The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor coactivator 1 (SRC1) and its homologues and the general coactivators CREB binding protein (CBP) and p300. SRC1 contains an activation domain (AD1) which functions via recruitment of CBP and and p300. In this study, we have used yeast two-hybrid and in vitro interaction-peptide inhibition experiments to map the AD1 domain of SRC1 to a 35-residue sequence potentially containing two ␣-helices. We also define a 72-amino-acid sequence in CBP necessary for SRC1 binding, designated the SRC1 interaction domain (SID). We show that in contrast to SRC1, direct binding of CBP to the estrogen receptor is weak, suggesting that SRC1 functions primarily as an adaptor to recruit CBP and p300. In support of this, we show that the ability of SRC1 to enhance ligand-dependent nuclear receptor activity in transiently transfected cells is dependent upon the integrity of the AD1 region. In contrast, the putative histone acetyltransferase domain, the Per-Arnt-Sim basic helix-loop-helix domain, the glutamine-rich domain, and AD2 can each be removed without loss of ligandinduced activity. Remarkably, a construct corresponding to residues 631 to 970, which contains only the LXXLL motifs and the AD1 region of SRC1, retained strong coactivator activity in our assays.The nuclear receptors (NRs) are ligand-regulated transcription factors that mediate the effects of steroids, retinoids, and other lipophilic hormones on gene expression (32). In common with other transcriptional activators, NRs stimulate transcription by promoting the local modification of chromatin structure and recruitment of a preinitiation complex (59). This is achieved via two transcriptional activation functions (AF1 and AF2) which provide molecular surfaces for the recruitment of transcriptional coactivator proteins (17,28,36,60).The AF2 surfaces of the ligand binding domains (LBDs) of NRs appear to be the principal sites for coactivator recruitment. Far-Western experiments detected two major classes of proteins in nuclear extracts (with apparent molecular masses of 160 and 140 kDa) which bind to the LBD of the estrogen receptor (ER) in the presence of ligand (5, 14). At least three distinct p160 proteins have been identified, including steroid receptor coactivator 1 (SRC1) (39), transcription intermediary factor 2 (TIF2) (54) and its murine homologue GRIP1 (18), and p300-CBP cointegrator-associated protein (pCIP) (50), which is the mouse homologue of the human protein AIB1 (1), also known as ACTR (8), RAC3 (29), or TRAM1 (49). These proteins appear to be bona fide coactivators, as they enhance the activity of NRs in both in vitro and in vivo experimental systems. The p140 class appears to consist chiefly of the nuclear protein RIP140 (6). The function of RIP140 is unknown, although it has been shown to down-regulate NR-mediated transcription in transient-reporter assays, possibly via competition with p160s for the LBD (15,27,35,51). Other AF2 binding proteins ...

CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the ␣-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ER␣/ SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by cotransfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the ␣-helix A␣1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic ␣-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP. CBP1 and p300 interact with a wide range of DNA-binding transcription factors and their cofactors (1, 2). Recruitment of CBP and associated factors permits acetylation and methylation of histones and other proteins at gene promoters, leading to chromatin remodeling, RNA polymerase II recruitment, and transcription. The ability of CBP and p300 to form contacts with multiple diverse factors assembled at gene promoters such as the IFN-␤ enhanceosome, facilitates synergistic activation of transcription (3). Conversely, competition between transcription factors for common binding sites on CBP/p300, which are in limiting concentrations in the nucleus, is likely to be important in negative cross-talk, as observed between nuclear receptors (NRs) and AP-1, NFB, or STAT proteins (4, 5) or hypoxia-inducible factor (HIF1␣) and CITED2 (6). Furthermore, CBP and p300 are important targets in viral infection, as they associate with viral proteins such as adenoviral E1A, SV40 large T antigen, and HTLV Tax (1, 2). Thus, CBP and p300 act as molecular integrators of signal transduction pathways regulating cellular processes such as proliferation, differentiation, apoptosis, and the response to viral infection.The interaction of CBP/p300 with a large number of functionally diverse proteins is facilitated by a series of modular protein-binding domains. These include the cysteine/histidinerich domains CH1 and CH3, also known as TAZ1 and ZZ/TAZ2, which are major sites of protein interaction. The CH3/TA...

G-protein modulation of neuronal nicotinic acetylcholine receptor (nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTP␥S increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh-and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTP␥S. In inside-out membrane patches, the open probability (NP o ) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2 mM GTP␥S relative to control but was unchanged in the presence of GDP␤S. The modulation of nAChR-mediated whole-cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTP␥S. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G o ␣ or G␤␥ subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nM G␤␥ increased the open probability of ACh-activated single-channel currents fivefold, whereas G o ␣ (50 nM) produced no significant increase in NP o . Neuronal nAChR subunits ␣3-␣5 and ␤2 exhibited a positive interaction with G o ␣ and G␤␥, whereas ␤4 and ␣7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3207 KEY WORDSCBP, CAS/CSE1p, importin, NUP93, HDAC inhibitor, acetyltransferase ACKNOWLEDGEMENTS Report Functional Interaction of CREB Binding Protein (CBP) with Nuclear Transport Proteins and Modulation by HDAC Inhibitors ABSTRACTNuclear transport proteins such as CSE1, NUP93 and Importin-α have recently been shown to be chromatin-associated proteins in yeast, which have unexpected functions in gene regulation. Here we report interactions between the mammalian histone acetyltransferase CBP with nuclear transport proteins CAS (a CSE1 homologue) and Importin-α (Impα) and NUP93. CAS was found to bind the SRC1 interaction domain (SID) of CBP via a leucine-rich motif in the N-terminus of the protein, that is conserved in other SID-binding proteins. Coimmunoprecipitation experiments also revealed that CBP and Impα proteins form a complex. As Impα is a known acetylation target of CBP/p300, and is recycled to the cytoplasm via the exportin CAS, we investigated whether HDAC inhibitors would alter the subcellular localization of these proteins. Treatment of COS-1 cells with the HDAC inhibitors trichostatin A or sodium butyrate resulted in sequestration of Impα in the nuclear envelope, accumulation of CAS in nuclear aggregates, and an increased number of CBP-containing PML bodies per cell. In addition, HDACi treatment appeared to enhance the association of Impα and CBP in coimmunoprecipitation experiments. Our results provide evidence for novel functional interactions between the chromatin modification enzyme CBP and nuclear transport proteins in mammalian cells.

The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor. The fibroblast growth factors (FGFs)2 and their receptor tyrosine kinases (FGFRs) include a signaling system that lies at the heart of metazoan development, homeostasis, and adult pathobiology. FGF/FGFR signaling is involved in a multitude of biological processes from cell division, migration, and differentiation to cell survival and control of metabolic homeostasis (for reviews see Refs. 1-4).The paradigm of FGF involvement in such diverse biological functions results from the tight regulation of the FGF/FGFR signaling system at various levels. The expression of the 22 vertebrate FGF genes and the four tyrosine kinase FGFR genes is spatially and temporally regulated throughout development.Additional tissue specificity is generated by alternative splicing of the FGF receptor genes. For example, splicing of the third immunoglobulin domain (D3) to B and C isoforms is tissue-dependent and determines a degree of the ligand binding specificity. Some of the FGF ligands, such as FGF1, are promiscuous and bind to all FGFRs, whereas other ligands bind only specific FGFR isoforms and/or splice variants (5, 6). In canonical FGF signaling, the growth factor is thought to assemble in a ternary complex with the FGFR and a glycosaminoglycan co-receptor, usually heparan sulfate. Specific heparan sulfate structures can either activate or inactivate FGFR signaling (7).Mutations in genes encoding FGF receptors give rise to a variety of human disorders and diseases (for review see Ref. 8). The activating mutations lead to excessive receptor signaling (9, 10), increased ligand binding affinity (11, 12), or altered ligand specificity (13), whereas inactivating mutations such as in Kallmann syndrome result in decreased signaling (14). So...