Accurate chromosome segregation during cell division maintains genomic integrity and requires the proper establishment of kinetochore-microtubule attachment in mitosis. As a key regulator of mitosis, Polo-like kinase 1 (Plk1) is essential for this attachment process, but the molecular mechanism remains elusive. Here we identify Sgt1, a cochaperone for Hsp90, as a novel Plk1 substrate during mitosis. We show that Sgt1 dynamically localizes at the kinetochores, which lack microtubule attachments during prometaphase. Plk1 is required for the kinetochore localization of Sgt1 and phosphorylates serine 331 of Sgt1 at the kinetochores. This phosphorylation event enhances the association of the Hsp90-Sgt1 chaperone with the MIS12 complex to stabilize this complex at the kinetochores and thus coordinates the recruitment of the NDC80 complex to form efficient microtubule-binding sites. Disruption of Sgt1 phosphorylation reduces the MIS12 and NDC80 complexes at the kinetochores, impairs stable microtubule attachment, and eventually results in chromosome misalignment to delay the anaphase onset. Our results demonstrate a mechanism for Plk1 in promoting kinetochore-microtubule attachment to ensure chromosome stability.
Glycogen synthase kinase 3β (GSK-3β) represses cell cycle progression by directly phosphorylating cyclin D1 and indirectly regulating cyclin D1 transcription by inhibiting Wnt signaling. Recently, we reported that the Epm2a-encoded laforin is a GSK-3β phosphatase and a tumor suppressor. The cellular mechanism for its tumor suppression remains unknown. Using ex vivo thymocytes and primary embryonic fibroblasts from Epm2a−/− mice, we show here a general function of laforin in the cell cycle regulation and repression of cyclin D1 expression. Moreover, targeted mutation of Epm2a increased the phosphorylation of Ser9 on GSK-3β while having no effect on the phosphorylation of Ser21 on GSK-3α. In the GSK-3β+/+ but not the GSK-3β−/− cells, Epm2a small interfering RNA significantly enhanced cell growth. Consistent with an increased level of cyclin D1, the phosphorylation of retinoblastoma protein (Rb) and the levels of Rb-E2F-regulated genes cyclin A, cyclin E, MCM3, and PCNA are also elevated. Inhibitors of GSK-3β selectively increased the cell growth of Epm2a+/+ but not of Epm2a−/− cells. Taken together, our data demonstrate that laforin is a selective phosphatase for GSK-3β and regulates cell cycle progression by GSK-3β-dependent mechanisms. These data provide a cellular basis for the tumor suppression activity of laforin.
Hydrogen sulfide (H2S) is the third endogenous signaling gasotransmitter, following nitric oxide and carbon monoxide. It is physiologically generated by cystathionine-γ-lyase, cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase. H2S has been gaining increasing attention as an important endogenous signaling molecule because of its significant effects on the cardiovascular and nervous systems. Substantial evidence shows that H2S is involved in aging by inhibiting free-radical reactions, activating SIRT1, and probably interacting with the age-related gene Klotho. Moreover, H2S has been shown to have therapeutic potential in age-associated diseases. This article provides an overview of the physiological functions and effects of H2S in aging and age-associated diseases, and proposes the potential health and therapeutic benefits of H2S.
Polo-like kinase 1 (Plk1) plays pivotal roles in mitosis; however, little is known about its function in S phase. In this study, we show that inhibition of Plk1 impairs DNA replication and results in slow S-phase progression in cultured cancer cells. We have identified origin recognition complex 2 (Orc2), a member of the DNA replication machinery, as a Plk1 substrate and have shown that Plk1 phosphorylates Orc2 at Ser188 in vitro and in vivo. Furthermore, Orc2-S188 phosphorylation is enhanced when DNA replication is under challenge induced by ultraviolet, hydroxyurea, gemcitabine, or aphidicolin treatment. Cells expressing the unphosphorylatable mutant (S188A) of Orc2 had defects in DNA synthesis under stress, suggesting that this phosphorylation event is critical to maintain DNA replication under stress. To dissect the mechanism pertinent to this observation, we showed that Orc2-S188 phosphorylation associates with DNA replication origin and that cells expressing Orc2-S188A mutant fail to maintain the functional pre-replicative complex (pre-RC) under DNA replication stress. Furthermore, the intra-S-phase checkpoint is activated in Orc2-S188A-expressing cells to cause delay of S-phase progress. Our study suggests a novel role of Plk1 in facilitating DNA replication under conditions of stress to maintain genomic integrity.
Mitochondria are highly dynamic organelles that play multiple roles in cells. How mitochondria cooperatively modulate embryonic stem (ES) cell function during development is not fully understood. Global disruption of Ptpmt1, a mitochondrial Pten-like phosphatidylinositol phosphate (PIP) phosphatase, resulted in developmental arrest and postimplantation lethality. Ptpmt1−/− blastocysts failed to outgrow, and inner-cell-mass cells failed to thrive. Depletion of Ptpmt1 in conditional knockout ES cells decreased proliferation without affecting energy homeostasis or cell survival. Differentiation of Ptpmt1-depleted ES cells was essentially blocked. This was accompanied by upregulation of cyclin-dependent kinase inhibitors and a significant cell cycle delay. Reintroduction of wild-type but not of catalytically deficient Ptpmt1 C132S or truncated Ptpmt1 lacking the mitochondrial localization signal restored the differentiation capabilities of Ptpmt1 knockout ES cells. Intriguingly, Ptpmt1 is specifically important for stem cells, as ablation of Ptpmt1 in differentiated embryonic fibroblasts did not disturb cellular function. Further analyses demonstrated that oxygen consumption of Ptpmt1-depleted cells was decreased, while glycolysis was concomitantly enhanced. In addition, mitochondrial fusion/dynamics were compromised in Ptpmt1 knockout cells due to accumulation of PIPs. These studies, while establishing a crucial role for Ptpmt1 phosphatase in embryogenesis, reveal a mitochondrial metabolic stress-activated checkpoint in the control of ES cell differentiation.
A new class of inflammatory CD4+ T cells that produce interleukin-17 (IL-17) (termed Th17) has been identified, which plays a critical role in numerous inflammatory conditions and autoimmune diseases. The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has a direct repressive effect on the expression of IL-17A in both human and mouse T cells. In vivo treatment of mice with ongoing experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis) diminishes paralysis and progression of the disease and reduces IL-17A-secreting CD4+ T cells in the periphery and central nervous system (CNS). The mechanism of 1,25(OH)2D3 repression of IL-17A expression was found to be transcriptional repression, mediated by the vitamin D receptor (VDR). Transcription assays, gel shifting, and chromatin immunoprecipitation (ChIP) assays indicate that the negative effect of 1,25(OH)2D3 on IL-17A involves blocking of nuclear factor for activated T cells (NFAT), recruitment of histone deacetylase (HDAC), sequestration of Runt-related transcription factor 1 (Runx1) by 1,25(OH)2D3/VDR, and a direct effect of 1,25(OH)2D3 on induction of Foxp3. Our results describe novel mechanisms and new concepts with regard to vitamin D and the immune system and suggest therapeutic targets for the control of autoimmune diseases.
Drosophila microRNAs (miRNAs) and small interfering RNAs (siRNAs) are generally produced by different Dicer enzymes (Dcr-1 and Dcr-2) and sorted to functionally distinct Argonaute effectors (AGO1 and AGO2). However, there is cross talk between these pathways, as highlighted by the recognition that Drosophila miRNA* strands (the partner strands of mature miRNAs) are generated by Dcr-1 but are preferentially sorted to AGO2. Here, we show that a component of the siRNA loading complex, R2D2, is essential both to load endogenously encoded siRNAs (endo-siRNAs) into AGO2 and to prevent endo-siRNAs from binding to AGO1. Northern blot analysis and deep sequencing showed that in the r2d2 mutant, all classes of endo-siRNAs were unable to load AGO2 and instead accumulated in the AGO1 complex. Such redirection was specific to endo-siRNAs and was not observed with miRNA* strands. We observed functional consequences of altered sorting in RNA interference (RNAi) mutants, since endo-siRNAs generated from cis-natural antisense transcripts (cis-NAT-siRNA) exhibited evidence for biased maturation as single strands in AGO1 according to thermodynamic asymmetry and a hairpin-derived endo-siRNA formed cleavage-competent complexes with AGO1 upon mutation of r2d2. Finally, we demonstrated a direct role for the R2D2/Dcr-2 heterodimer in sensing central mismatch positions that direct miRNA* strands to AGO2. Together, these data reveal new roles of R2D2 in organizing small RNA networks in Drosophila.
Cwc22 was previously identified to associate with the pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex [NTC]) involved in spliceosome activation. We show here that Cwc22 is required for pre-mRNA splicing both in vivo and in vitro but is neither tightly associated with the NTC nor required for spliceosome activation. Cwc22 is associated with the spliceosome prior to catalytic steps and remains associated throughout the reaction. The stable association of Cwc22 with the spliceosome requires the presence of the NTC but is independent of Prp2. Although Cwc22 is not required for the recruitment of Prp2 to the spliceosome, it is essential for the function of Prp2 in promoting the release of the U2 components SF3a and SF3b. In the absence of Cwc22, Prp2 can bind to the spliceosome but is dissociated upon ATP hydrolysis without promoting the release of SF3a/b. Thus, Cwc22 represents a novel ATP-dependent step one factor besides Prp2 and Spp2 and has a distinct role from that of Spp2 in mediating the function of Prp2.
The Mohawk homeobox (Mkx) gene encodes a new atypical homeodomain-containing protein with transcriptional repressor activity. Mkx mRNA exhibited dynamic expression patterns during development of the palate, somite, kidney, and testis, suggesting that it may be an important regulator of multiple developmental processes. To investigate the roles of Mkx in organogenesis, we generated mice carrying a null mutation in this gene. Mkx−/− mice survive postnatally and exhibit a unique wavy-tail phenotype. Close examination revealed that the mutant mice had smaller tendons than wild-type littermates and that the rapid postnatal growth of collagen fibrils in tendons was disrupted in Mkx−/− mice. Defects in tendon development were detected in the mutant mouse embryos as early as embryonic day 16.5 (E16.5). Although collagen fibril assembly initially appeared normal, the tendons of Mkx−/− embryos expressed significantly reduced amounts of collagen I, fibromodulin, and tenomodulin in comparison with control littermates. We found that Mkx mRNA was strongly expressed in differentiating tendon cells during embryogenesis and in the tendon sheath cells in postnatal stages. In addition to defects in tendon collagen fibrillogenesis, Mkx−/− mutant mice exhibited abnormal tendon sheaths. These results identify Mkx as an important regulator of tendon development.
Virus infection induces host antiviral responses, including induction of type I interferons. Transcription factor interferon regulatory factor 3 (IRF3) plays a pivotal role and is tightly regulated in this process. Here, we identify HERC5 (HECT domain and RLD 5) as a specific binding protein of IRF3 by immunoprecipitation. Ectopic expression or knockdown of HERC5 could, respectively, enhance or impair IRF3-mediated gene expression. Mechanistically, HERC5 catalyzes the conjugation of ubiquitin-like protein ISG15 onto IRF3 (Lys193, -360, and -366), thus attenuating the interaction between Pin1 and IRF3, resulting in sustained IRF3 activation. In contrast to results for wild-type IRF3, the mutant IRF3(K193,360,366R) interacts tightly with Pin1, is highly polyubiquitinated, and becomes less stable upon Sendai virus (SeV) infection. Consistently, host antiviral responses are obviously boosted or crippled in the presence or absence of HERC5, respectively. Collectively, this study characterizes HERC5 as a positive regulator of innate antiviral responses. It sustains IRF3 activation via a novel posttranslational modification, ISGylation.
The loss of E-cadherin gene expression can cause the dysfunction of the cell-cell junction to trigger tumor metastasis. Members of the Snail family of transcription factors are repressors of the expression of the E-cadherin gene. In this study, we showed that the activated androgen receptor (AR) is a novel repressor of E-cadherin gene expression and can promote metastasis. Our results demonstrated that the activated AR could bind to the E-cadherin promoter in vitro and in vivo. The activated AR and HDAC1 had synergistic effects in downregulating E-cadherin gene expression. Treating cells with the AR ligand, dihydrotestosterone (DHT), triggered the reduction of E-cadherin expression and induced changes in cell morphology from an epithelial-like to a mesenchymal-like appearance. When nonmetastatic breast cancer cells expressing cytoplasmic AR were transplanted into mice and the mice were treated with DHT, tumors were detected at metastatic sites, whereas no tumors were detected in transplanted mice without DHT treatment. Furthermore, clinical data from breast cancer patients with invasive ductal carcinomas showed high levels of AR expression in the nuclei and low levels of E-cadherin expression. These results suggest that, similarly to Snail and Twist, the activated AR can downregulate E-cadherin expression to promote the activation of epithelial-mesenchymal transition and tumor metastasis.
Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxide-induced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation.
When recognized by the RNA interference (RNAi) pathway, double-stranded RNA (dsRNA) produced in eukaryotic cells results in posttranscriptional gene silencing. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not short interfering RNA (siRNA), induces the expression of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for normal gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, a genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. Together, our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections.
RNA polymerases can be shared by a particular group of genes in a transcription “factory” in nuclei, where transcription may be coordinated in concert with the distribution of coexpressed genes in higher-eukaryote genomes. Moreover, gene expression can be modulated by regulatory elements working over a long distance. Here, we compared the conformation of a 130-kb chromatin region containing the mouse α-globin cluster and their flanking housekeeping genes in 14.5-day-postcoitum fetal liver and brain cells. The analysis of chromatin conformation showed that the active α1 and α2 globin genes and upstream regulatory elements are in close spatial proximity, indicating that looping may function in the transcriptional regulation of the mouse α-globin cluster. In fetal liver cells, the active α1 and α2 genes, but not the inactive ζ gene, colocalize with neighboring housekeeping genes C16orf33, C16orf8, MPG, and C16orf35. This is in sharp contrast with the mouse α-globin genes in nonexpressing cells, which are separated from the congregated housekeeping genes. A comparison of RNA polymerase II (Pol II) occupancies showed that active α1 and α2 gene promoters have a much higher RNA Pol II enrichment in liver than in brain. The RNA Pol II occupancy at the ζ gene promoter, which is specifically repressed during development, is much lower than that at the α1 and α2 promoters. Thus, the mouse α-globin gene cluster may be regulated through moving in or out active globin gene promoters and regulatory elements of a preexisting transcription factory in the nucleus, which is maintained by the flanking clustered housekeeping genes, to activate or inactivate α-globin gene expression.
G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.
The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha IFN (IFN-α). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling of the promoter upon IFN-α induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3 promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling BAF complex.
RhoB is an endosomal small GTPase that is implicated in the response to growth factors, genotoxic stress, and farnesyltransferase inhibitors. To gain insight into its physiological functions we examined the consequences of homozygous gene deletion in the mouse. Loss of RhoB did not adversely affect mouse development, fertility, or wound healing. However, embryo fibroblasts cultured in vitro exhibited a defect in motility, suggesting that RhoB has a role in this process that is conditional on cell stress. Neoplastic transformation by adenovirus E1A and mutant Ras yielded differences in cell attachment and spreading that were not apparent in primary cells. In addition, transformed −/− cells displayed altered actin and proliferative responses to transforming growth factor β. A negative modifier role in transformation was suggested by the increased susceptibility of −/− mice to 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis and by the increased efficiency of intraperitoneal tumor formation by −/− cells. Our findings suggest that RhoB is a negative regulator of integrin and growth factor signals that are involved in neoplastic transformation and possibly other stress or disease states.
Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P2 and PI-3,4,5-P3 in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-γ1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.
The winged-helix (WH) BF-1 gene, which encodes brain factor 1 (BF-1) (also known as foxg1), is essential for the proliferation of the progenitor cells of the cerebral cortex. Here we show that BF-1-deficient telencephalic progenitor cells are more apt to leave the cell cycle in response to transforming growth factor β (TGF-β) and activin. We found that ectopic expression of BF-1 in vitro inhibits TGF-β mediated growth inhibition and transcriptional activation. Surprisingly, we found that the ability of BF-1 to function as a TGF-β antagonist does not require its DNA binding activity. Therefore, we investigated whether BF-1 can inhibit Smad-dependent transcriptional responses by interacting with Smads or Smad binding partners. We found that BF-1 does not interact with Smads. Because the identities of the Smad partners mediating growth inhibition by TGF-β are not clearly established, we examined a model reporter system which is known to be activated by activin and TGF-β through Smads and the WH factor FAST-2. We demonstrate that BF-1 associates with FAST-2. This interaction is dependent on the same region of protein which mediates its ability to interfere with the antiproliferative activity of TGF-β and with TGF-β-dependent transcriptional activation. Furthermore, the interaction of FAST-2 with BF-1 is mediated by the same domain which is required for FAST-2 to interact with Smad2. We propose a model in which BF-1 interferes with transcriptional responses to TGF-β by interacting with FAST-2 or with other DNA binding proteins which function as Smad2 partners and which have a common mode of interaction with Smad2.
Farnesyltransferase inhibitors (FTIs) are in clinical trials, but how they selectively inhibit malignant cell growth remains uncertain. One important player in this process appears to be RhoB, an endosomal Rho protein that regulates receptor trafficking. FTI treatment elicits a gain of the geranylgeranylated RhoB isoform (RhoB-GG) that occurs due to modification of RhoB by geranylgeranyltransferase I in drug-treated cells. Notably, this event is sufficient to mediate antineoplastic effects in murine models and human carcinoma cells. To further assess this gain-of-function mechanism and determine whether RhoB-GG has a necessary role in drug action, we examined the FTI response of murine fibroblasts that cannot express RhoB-GG due to homozygous deletion of the rhoB gene. Nullizygous (−/−) cells were susceptible to cotransformation by adenovirus E1A plus activated H-Ras but defective in their FTI response, despite complete inhibition of H-Ras prenylation. Actin cytoskeletal and phenotypic events were disrupted in −/− cells, implicating RhoB-GG in these effects. Interestingly, −/− cells were resistant to FTI-induced growth inhibition under anchorage-dependent but not anchorage-independent conditions, indicating that, while RhoB-GG is sufficient, it is not necessary for growth inhibition under all conditions. In contrast, −/− cells were resistant to FTI-induced apoptosis in vitro and in vivo. Significantly, the apoptotic defect of −/− cells compromised the antitumor efficacy of FTI in xenograft assays. This study offers genetic proof of the hypothesis that RhoB-GG is a crucial mediator of the antineoplastic effects of FTIs.
Nucleocytoplasmic shuttling of class IIa of histone deacetylases (HDACs) is a key mechanism that controls cell fate and animal development. We have identified the filamin B (FLNB) as a novel HDAC7-interacting protein that is required for temporal and spatial regulation of vascular endothelial growth factor (VEGF)-mediated HDAC7 cytoplasmic sequestration. This interaction occurs in the cytoplasm and requires monoubiquitination of an evolutionarily conserved lysine 1147 (K1147) in the immunoglobulin (Ig)-like repeat 10 (R10) of FLNB and the nuclear localization sequence of HDAC7. Inhibition of protein kinase C (PKC) blocks VEGF-induced ubiquitination of FLNB and its interaction with HDAC7. Small interfering RNA (siRNA) knockdown of FLNB or ubiquitin (Ub) in human primary endothelial cells blocks VEGF-mediated cytoplasmic accumulation of HDAC7, reduces VEGF-induced expression of the HDAC7 target genes Mmp-10 and Nur77, and inhibits VEGF-induced vascular permeability. Using dominant negative mutants and rescue experiments, we demonstrate the functional significance of FLNB K1147 to interfere with the ability of phorbol myristate acetate (PMA) to promote FLNB-mediated cytoplasmic accumulation of HDAC7. Taken together, our data show that VEGF and PKC promote degradation-independent protein ubiquitination of FLNB to control intracellular trafficking of HDAC7.
TRIP6 is an adaptor protein that regulates cell motility and antiapoptotic signaling. Although it has been implicated in tumorigenesis, the underlying mechanism remains largely unknown. Here we provide evidence that TRIP6 promotes tumorigenesis by serving as a bridge to promote the recruitment of p27KIP1 to AKT in the cytosol. TRIP6 regulates the membrane translocation and activation of AKT and facilitates AKT-mediated recognition and phosphorylation of p27KIP1 specifically at T157, thereby promoting the cytosolic mislocalization of p27KIP1. This is required for p27KIP1 to enhance lysophosphatidic acid (LPA)-induced ovarian cancer cell migration. TRIP6 also promotes serum-induced reduction of nuclear p27KIP1 expression levels through Skp2-dependent and -independent mechanisms. Consequently, knockdown of TRIP6 in glioblastoma or ovarian cancer xenografts restores nuclear p27KIP1 expression and impairs tumor proliferation. As TRIP6 is upregulated in gliomas and its levels correlate with poor clinical outcomes in a dose-dependent manner, it may represent a novel prognostic marker and therapeutic target in gliomas.
Conversion of mesenchymal stem cells into terminally differentiated adipocytes progresses sequentially through regulated transcriptional steps. While it is clear that the late phases of adipocyte maturation are governed by the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), less is known about the transcriptional control of the initial stages of differentiation. To identify early regulators, we performed a small interfering RNA (siRNA) screen of Forkhead-box genes in adipocytes and show here for the first time that the winged helix factor Foxa3 promotes adipocyte differentiation by cooperating with C/EBPβ and -δ to transcriptionally induce PPARγ expression. Furthermore, we demonstrate that mice with genetic ablation of Foxa3 have a selective decrease in epididymal fat depot and a cell-autonomous defect to induce PPARγ specifically in their visceral adipocytes. In obese subjects, FOXA3 is differentially expressed in visceral and subcutaneous adipose depots. Overall, our study implicates Foxa3 in the regulation of adipocyte differentiation and depot-selective adipose tissue expansion.
(CTG)n · (CAG)n trinucleotide repeat (TNR) expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)n · (CAG)n structures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)45 · (CAG)45 causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)45 · (CAG)45 lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)45 · (CAG)45 expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linked in vivo.
The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a
divergent C-terminal tail. While the function and structure of the Asf1 core are well
known, the function of the tail is less well understood. Here, we have explored the role
of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a
photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3
in vivo and, further, that loss of C-terminal tail residues 211 to 279
weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via
several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that
truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail
partially substitutes for full-length yAsf1 with respect to silencing but that full-length
hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the
C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation
site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts,
whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally,
we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence
yAsf1's function in silencing.