Forkhead box class O (FOXO) proteins are transcription factors that function downstream of the PTEN tumor suppressor and directly control the expression of genes involved in apoptosis, cell cycle progression, and stress responses. In the present study, we show that FOXO1 interacts with four and a half LIM 2 (FHL2) in prostate cancer cells. This interaction occurred in the nucleus and was enhanced by lysophosphatic acid. FHL2 decreased the transcriptional activity of FOXO1 and the expression of known FOXO target genes and inhibited FOXO1-induced apoptosis. Interestingly, SIRT1, a mammalian homolog of yeast Sir2, bound to and deacetylated FOXO1 and inhibited its transcriptional activity. FHL2 enhanced the interaction of FOXO1 and SIRT1 and the deacetylation of FOXO1 by Sirtuin-1 (SIRT1). Overall, our data show that FHL2 inhibits FOXO1 activity in prostate cancer cells by promoting the deacetylation of FOXO1 by SIRT1.
SIRT1 is the closest mammalian homologue of yeast SIR2, an important ageing regulator that prolongs lifespan in response to caloric restriction. Despite its importance, the mechanisms that regulate SIRT1 activity are unclear. Our study identifies a novel post-translational modification of SIRT1, namely sumoylation at Lys 734. In vitro sumoylation of SIRT1 increased its deacetylase activity. Conversely, mutation of SIRT1 at Lys 734 or desumoylation by SENP1, a nuclear desumoylase, reduced its deacetylase activity. Stress-inducing agents promoted the association of SIRT1 with SENP1 and cells depleted of SENP1 (but not of SENP1 and SIRT1) were more resistant to stress-induced apoptosis than control cells. We suggest that stress-inducing agents counteract the anti-apoptotic activity of SIRT1 by recruiting SENP1 to SIRT1, which results in the desumoylation and inactivation of SIRT1 and the consequent acetylation and activation of apoptotic proteins.Sirtuins (SIRTs) are mammalian NAD + -dependent histone deacetylases (HDACs) 1 . Of the seven SIRTs, SIRT1 is the closest homologue of yeast SIR2 (ref. 2,3), which has important roles in diverse cellular processes, including transcriptional silencing 4 , rDNA recombination 5 , glucose metabolism and energy homeostasis 6 , DNA repair and cell survival [7][8][9] . Because of its dependency on NAD + , the activity of SIRT1 is regulated by the NAD + :NADH ratio and, is therefore sensitive to the status of redox and cellular metabolism. Author Contributions: Y.Y. designed (with W.B.) and performed (with W.F.) the included studies. J.C., X.Z. and K.B. contributed scientifically to the revision. The manuscript was written by W.B., edited by N.O. and S.V.N. and read by all authors. The senior author (W.B.) designed the project, helped with the analyses and the organization of the data, and provided the financial support. All authors have discussed the data and had scientific input into the manuscript.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Similar to SIR2 in lower organisms, SIRT1 is potentially a nutrient sensor that regulates the lifespan of mammals in response to caloric restriction or nutrient starvation [9][10][11][12][13] . By deacetylating and inactivating apoptotic proteins (such as the p53 tumour suppressor 14-17 ), SIRT1 also protects cancer cells from apoptosis induced by DNA damage. The expression of SIRT1 is known to be controlled at both transcriptional and post-transcriptional levels, but post-translational mechanisms that regulate SIRT1 activity have yet to be defined.Sumoylation is a reversible post-translational modification in which proteins termed small ubiquitin-related modifiers (SUMOs) are covalently linked to lysine residues of target proteins. Similar to ubiquitination, sumoylation is catalysed by a three-step enzymatic reaction involving an E1 activating enzyme, an E2 conjugating enzyme and E3 ligases. The reverse reaction is catalysed by SENP desumoylases, a famil...
Cortactin binds F-actin and promotes cell migration. We showed earlier that cortactin is acetylated. Here, we identify SIRT1 (a class III histone deacetylase) as a cortactin deacetylase and p300 as a cortactin acetylase. We show that SIRT1 deacetylates cortactin in vivo and in vitro and that the SIRT1 inhibitor EX-527 increases amounts of acetylated cortactin in ovarian cancer cells. We also show that p300 acetylates cortactin in vivo and that cells lacking or depleted of p300 express lessacetylated cortactin than do control cells. Deletion analysis mapped the SIRT1-binding domain of cortactin to its repeat region, which also binds F-actin. Mouse embryo fibroblasts (MEFs) lacking sir2a (the mouse homolog of SIRT1) migrated more slowly than did wildtype cells. The expression of SIRT1 in sir2a-null cells restored migratory capacity, as did expression of a deacetylation-mimetic mutant of cortactin. SIRT1 and cortactin were more abundant in breast tumor tissue than in their normal counterparts, whereas SIRT1 expression inversely correlates with the ratio of acetylation cortactin versus total cortactin. These data suggest that deacetylation of cortactin is associated with high levels of SIRT1 and tumorigenesis. Finally, breast and ovarian cancer cell lines expressing an acetylation mimetic mutant of cortactin are less motile than that of control cells, whereas cells expressing the deacetylation mimetic mutant of cortactin migrate faster than that of control cells in Transwell migration assays. In summary, our results suggest that cortactin is a novel substrate for SIRT1 and p300 and, for the first time, a possible role for SIRT1 in cell motility through deacetylation of cortactin.
Estrogen receptors are phosphoproteins which can be activated by ligands, kinase activators, or phosphatase inhibitors. Our previous study showed that p38 mitogen-activated protein kinase was involved in estrogen receptor activation by estrogens and MEKK1. Here, we report estrogen receptor-dependent p38 activation by estrogens in endometrial adenocarcinoma cells and in vitro and in vivo phosphorylation of the estrogen receptor ␣ mediated through p38. The phosphorylation site was identified as threonine-311 (Thr 311 ), located in helix 1 of the hormone-binding domain. The mutation of threonine-311 to alanine did not affect estrogen binding of the receptor but compromised its interaction with coactivators. Suppression of p38 activity or mutation of the site inhibited the estrogen-induced receptor nuclear localization as well as its transcriptional activation by estrogens and MEKK1. The inhibition of the p38 signal pathway by a specific chemical inhibitor blocked the biological activities of estrogens in regulating endogenous gene expression as well as endometrial cancer cell growth. Our studies demonstrate the role of estrogen receptor phosphorylation induced by the natural ligand in estrogen receptor's cellular distribution and its significant contribution to the growthstimulating activity of estrogens in endometrial cancer cells.Estrogens are female sex steroid hormones that control development, maintenance, and regulation of the female reproductive phenotype and behavior. They also stimulate the growth of normal and transformed epithelial cells of the female reproductive systems. The effect of estrogens is mediated through both estrogen receptors ␣ and  (ER␣ and ER), which belong to the nuclear hormone receptor superfamily, a group of ligand-regulated, zinc finger-containing transcription factors (11,40). The superfamily includes not only receptors for classical steroids such as estrogens, androgens, progesterones, and glucocorticoids, but also receptors for steroid analogues and nonsteroid ligands such as vitamin D, thyroid, and retinoic acids, as well as orphan receptors for which the ligand is unknown.Unlike the thyroid and vitamin D receptors, which reside in the nucleus in the absence of ligands, receptors for classical steroids such as ER␣ are targeted to the nucleus after binding with estrogens or selective estrogen receptor modulators such as tamoxifen. In contrast, the pure ER␣ antagonist ICI 182,780 directs the ER␣ to the cytoplasm (6). Of importance in this respect are three clusters of basic amino acids, similar to the nuclear localization signals found in simian virus 40 large T antigen, which were identified in the DNA binding domain and the hinge region of ER␣ (44). The nuclear localization signals are constitutively active and do not seem to explain the estrogen effect on the ER␣ nuclear localization (44). It is therefore possible that estrogen-induced targeting of ER␣ to the nucleus is mediated through other mechanisms.Studies in recent years have provided increasing evidence that nuclear loc...
SUMMARY MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSβ) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.
Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasomemediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.FOXO (forkhead O) proteins belong to the family of forkhead transcriptional factors, which are characterized by a conserved DNA binding domain termed the "Forkhead box" (1). Mammalian FOXO factors include FOXO1 (previously known as FKHR), FOXO3A (previously known as FKHRL1), FOXO4 (previously known as AFX), and FOXO6. These factors control the expression of a variety of genes that regulate essential cellular processes, such as cell cycle (2-4), apoptosis (5), oxidative stress (6, 7), atrophy (8), energy homeostasis, and glucose metabolism (9, 10). Whole organism studies in worms and flies show that FOXO factors have conserved ability to increase the organismal longevity (11). Genetic and functional analysis identifies FOXO1 as a tumor suppressor in the prostate (12). Knock-out studies show that mammalian FOXO factors act redundantly to suppress tumorigenesis in a lineage-specific fashion (13) and to maintain the long term regenerative potential of hematopoietic stem cells by regulating genes involved in the cellular response to physiological oxidative stresses (14).The transcription of FOXO factors is regulated by posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. Multiple kinases, including AKT (15, 16), serum-and glucocorticoid-induced kinase (17), casein kinase 1 (18), mammalian Ste20-like kinase 1 (19), I B kinase (20), and cyclin-dependent kinase 2 (21), catalyze FOXO phosphorylation and often promote FOXO nuclear exportation. In response to insulin and growth factors, FOXO1 and FOXO3A are ubiquitinated and degraded by the proteasome pathway after phosphorylation at known AKT sites (15,22,23). Acetyltransferases, p300 (24) and CBP (25), and SIRT1 deacetylase (26, 27) regulate the activity of FOXO through acetylation/deacetylation. The role of FOXO acetylation is controversial, but it could affect their nuclear retention (28), phosphorylation (25), and ubiquitination-medi...
The maintenance of telomere length is required for continued cell proliferation, and ϳ85-90% of human cancers, including ovarian epithelial cancers (OCa), show high activity of telomerase. In the present study we report that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 Vitamin D is a lipophilic hormone, and its effects are mediated by the vitamin D receptor (VDR) 1 (1), which is a member of the steroid/thyroid nuclear receptor superfamily. This superfamily includes receptors for steroids such as progesterone, androgens, estrogens, glucocorticoids and mineralocorticoids, receptors for non-steroid hormones like vitamin D, thyroid hormones, all-trans-retinoic acid, and 9-cis-retinoic acid as well as orphan receptors for which the ligand is unknown. The VDR acts as a ligand-inducible transcription factor that in most cases forms heterodimers with the retinoid X receptor (RXR). The activated receptors bind vitamin D response elements (VDREs) to regulate the expression of the target genes through activation or repression of transcription.In addition to classic effects on calcium homeostasis, bone density, and mineral metabolism, the active metabolite of vitamin D, 1,25(OH) 2 VD 3 , modulates cellular proliferation (2, 3), differentiation (4), and apoptosis (5) of both normal and malignant cells. Our recent studies have shown that the growth of multiple OCa cell lines is suppressed by 1,25(OH) 2 VD 3 (6), suggesting that active vitamin D compounds are potential therapeutic agents for OCa treatment and prevention. Molecular analyses have identified GADD45 as a primary target gene that mediates the effect of 1,25(OH) 2 VD 3 on G 2 /M arrest (7) and the p27 CDK inhibitor as the mediator for the arrest at G 1 /S checkpoint (6).The ends of chromosomes, telomeres, are subject to progressive shortening in normal somatic cells, leading ultimately to irreversible growth arrest, known as senescence (8). In contrast, the telomere length in cancer cells is stabilized by telomerase, an enzyme that catalyzes the synthesis of telomeric DNA repeats. Telomerase is a ribonucleoprotein complex containing three essential components, the telomerase RNA, which contains a sequence complementary to the telomeric TTAGGG repeat, the catalytic subunit, telomerase reverse transcriptase, which catalyzes the reverse transcription of TTAGGG repeats in the telomerase RNA, and the telomerase-associated protein 1, which helps the reverse transcription. Whereas all human somatic cells express the telomerase RNA constitutively (9, 10), the level of hTERT is increased in most human cancers, and its expression determines the cellular activity of telomerase (11). Thus, the expression of the hTERT gene appears to be the rate-limiting factor for telomerase activity in human cancer cells.The reactivation of telomerase has been shown to be one of the three events required to transform a normal human epithelial cell into a cancer cell (12). An anti-apoptotic function of telomerase has been described in human fibroblasts and neuronal cells (13)(14)(15). Studies in epid...
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