27 in cells of the mutant mice at G0 phase was prevented by a proteasome inhibitor. These data indicate that the physiological role of p27 phosphorylation on Ser10 is to stabilize the protein in G0 phase. Unexpectedly, the nuclear export of p27 at the G0-G1 transition occurred normally in p27(S10A/S10A) mouse embryonic fibroblasts, indicating that phosphorylation of Ser10 is dispensable for this process.