Steroid, retinoid, vitamin D 3 , and thyroid hormones signal through ligand-dependent transcription factors that collectively comprise a superfamily of intracellular, soluble receptors (hereafter collectively called nuclear receptors) that reside in the nucleus or translocate there in response to hormonal signals. As the largest known family of eukaryotic transcriptional regulators, nuclear receptors are implicated via the target genes they modulate in the control of cell growth and differentiation, homeostasis, development, and several physiological processes (for review, see Freedman 1997 and references therein). Moreover, because they are regulated tightly by small lipophilic molecules, they are extremely attractive as pharmacologic targets.Nuclear receptors all share a common organization in functional domains and extensive homologies in structure. A DNA-binding domain allows the receptors to bind as homodimers, or heterodimers with a common partner, retinoid X receptor (RXR), to specific DNA response elements typically composed of two hexameric half-sites organized as direct or inverted repeats. The carboxy-terminal half of the prototype nuclear receptor includes a ligand-binding domain (LBD) with a superimposed dimerization surface, and a ligand-dependent transcriptional activation function called AF-2, located at the extreme carboxyl terminus of the receptor (Danielian et al. 1992;Barettino et al. 1994;Durand et al. 1994). Crystallographic analyses have revealed that binding of a specific ligand, all-trans retinoic acid (ATRA), to retinoic acid receptor ␥ (RAR␥) induces a conformational change in its structure that modifies the orientation of the AF-2 core motif, contained within the last of 12 ␣-helices that
Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D 3 receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.Nuclear receptors, including vitamin D 3 , thyroid hormone, and retinoic acid receptors (VDR, TR, and RAR, respectively), are intracellular factors that can transduce the signals of small lipophilic hormonal ligands by binding to target DNA sequences and regulating gene transcription in direct response to such ligands (13,27). The VDR/TR/RAR subgroup typically acts in conjunction with a common partner, the retinoid X receptor (RXR), by recognizing and binding as heterodimers to specific DNA response elements composed of direct hexameric repeats of the (A/G)G(G/T)TCA consensus sequence separated by three, four, or five nucleotides (VDRE, TRE, or RARE, respectively). Nuclear receptors can be dissected into discrete functional regions, including a DNA binding domain and a ligand binding domain (LBD). The LBD contains at its C terminus a short ␣-helical motif called AF-2 (3, 8, 11) that is required for ligand-dependent transactivation, and it is a key determinant in interactions with other proteins, generally called coactivators, that mediate connections to the transcription machinery.Transcriptional activation of genes regulated by nuclear receptors and other transcription factors involves both direct DNA binding of activators to their specific response elements and protein-protein interactions with components of the basal transcription machinery, ultimately targeting RNA polymerase II (RNA Pol II) or an RNA Pol I...
Two functionally distinct classes of coactivators are recruited by liganded estrogen receptor, the DRIP/Mediator complex and p160 proteins, although the relative dynamics of recruitment is unclear. Previously, we have shown a direct, estradiol-dependent interaction between the DRIP205 subunit of the DRIP complex and the estrogen receptor (ER) AF2 domain. Here we demonstrate the in vivo recruitment of other endogenous DRIP subunits to ER in response to estradiol treatment in MCF-7 cells. To explore the relationship between DRIP and p160 coactivators, we examined the kinetics of coactivator recruitment to the ER target promoter, pS2, by chromatin immunoprecipitation. We observed a cyclic association and dissociation of coactivators with the promoter, with recruitment of p160s and DRIPs occurring in opposite phases, suggesting an exchange between these coactivator complexes at the target promoter.The actions of estradiol are mediated by two isoforms of the estrogen receptor (ER), 1 ER␣ and ER, which function as ligand-regulated transcription factors. The liganded ER homodimer binds the promoters of target genes and interacts with coactivators to facilitate transcriptional activation (1). A number of coactivators interact with the C-terminal activation domain (AF2) in a ligand-dependent manner and have been implicated in ER-mediated transcription. One class of coactivators, collectively termed the p160 family, includes SRC1/ NCoA-1, TIF2/GRIP1/NCoA-2, and pCIP/ACTR/AIB1 (reviewed in Refs. 16 and 23). The p160 coactivators not only possess weak histone acetyltransferase activity but also recruit CBP/p300 (2), presumably leading to the generation of an open chromatin structure at the promoter (3). A second distinct class of coactivators, alternatively called DRIP, ARC, or TRAP (4, 5), comprises a multi-protein complex that interacts with liganded nuclear receptors, including ER␣ and ER, via the DRIP205/ TRAP220 subunit (6 -10). The DRIP complex shares several subunits with the mammalian Mediator complex, suggesting that it functions in the direct recruitment of RNA polymerase II to the promoter (11).It is evident that both p160s and DRIP205 play a key role in ER-mediated transcription (12-15), and despite the functional distinction between the p160 coactivators and the DRIP complex, the molecular determinants of the interactions between these coactivators and ER appear to be very similar. Both classes of coactivators interact with the receptor AF2 via LXXLL signature motifs (16), and the same residues in the ER␣-AF2 are critical for interactions with both p160s and DRIP205 (6), raising the question of whether these complexes are utilized by the receptor dimer simultaneously or sequentially. It has been reported that ACTR can be acetylated by CBP/p300, leading to the dissociation of p160 coactivator complexes from the promoter-bound ER (17), suggesting a mechanism whereby coactivator exchange may take place. Recently, spectroscopic methods have been used to demonstrate that the stoichiometry of the SRC1/ER␣/E 2 compl...
Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)2D3)], controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the vitamin D receptor (VDR)-interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the VDR. However, the relationship between these two classes of coactivators is not clear. Using glutathione-S-transferase-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP and SRC proteins were expressed in keratinocytes. DRIP205 expression decreased during differentiation, although SRC-3 levels increased. Both DRIP205 and SRC-3 potentiated vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are sequentially involved in vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.
Cancer cells constantly adapt to oxidative phosphorylation (OXPHOS) suppression resulting from hypoxia or mitochondria defects. Under the OXPHOS suppression, AMP-activated protein kinase (AMPK) regulates global metabolism adjustments, but its activation has been found to be transient. Whether cells can maintain cellular ATP homeostasis and survive beyond the transient AMPK activation is not known. Here, we study the bioenergetic adaptation to the OXPHOS inhibitor oligomycin in a group of cancer cells. We found that oligomycin at 100 ng/ml completely inhibits OXPHOS activity in 1 h and induces various levels of glycolysis gains by 6 h, from which we calculate the bioenergetic organizations of cancer cells. In glycolysis-dominant cells, oligomycin does not induce much energy stress as measured by glycolysis acceleration, ATP imbalance, AMPK activation, AMPK substrate acetyl-CoA carboxylase phosphorylation at Ser 79 , and cell growth inhibition. In OXPHOS-dependent LKB1 wild type cells, oligomycin induces 5-8% ATP drops and transient AMPK activation during the initial 1-2 h. After AMPK activation is completed, oligomycininduced increase of acetyl-CoA carboxylase phosphorylation at Ser 79 is still detected, and cellular ATP is back at preoligomycin treatment levels by sustained elevation of glycolysis. Cell growth, however, is inhibited without an increase in cell death and alteration in cell cycle distribution. In OXPHOS-dependent LKB1-null cells, no AMPK activation by oligomycin is detected, yet cells still show a similar adaptation. We also demonstrate that the adaptation to oligomycin does not invoke activation of hypoxia-induced factor. Our data suggest that cancer cells may grow and survive persistent OXPHOS suppression through an as yet unidentified regulatory mechanism.The bioenergetic organization, the fraction of cellular ATP produced by glycolysis and mitochondrial OXPHOS, 2 determines the bioenergetic homeostasis in cells. Tumors have a bioenergetic organization distinct from that of normal cells, in which the burden of ATP production increasingly shifts from OXPHOS to glycolysis, the so-called Warburg effect (1). Deregulation of multiple oncogenes and tumor suppressors during tumorigenesis contributes to this distinct neoplastic metabolism alteration because glycolysis is the downstream target of the altered pathways (2, 3). In addition, mitochondrial defects can also drive up the aerobic glycolysis to bioenergetically compensate for the loss of OXPHOS ATP production. The reduction in OXPHOS activity has been identified in widely spread cancer cells (4 -9). The switch of bioenergetic dependence from OXPHOS to glycolysis is proposed for cancer cells to cope with intermittent and chronicle hypoxia microenvironments, reduce mitochondrial-initiated cell death (3, 10), and promote invasion and metastasis (10 -12).Suppression of OXPHOS activity activates master energy stress sensor AMPK that reprograms the global cellular metabolism for the stress adaptation (13,14). In compensating for the loss of OXPHOS ATP...
Glued phosphorylation may positively regulate mitotic processes, such as spindle assembly or orientation, or negatively regulate interphase processes such as minus-end-directed organelle trafficking.Fully grown (stage VI) Xenopus oocytes are arrested in a G 2 -like state. Exposure to progesterone releases oocytes from this arrest and causes meiotic maturation. The maturing oocyte undergoes germinal vesicle breakdown (GVBD), 1 forms a meiotic spindle, segregates its homologous chromosomes, completes the first meiotic division, enters meiosis 2, and then arrests in metaphase of meiosis 2.Oocyte maturation depends upon the activation of Cdc2-cyclin B complexes (1), just as entry into mitotic M phase does (reviewed in Refs. 2-4). In the Xenopus oocyte, Cdc2 activation is tightly linked to activation of p42 mitogen-activated protein kinase (MAPK) and its upstream activators Mek-1 (a MAPK kinase) and Mos (a MAPK kinase kinase) (5-7). Interfering with the activation of Mos (8), Mek-1 (9), or p42 MAPK (10) inhibits progesterone-induced Cdc2 activation, and overexpression of Mos (11,12) or introduction of active forms of Mek-1 (13) or p42 MAPK (14) can cause activation in the absence of progesterone. p42 MAPK is activated in an all-or-none fashion (15), ensuring that the oocyte's decision to carry out maturation is decisive and irrevocable.Relatively little is known about how the activation of MAPK and Cdc2 brings about the dramatic cell biological changes of maturation. Some of the relevant substrates of these kinases are undoubtedly involved in entry into mitosis as well; others must be specific for meiosis or maturation. Expression cloning studies have identified a number of proteins that become phosphorylated at the onset of mitosis (16 -18), and work is under way to determine whether and how their mitotic phosphorylation affects their function.Here we have approached the identification of meiotic phosphoproteins by antiphosphotyrosine immunoblotting, the strategy that implicated p42 MAPK in Xenopus oocyte maturation (19 -22). We looked for immunoreactive bands that increased in intensity during oocyte maturation (as does p42 MAPK), decreased in intensity (as does Cdc2), or changed in their electrophoretic mobility. This paper focuses on the first of these proteins, p83, a band that shifts up in its apparent molecular weight during oocyte maturation but does not change substantially in intensity. Through purification and peptide sequencing, we have identified p83 as a component of the minus-end-directed microtubule motor dynein, the cytoplasmic dynein intermediate chain (dynein IC). We found that dynein IC comigrates with p83 and undergoes mobility shifts that exactly parallel those of p83; that the mobility shift of dynein IC is due to phosphorylation; and that dynein IC cross-reacts with various phosphotyrosine antisera but is phosphorylated mainly at serine in vivo. Dynein IC was found to undergo hyperphosphorylation just prior to germinal vesicle breakdown and to remain hyperphosphorylated throughout maturation a...
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