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D W Rose

Publications and source records attributed to D W Rose.

At least 37 records · Page 2Linked to original sources
PubMed1999

Transcriptional activation by NF-kappaB requires multiple coactivators.

Nuclear factor-kappaB (NF-kappaB) plays a role in the transcriptional regulation of genes involved in inflammation and cell survival. In this report we demonstrate that NF-kappaB recruits a coactivator complex that has striking similarities to that recruited by nuclear receptors. Inactivation of either cyclic AMP response element binding protein (CREB)-binding protein (CBP), members of the p160 family of coactivators, or the CBP-associated factor (p/CAF) by nuclear antibody microinjection prevents NF-kappaB-dependent transactivation. Like nuclear receptor-dependent gene expression, NF-kappaB-dependent gene expression requires specific LXXLL motifs in one of the p160 family members, and enhancement of NF-kappaB activity requires the histone acetyltransferase (HAT) activity of p/CAF but not that of CBP. This coactivator complex is differentially recruited by members of the Rel family. The p50 homodimer fails to recruit coactivators, although the p50-p65 heterodimeric form of the transcription factor assembles the integrator complex. These findings provide new mechanistic insights into how this family of dimeric transcription factors has a differential effect on gene expression.

Acetyltransferases↗
PubMed1998

Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation.

Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3') in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARgamma activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.

Amino Acid Sequence↗
PubMed1998

Signal-specific co-activator domain requirements for Pit-1 activation.

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes. Here we show that whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT, mSin3A/B and histone deacetylases, and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, the histone acetyltransferase functions of CBP or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors.

Acetyltransferases↗
PubMed1998

Interactions controlling the assembly of nuclear-receptor heterodimers and co-activators.

Retinoic-acid receptor-alpha (RAR-alpha) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) are members of the nuclear-receptor superfamily that bind to DNA as heterodimers with retinoid-X receptors (RXRs). PPAR-RXR heterodimers can be activated by PPAR or RXR ligands, whereas RAR-RXR heterodimers are selectively activated by RAR ligands only, because of allosteric inhibition of the binding of ligands to RXR by RAR. However, RXR ligands can potentiate the transcriptional effects of RAR ligands in cells. Transcriptional activation by nuclear receptors requires a carboxy-terminal helical region, termed activation function-2 (AF-2), that forms part of the ligand-binding pocket and undergoes a conformational change required for the recruitment of co-activator proteins, including NCoA-1/SRC-1. Here we show that allosteric inhibition of RXR results from a rotation of the RXR AF-2 helix that places it in contact with the RAR coactivator-binding site. Recruitment of an LXXLL motif of SRC-1 to RAR in response to ligand displaces the RXR AF-2 domain, allowing RXR ligands to bind and promote the binding of a second LXXLL motif from the same SRC-1 molecule. These results may partly explain the different responses of nuclear-receptor heterodimers to RXR-specific ligands.

Allosteric Regulation↗
PubMed1998

Prolonged vs transient roles for early cell cycle signaling components.

Both p21ras and phosphatidylinositol 3-kinase (PI 3-k) are critical elements in signaling pathways mediating insulin/IGF-I induced cell cycle progression. For example, microinjection of antibodies, peptides, or recombinant proteins which block the interaction of the SH2 domains of the PI 3-k p85alpha subunit with tyrosine phosphorylated intracellular targets blocks insulin mediated DNA synthesis. We report here that this inhibitory phenotype is observed whether the injections are made into quiescent cells (the standard approach), or at any time point during G1 phase subsequent to stimulation. This observation is not true, however, for the major substrate of the insulin/IGF-I receptor (IRS-1) despite the well known interaction of p85 with IRS-1. Antibodies to IRS-1 are inhibitory only when injected during the first 15 min of G1 phase, as are antibodies to another major IRS-1 binding protein, the tyrosine phosphatase SHP2. We also have microinjected reagents which target proteins involved in the formation of rasGTP and which mediate some of the downstream effects of ras activation. Reagents which target the formation of rasGTP (Shc and dominant negative ras protein) inhibit DNA synthesis only at points early in G1, as do reagents which target components of the MAP kinase pathway. Injection of antibodies to p21ras itself, or a recombinant Raf-1 protein domain which binds to the effector region of ras in a GTP-dependent manner, results in the inhibition of cell cycle progression throughout G1 phase. The results point to a continuous requirement for both PI 3-k and ras activity until cellular commitment to DNA synthesis, although some of the molecules which are both upstream and downstream of these activities are only required transiently. Our results are also consistent with a Raf-1 independent ras activity late in G1, as well as IRS-1 independent effects of PI 3-kinase.

3T3 Cells↗
PubMed1998

Grb10 interacts differentially with the insulin receptor, insulin-like growth factor I receptor, and epidermal growth factor receptor via the Grb10 Src homology 2 (SH2) domain and a second novel domain located between the pleckstrin homology and SH2 domains.

The Grb10 protein appears to be an adapter protein of unknown function that has been implicated in insulin receptor (IR) signaling. The interaction of this protein with the IR has been shown to be mediated in part by the Src homology 2 (SH2) domain of Grb10. Here we demonstrate the existence of a second novel domain within Grb10 that interacts with the IR and insulin-like growth factor receptor in a kinase-dependent manner. This domain was localized to a region of approximately 50 amino acids, and we term it the BPS domain to denote its location between the PH and SH2 domains. The BPS domain does not bear any obvious resemblance to other known protein interaction domains but is highly conserved among the Grb10-related proteins Grb7 and Grb14. We show that the BPS domain interaction is dependent upon receptor tyrosine kinase activity. Furthermore, interaction of the BPS domain requires the kinase domain of the IR, since mutation of the paired tyrosine residues (Y1150F/Y1151F) within the IR activation loop dramatically reduced the interaction. Last, our data suggest that the presence of two distinct protein interaction domains may help to determine the specificity by which Grb10 interacts with different receptors. Specifically, the IR, which appears to interact most strongly with Grb10, interacts well with both the SH2 and BPS domains. Conversely, the insulin-like growth factor receptor and EGFR, which interact less avidly with Grb10, interact well only with the BPS domain or the SH2 domain, respectively. In summary, our findings demonstrate the existence of a previously unidentified tyrosine kinase activity-dependent binding domain located between the Pleckstrin homology and SH2 domains of Grb10.

Amino Acid Sequence↗
PubMed1998

Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes.

Several lines of evidence indicate that the nuclear receptor corepressor (N-CoR) complex imposes ligand dependence on transcriptional activation by the retinoic acid receptor and mediates the inhibitory effects of estrogen receptor antagonists, such as tamoxifen, suppressing a constitutive N-terminal, Creb-binding protein/coactivator complex-dependent activation domain. Functional interactions between specific receptors and N-CoR or SMRT corepressor complexes are regulated, positively or negatively, by diverse signal transduction pathways. Decreased levels of N-CoR correlate with the acquisition of tamoxifen resistance in a mouse model system for human breast cancer. Our data suggest that N-CoR- and SMRT-containing complexes act as rate-limiting components in the actions of specific nuclear receptors, and that their actions are regulated by multiple signal transduction pathways.

Animals↗
PubMed1998

Transcription factor-specific requirements for coactivators and their acetyltransferase functions.

Different classes of mammalian transcription factors-nuclear receptors, cyclic adenosine 3',5'-monophosphate-regulated enhancer binding protein (CREB), and signal transducer and activator of transcription-1 (STAT-1)-functionally require distinct components of the coactivator complex, including CREB-binding protein (CBP/p300), nuclear receptor coactivators (NCoAs), and p300/CBP-associated factor (p/CAF), based on their platform or assembly properties. Retinoic acid receptor, CREB, and STAT-1 also require different histone acetyltransferase (HAT) activities to activate transcription. Thus, transcription factor-specific differences in configuration and content of the coactivator complex dictate requirements for specific acetyltransferase activities, providing an explanation, at least in part, for the presence of multiple HAT components of the complex.

Acetyltransferases↗
PubMed1998

SAP30, a component of the mSin3 corepressor complex involved in N-CoR-mediated repression by specific transcription factors.

The transcriptional corepressor mSin3 is found in a large multiprotein complex containing the histone deacetylases HDAC1 and HDAC2, in addition to at least five tightly associated polypeptides. We have cloned and characterized a novel component of the mSin3 complex, SAP30, SAP30 binds to mSin3 and is capable of mediating transcriptional repression via histone deacetylases. SAP30 also binds the N-CoR corepressor and is required for N-CoR-mediated repression by antagonist-bound estrogen receptor and the homeodomain protein Rpx, as well as N-CoR suppression of transactivation by the POU domain protein Pit-1. However, SAP30 is not required for N-CoR-mediated repression by unliganded retinoic acid receptor or thyroid hormone receptor, suggesting that SAP30 is involved in the functional recruitment of the mSin3-histone deacetylase complex to a specific subset of N-CoR corepressor complexes.

Amino Acid Sequence↗
PubMed1998

Ligand-independent GLUT4 translocation induced by guanosine 5'-O-(3-thiotriphosphate) involves tyrosine phosphorylation.

To delineate the signaling pathway leading to glucose transport protein (GLUT4) translocation, we examined the effect of microinjection of the nonhydrolyzable GTP analog, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mM GTPgammaS, 40% of injected cells displayed surface GLUT4 staining indicative of GLUT4 translocation compared with 55% for insulin-treated cells and 10% in control IgG-injected cells. Treatment of the cells with the phosphatidylinositol 3-kinase inhibitor wortmannin or coinjection of GST-p85 SH2 fusion protein had no effect on GTPgammaS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPgammaS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPgammaS led to the appearance of tyrosine-phosphorylated proteins around the periphery of the plasma membrane, as observed by immunostaining with PY20. Treatment of the cells with insulin caused a similar phosphotyrosine-staining pattern. Electroporation of GTPgammaS stimulated 2-deoxy-D-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPgammaS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPgammaS acts upon a signaling pathway either downstream of or parallel to activation of phosphatidylinositol 3-kinase and that this pathway involves tyrosine-phosphorylated protein(s).

3T3 Cells↗
PubMed1997

The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function.

The functionally conserved proteins CBP and p300 act in conjunction with other factors to activate transcription of DNA. A new factor, p/CIP, has been discovered that is present in the cell as a complex with CBP and is required for transcriptional activity of nuclear receptors and other CBP/p300-dependent transcription factors. The highly related nuclear-receptor co-activator protein NCoA-1 is also specifically required for ligand-dependent activation of genes by nuclear receptors. p/CIP, NCoA-1 and CBP all contain related leucine-rich charged helical interaction motifs that are required for receptor-specific mechanisms of gene activation, and allow the selective inhibition of distinct signal-transduction pathways.

Amino Acid Sequence↗
PubMed1997

A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression.

Transcriptional repression by nuclear receptors has been correlated to binding of the putative co-repressor, N-CoR. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity.

Animals↗
PubMed1997

Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300.

We report that interferon gamma (IFN-gamma) inhibits transcription of the macrophage scavenger receptor gene by antagonizing the Ras-dependent activities of AP-1 and cooperating ets domain transcription factors, apparently as a result of competition between AP-1/ets factors and activated STAT1 for limiting amounts of CBP and p300. Consistent with this model, STAT1 alpha interacts directly with CBP in cells, and microinjection of anti-CBP and anti-p300 antibodies blocks transcriptional responses to IFN-gamma. Cells lacking STAT1 fail to inhibit AP-1/ets activity, and overexpression of CBP both potentiates IFN-gamma-dependent transcription and relieves AP-1/ets repression. Thus, CBP and p300 integrate both positive and negative effects of IFN-gamma on gene expression by serving as essential coactivators of STAT1 alpha, modulating gene-specific responses to simultaneous activation of two or more signal transduction pathways.

Acetyltransferases↗
PubMed1997

Nuclear receptor coactivators.

Retinoic acid, steroid and thyroid hormones regulate complex programs of gene expression by binding to intracellular receptors that are members of the nuclear receptor superfamily of ligand-dependent transcription factors. Recent studies have led to the identification and cloning of genes encoding coactivator molecules that appear to play important roles in mediating ligand-dependent transcription by members of this family. The identification of these coactivator molecules suggests a point of entry into the general transcriptional machinery that is common to several other classes of regulated transcription factors.

Allosteric Regulation↗
PubMed1997

The small guanosine triphosphate-binding protein Rab4 is involved in insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-L1 cells.

Insulin's stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter GLUT4 to the plasma membrane. Small GTP-binding proteins have been implicated in the regulation of vesicular traffic. We studied the effects of microinjection of wild-type Rab4 glutathione S-transferase fusion protein (WT Rab4), a GTP-binding defective mutant (Rab4 N121I), a guanosine triphosphatase-defective mutant (Rab4 Q67L), and a Rab4 antibody on insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. Microinjection of Rab4 N121I and Rab4 antibodies had no effect on basal GLUT4 staining, but inhibited insulin-induced GLUT4 translocation by 50% compared with that in control IgG-injected cells. WT Rab4 and Rab4 Q67L microinjection had no effect on either basal or insulin-induced GLUT4 translocation. Premixing and coinjection of the Rab4 antibody with WT Rab4 almost completely abolished its inhibitory effect on insulin-induced GLUT4 translocation. In contrast, microinjection of an antibody directed against the highly conserved region of Rab3 proteins had no effect on insulin-induced GLUT4. These results point to a direct role of Rab4 in insulin-induced GLUT4 translocation, and that this effect is dependent on nucleotide binding to the protein. We also studied the effect of microinjection of the same proteins on insulin-induced actin filament rearrangement (membrane ruffling) in the same cell line. Microinjection of Rab4 N121I and Rab4 antibodies inhibited insulin-induced membrane ruffling by 40%, whereas WT Rab4 or a Rab3 antibody injection had no effect on cytoskeletal rearrangement. In summary, 1) Rab4 is a necessary component of the insulin/GLUT4 translocation signaling pathway; 2) the function of Rab4 in this pathway requires GTP binding; 3) Rab4 also participates in the process of insulin-induced membrane ruffling; and 4) Rab3 proteins do not seem to be involved in these processes.

3T3 Cells↗
PubMed1996

Functional roles of the Shc phosphotyrosine binding and Src homology 2 domains in insulin and epidermal growth factor signaling.

Shc is involved in the activation of Ras in response to many growth factors. Shc contains two phosphotyrosine binding domains, an Src homology 2 (SH2) domain in the carboxyl terminus of the protein and a phosphotyrosine binding (PTB) domain in the amino terminus. Since functional roles for these two domains have not been established, we microinjected glutathione S-transferase fusion proteins of either the Shc PTB or SH2 domains into fibroblasts expressing insulin and epidermal growth factor receptors and measured their effects on DNA synthesis. We found that the Shc PTB was necessary for insulin-induced mitogenic signaling, whereas the SH2 domain was not. In contrast, for epidermal growth factor signaling, the Shc SH2 was functionally more important. These differential modes of signal transduction may be an important factor in determining the specificity of the response of a cell to external stimuli.

Animals↗
PubMed1996

Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding.

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.

3T3 Cells↗