The transcriptional co-activator PCAF regulates cdk2 activity.
ABSTRACT: Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G(2)/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G(2)/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.
Project description:The activity of p53 as a tumor suppressor primarily depends on its ability to transactivate specific target genes in response to genotoxic and other potentially mutagenic stresses. Several histone acetyl transferases (HATs), including p300, CBP, PCAF and GCN5 have been implicated in the activation of p53-dependent transcription of the cyclin-dependent kinase (cdk) inhibitor p21 as well as other target genes. Here we show that PCAF, but not CBP or p300, is a critical regulator of p53-dependent p21 expression in response to multiple p53-activating stresses. PCAF was required for the transcriptional activation of p21 in response to exogenous p53 in p53-null cells, nutlin-3, DNA damaging agents and p14(ARF) expression, suggesting a broad requirement for PCAF in p53 signaling to p21 after stress. Importantly, cells lacking PCAF failed to undergo cell cycle arrest in response to nutlin-3 treatment or p14(ARF) expression, consistent with a physiologically important role for PCAF in this p53 function. Surprisingly, the role for PCAF in induction of p21 was independent of p53 lysine 320 acetylation, a previously suggested target of PCAF-mediated acetylation. Though p21 promoter occupancy by p53 was not altered by PCAF knockdown, activation of p21 transcription required an intact PCAF HAT domain, and induction of chromatin marks acetyl-H3K9 and acetyl-H3K14 at the p21 promoter by p53 was dependent upon physiologic levels of PCAF. Together, our experiments indicate that PCAF is required for stress-responsive histone 3 acetylation at the p21 promoter, p53-directed transcription of p21 and the resultant growth arrest.
Project description:Sirtuin 7 (SIRT7), an NAD+-dependent deacetylase, plays vital roles in energy sensing, but the underlying mechanisms of action remain less clear. Here, we report that SIRT7 is required for p53-dependent cell-cycle arrest during glucose deprivation. We show that SIRT7 directly interacts with p300/CBP-associated factor (PCAF) and the affinity for this interaction increases during glucose deprivation. Upon binding, SIRT7 deacetylates PCAF at lysine 720 (K720), which augments PCAF binding to murine double minute (MDM2), the p53 E3 ubiquitin ligase, leading to accelerated MDM2 degradation. This effect results in upregulated expression of the cell-cycle inhibitor, p21Waf1/Cip1, which further leads to cell-cycle arrest and decreased cell viability. These data highlight the importance of the SIRT7-PCAF interaction in regulating p53 activity and cell-cycle progression during conditions of glucose deprivation. This axis may represent a new avenue to design effective therapeutics based on tumor starvation.
Project description:Oncogene-induced senescence represents a key tumor suppressive mechanism. Here, we show that Ras oncogene-induced senescence can be mediated by the recently identified haploinsufficient tumor suppressor apoptosis-stimulating protein of p53 (ASPP) 2 through a novel and p53/p19(Arf)/p21(waf1/cip1)-independent pathway. ASPP2 suppresses Ras-induced small ubiquitin-like modifier (SUMO)-modified nuclear cyclin D1 and inhibits retinoblastoma protein (Rb) phosphorylation. The lysine residue, K33, of cyclin D1 is a key site for this newly identified regulation. In agreement with the fact that its nuclear localization is required for its oncogenic activity, we show that nuclear cyclin D1 is far more potent than wild-type (WT) cyclin D1 in bypassing Ras-induced senescence. Thus, this study identifies SUMO modification as a positive regulator of nuclear cyclin D1, and reveals a new way by which cell cycle entry and senescence are regulated.
Project description:Gastric cancer (GC) is a leading cause of cancer-related death worldwide and the pathogenesis of GC remains largely unknown. Here, we demonstrate a novel mechanism by which P300/CBP associating factor (PCAF) acts as a tumor suppressor in GC cells. We showed that both PCAF mRNA and protein were downregulated in GC cells, and that this downregulation correlated with poor survival. Meanwhile, the interaction between human anion exchanger 1 (AE1) and p16 is a key event in GC development. We found that PCAF inhibited GC growth by interacting with AE1 and p16 to promote ubiquitin-mediated degradation of AE1 and p16 upregulation and translocation into the nucleus. Binding of nuclear p16 to CDK4 prevented the CDK4-Cyclin D1 interaction to inhibit GC proliferation. Furthermore, reduced PCAF levels in GC cells were associated with intracellular alkalinization and decreased immunity. Together these results suggest that PCAF acts as a GC suppressor through a novel PCAF-p16-CDK4 axis. The downregulation of PCAF expression in GC cells that follows intracellular alkalinization and decreased immune response, indicates that GC therapies should focus on restoring PCAF levels.
Project description:KAT2B (PCAF) belongs to GNAT family of lysine acetyltransferases (KAT), which specifically acetylates histone H3K9 residue and several nonhistone proteins. PCAF is also a transcription coactivator. Due to the lack of PCAF, KAT specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here we report that a hydroxy benzoquinone class of natural compound, embelin, specifically inhibits H3Lys9 acetylation in mice and recombinant PCAF mediated acetylation with near complete specificity in vitro. Further, using embelin, we have identified the PCAF regulated gene networks during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation, apart from regulation through MyoD acetylation. C2C12 cells were treated with either DMSO or 10uM embelin in GM/DM for either 24 or 48h.
Project description:P300/CBP-associated factor (PCAF), a histone acetyltransferase (HAT), has been found to regulate numerous cell signaling pathways controlling cell fate by acetylating both histone and non-histone proteins. We previously reported that PCAF upregulates cell apoptosis by inactivating Serine/Threonine Protein Kinase 1 (AKT1) signaling and consequently inhibits hepatocellular carcinoma (HCC) cell growth. Here, we show that PCAF can directly acetylate cytoplasmic GLI1 protein at lysine 518, preventing its nuclear translocation and promoter occupancy, and consequently suppressing Hedgehog (Hh) signaling in HCC. Further, our results show that GLI1 can increase Bcl-2 expression and downregulate BAX. Interestingly, forced expression of PCAF reduced Bcl-2 expression, upregulated BAX and repressed cell apoptosis. Further, we provide evidence that knockdown of GLI1 abrogates the inhibitory effect of PCAF on the growth of HCC in vitro. PCAF was also found to sensitize HCC cells to 5-fluorouracil (5-FU) treatment by regulating GLI1/Bcl-2/BAX axis-dependent apoptosis. In vivo experiments also confirmed the regulatory effect of PCAF on the GLI1/Bcl-2/BAX axis and its synergistic antitumor effects with 5-FU. Gene expression microarray studies showed that PCAF was downregulated in HCC tissues compared with adjacent liver tissues and that PCAF expression was significantly associated with longer overall survival and recurrence-free survival after surgery. Together, these results show that PCAF can induce cell apoptosis by modulating a GLI1/Bcl-2/BAX axis that in turn suppresses HCC progression, and suggest that 5-FU may exert a stronger anti-tumor effect in patients with PCAF expression in HCC tumors.
Project description:Histone acetylation plays crucial roles in transcriptional regulation and chromatin organization. Viral RNA of the influenza virus interacts with its nucleoprotein (NP), whose function corresponds to that of eukaryotic histones. NP regulates viral replication and has been shown to undergo acetylation by the cAMP-response element (CRE)-binding protein (CBP) from the host. However, whether NP is the target of other host acetyltransferases is unknown. Here, we show that influenza virus NP undergoes acetylation by the two host acetyltransferases GCN5 and P300/CBP-associated factor (PCAF) and that this modification affects viral polymerase activities. Western blot analysis with anti-acetyl-lysine antibody on cultured A549 human lung adenocarcinoma epithelial cells infected with different influenza virus strains indicated acetylation of the viral NP. A series of biochemical analyses disclosed that the host lysine acetyltransferases GCN5 and PCAF acetylate NP in vitro MS experiments identified three lysine residues as acetylation targets in the host cells and suggested that Lys-31 and Lys-90 are acetylated by PCAF and GCN5, respectively. RNAi-mediated silencing of GCN5 and PCAF did not change acetylation levels of NP. However, interestingly, viral polymerase activities were increased by the PCAF silencing and were decreased by the GCN5 silencing, suggesting that acetylation of the Lys-31 and Lys-90 residues has opposing effects on viral replication. Our findings suggest that epigenetic control of NP via acetylation by host acetyltransferases contributes to regulation of polymerase activity in the influenza A virus.
Project description:Genetic P300/CBP-associated factor (PCAF) variation affects restenosis-risk in patients. PCAF has lysine acetyltransferase activity and promotes nuclear factor kappa-beta (NF?B)-mediated inflammation, which drives post-interventional intimal hyperplasia development. We studied the contributing role of PCAF in post-interventional intimal hyperplasia.PCAF contribution to inflammation and intimal hyperplasia was assessed in leukocytes, macrophages and vascular smooth muscle cells (vSMCs) in vitro and in a mouse model for intimal hyperplasia, in which a cuff is placed around the femoral artery. PCAF deficiency downregulate CCL2, IL-6 and TNF-alpha expression, as demonstrated on cultured vSMCs, leukocytes and macrophages. PCAF KO mice showed a 71.8% reduction of vSMC-rich intimal hyperplasia, a 73.4% reduction of intima/media ratio and a 63.7% reduction of luminal stenosis after femoral artery cuff placement compared to wild type (WT) mice. The association of PCAF and vascular inflammation was further investigated using the potent natural PCAF inhibitor garcinol. Garcinol treatment reduced CCL2 and TNF-alpha expression, as demonstrated on cultured vSMCs and leukocytes. To assess the effect of garcinol treatment on vascular inflammation we used hypercholesterolemic ApoE*3-Leiden mice. After cuff placement, garcinol treatment resulted in reduced arterial leukocyte and macrophage adherence and infiltration after three days compared to untreated animals.These results identify a vital role for the lysine acetyltransferase PCAF in the regulation of local inflammation after arterial injury and likely the subsequent vSMC proliferation, responsible for intimal hyperplasia.
Project description:KAT2B (PCAF) belongs to GNAT family of lysine acetyltransferases (KAT), which specifically acetylates histone H3K9 residue and several nonhistone proteins. PCAF is also a transcription coactivator. Due to the lack of PCAF, KAT specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here we report that a hydroxy benzoquinone class of natural compound, embelin, specifically inhibits H3Lys9 acetylation in mice and recombinant PCAF mediated acetylation with near complete specificity in vitro. Further, using embelin, we have identified the PCAF regulated gene networks during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation, apart from regulation through MyoD acetylation. HEK 293T cells were treated with DMSO (solvent control) and 20uM embelin for 24h. Cells were harvested and total RNA was extracted by Trizol method. RNA was cleaned up using RNeasy MinElute Cleanup Kit (Qiagen) and reverse transcribed, labelled and subsequently hybridized to Agilent human 4X 44k array slide.
Project description:The binding between the HIV-1 trans-activator of transcription (Tat) and p300/(CREB-binding protein)-associated factor (PCAF) bromodomain is a crucial step in the HIV-1 life cycle. However, the structure of the full length acetylated Tat bound to PCAF has not been yet determined experimentally. Acetylation of Tat residues can play a critical role in enhancing HIV-1 transcriptional activation. Here, we have combined a fully flexible protein-protein docking approach with molecular dynamics simulations to predict the structural determinants of the complex for the common HIV-1BRU variant. This model reproduces all the crucial contacts between the Tat peptide 46SYGR(AcK)KRRQRC56 and the PCAF bromodomain previously reported by NMR spectroscopy. Additionally, inclusion of the entire Tat protein results in additional contact points at the protein-protein interface. The model is consistent with the available experimental data reported and adds novel information to our previous structural predictions of the PCAF bromodomain in complex with the rare HIVZ2 variant, which was obtained with a less accurate computational method. This improved characterization of Tat.PCAF bromodomain binding may help in defining the structural determinants of other protein interactions involving lysine acetylation.