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R H Goodman

Publications and source records attributed to R H Goodman.

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

Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling.

Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus (ci) encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that CI exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. In this paper, we demonstrate that the exogenous expression of CI that is mutant for four consensus PKA sites [CI(m1-4)], causes ectopic expression of wingless (wg) in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not CI(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of CI(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, we demonstrate that, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of CI. We also show that PKA directly phosphorylates the four consensus phosphorylation sites in vitro. Taken together, our results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of CI phosphorylation.

Animals↗
PubMed1998

The coactivator CBP stimulates human T-cell lymphotrophic virus type I Tax transactivation in vitro.

Tax interacts with the cellular cyclic AMP-responsive element binding protein (CREB) and facilitates the binding of the coactivator CREB binding protein (CBP), forming a multimeric complex on the cyclic AMP-responsive element (CRE)-like sites in the human T-cell lymphotrophic virus type I (HTLV-I) promoter. The trimeric complex is believed to recruit additional regulatory proteins to the HTLV-I long terminal repeat, but there has been no direct evidence that CBP is required for Tax-mediated transactivation. We present evidence that Tax and CBP activate transcription from the HTLV-I 21 base pair repeats on naked DNA templates. Transcriptional activation of the HTLV-I sequences required both Tax and CBP and could be mediated by either the N-terminal activation domain of CBP or the full-length protein. Fluorescence polarization binding assays indicated that CBP does not markedly enhance the affinity of Tax for the trimeric complex. Transcription analyses suggest that CBP activates Tax-dependent transcription by promoting transcriptional initiation and reinitiation. The ability of CBP to activate the HTLV-I promoter does not involve the stabilization of Tax binding, but rather depends upon gene activation properties of the co-activator that function in the context of a naked DNA template.

Acetyltransferases↗
PubMed1998

The human T-cell leukemia virus-1 transcriptional activator Tax enhances cAMP-responsive element-binding protein (CREB) binding activity through interactions with the DNA minor groove.

Tax-1, the transcriptional activation protein of human T-cell leukemia virus-1, increases transcription from the human T-cell leukemia virus-1 long terminal repeat and specific cellular promoters through interactions with cellular DNA-binding proteins. The Tax response elements (TxREs) of the long terminal repeat resemble cAMP response elements (CREs), the target of cAMP-responsive element-binding protein (CREB). CREB binds the TxRE with reduced affinity; however, the interaction is specifically enhanced by Tax. Using a fluorescence quenching method, we determined that CREB dimerizes in the absence of DNA, and that Tax does not enhance dimerization. DNA footprinting of the TxRE with 1, 10-phenanthroline-copper complex demonstrates that Tax contacts DNA and extends the footprint of CREB to GC-rich sequences flanking the core CRE-like element. The minor groove-binding drug chromomycin A3, but not distamycin A, disrupted Tax-enhanced CREB binding to the TxRE. Substitution of the guanine-rich sequences flanking the core of the TxRE with inosine residues also blocked the Tax effect. Finally, the IC-substituted TxRE binds CREB with increased affinity, suggesting flanking DNA influences the binding of CREB to the core CRE-like element. These data indicate that Tax does not regulate DNA binding of CREB by altering dimerization, but rather enhances DNA binding by additionally interacting with the minor groove of flanking DNA sequences.

Cyclic AMP Response Element-Binding Protein↗
PubMed1998

Protein kinase A directly regulates the activity and proteolysis of cubitus interruptus.

Cubitus interruptus (Ci) is a transcriptional factor that is positively regulated by the hedgehog (hh) signaling pathway. Recent work has shown that a 75-kDa proteolytic product of the full-length CI protein translocates to the nucleus and represses the transcription of CI target genes. In cells that receive the hh signal, the proteolysis of CI is inhibited and the full-length protein can activate the hh target genes. Because protein kinase A (PKA) inhibits the expression of the hh target genes in developing embryos and discs and the loss of PKA activity results in elevated levels of full-length CI protein, PKA might be involved directly in the regulation of CI proteolysis. Here we demonstrate that the PKA pathway antagonizes the hh pathway by phosphorylating CI. We show that the PKA-mediated phosphorylation of CI promotes its proteolysis from the full-length active form to the 75-kDa repressor form. The PKA catalytic subunit increases the proteolytic processing of CI and the PKA inhibitor, PKI, blocks the processing. In addition, cells do not process the CI protein to the 75-kDa repressor when all of the PKA sites in CI are mutated. Mutant CI proteins that cannot be phosphorylated by PKA have increased transcriptional activity compared with wild-type CI. In addition, exogenous PKA can increase further the transcriptional activity of the CI mutant, suggesting that PKA has a second positive, indirect effect on CI activity. In summary, we show that the modulation of the hh signaling pathway by PKA occurs directly at the level of CI phosphorylation.

Animals↗
PubMed1998

Activation and repression in the nervous system.

The mechanisms underlying transcriptional activation and repression have become much clearer. Recent evidence suggests that transcription factors that do not bind DNA directly, the co-activators and co-repressors, mediate a large number of cell signaling events. Their association with histone acetylases, to mediate activation, or deacetylases, to mediate repression, provide a model for explaining how gene expression is regulated.

CREB-Binding Protein↗
PubMed1997

The beta-adrenergic receptor kinase interacts with the amino terminus of the G protein beta subunit.

Desensitization of G protein-coupled receptors involves phosphorylation of the receptors by G protein-coupled receptor kinases, such as the beta-adrenergic receptor kinase (beta ARK). beta ARK activity depends upon its translocation from the cytoplasm to the membrane. The beta gamma subunits of G proteins bind to beta ARK and recruit the kinase to the membrane. The G beta gamma binding domain is localized to a carboxyl terminal region of beta ARK but the beta ARK binding domain of G beta gamma is not known. We used the yeast two-hybrid assay to characterize the interaction between G beta and beta ARK. We demonstrate an interaction between the carboxyl terminus of beta ARK and G beta 2. The strength of this interaction is increased when the VP16 transactivation domain is placed on the carboxyl end of G beta 2, indicating that an accessible G beta 2 amino terminus is important for its interaction with beta ARK. In addition, we show that amino acids 1 to 145 of G beta 2 are sufficient for beta ARK binding.

Amino Acid Sequence↗
PubMed1997

Construction of a 1.2-Mb contig surrounding, and molecular analysis of, the human CREB-binding protein (CBP/CREBBP) gene on chromosome 16p13.3.

In the interest of cloning and analyzing the genes responsible for two very different diseases, the Rubinstein-Taybi syndrome (RTS) and acute myeloid leukemia (AML) associated with the somatic translocation t(8;16)(p11;p13.3), we constructed a high-resolution restriction map of contiguous cosmids (contig) covering 1.2 Mb of chromosome 16p13.3. By fluorescence in situ hybridization and Southern blot analysis, we assigned all tested RTS and t(8;16) translocation breakpoints to a 100-kb region. We have previously reported exact physical locations of these 16p breakpoints, which all disrupt one gene we mapped to this interval: the CREB-binding protein (CBP or CREBBP) gene. Intriguingly, mutations in the CBP gene are responsible for RTS as well as the t(8;16)-associated AML. CBP functions as an integrator in the assembly of various multiprotein regulatory complexes and is thus necessary for transcription in a broad range of transduction pathways. We report here the cloning, physical mapping, characterization, and full cDNA nucleotide sequence of the human CBP gene.

Amino Acid Sequence↗
PubMed1997

Drosophila CBP is a co-activator of cubitus interruptus in hedgehog signalling.

The transcription factor CBP, originally identified as a coactivator for CREB, enhances transcription mediated by many other transcription factors. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haploinsufficiency disorder characterized by abnormal pattern formation, but the mechanism by which decreased CBP levels affect pattern formation is unclear. The hedgehog (hh) signalling pathway is an important determinant of pattern formation. cubitus interruptus (ci), a component in hh signalling, encodes a transcription factor homologous to the Gli family of proteins and is required for induction of the hh-dependent expression of patched (ptc), decapentaplegic (dpp) and wingless (wg). Haploinsufficiency for the ci-related transcription factor Gli3 causes phenotypic changes in mice (known as 'extra-toes) and humans (Greig's cephalopolysyndactyly syndrome) that have similarities to Rubinstein-Taybi syndrome. Here we show that Drosophila CBP (dCBP) functions as a coactivator of Ci, suggesting that the dCBP-Ci interaction may shed light on the contribution of CBP to pattern formation in mammals.

Animals↗
PubMed1997

Differential activation of viral and cellular promoters by human T-cell lymphotropic virus-1 tax and cAMP-responsive element modulator isoforms.

We have previously proposed that cAMP-responsive element-binding protein (CREB) activity is stimulated by human T-cell lymphotropic virus-1 (HTLV-1) Tax through two mechanisms that are differentially dependent upon CREB phosphorylation. We have tested this model by examining how Tax affects transcriptional activation mediated by the cAMP-responsive element (CRE) modulator (CREM). The CREM proteins are highly homologous to CREB, particularly in their DNA-binding domains and the kinase-inducible domain (KID), a region that interacts with the coactivator CREB-binding protein (CBP) in a phosphorylation-dependent manner. Despite this similarity, most CREM isoforms are transcriptional repressors. CREMalpha lacks the glutamine-rich domains found in CREB that are essential for transcriptional activation. We show that the normally repressive CREMalpha activates the HTLV-1 and cellular CREs in the presence of Tax; activation of the viral element is phosphorylation-independent, and activation of the cellular CRE is phosphorylation-dependent. CREMDelta(C-G) lacks both the KID and the glutamine-rich regions. This isoform activates the HTLV-1 long terminal repeat in a phosphorylation-independent manner, but does not activate the cellular CRE. This study suggests that Tax, interacting with the basic/zipper region of CREM, recruits CBP to the viral promoter. Tax activation of the cellular CRE depends on the KID and its ability to interact with CBP in a phosphorylation-dependent manner.

Animals↗
PubMed1997

The CRE-binding protein dCREB-A is required for Drosophila embryonic development.

We have previously described the cloning of a cyclic AMP response-element (CRE)-binding protein, dCREB-A, in Drosophila melanogaster that is similar to the mammalian CRE-binding protein CREB. dCREB-A is a member of the bZIP family of transcription factors, shows specific binding to the (CRE), and can activate transcription in cell culture. In this report, we describe the gene structure for dCREB-A, protein expression patterns throughout development and the necessary role for this gene in embryogenesis. The 4.5-kb transcript is encoded in six exons that are distributed over 21 kb of DNA. There are seven start sites and no TATA consensus sequences upstream. The dCREB-A protein is expressed in the nuclei of the embryonic salivary gland, proventriculus and stomadeum. Late in embryogenesis, tracheal cell nuclei and specific nuclei within the segments show staining with anti-dCREB-A antibodies. In adult female ovaries, dCREB-A is expressed in the stage 9 through stage 11 follicle cell nuclei. Null mutations of the dCREB-A gene give rise to animals that no longer express dCREB-A protein and die late in embryogenesis before or at hatching. The absolute requirement of dCREB-A for embryogenesis demonstrates a nonredundant function for a CRE-binding protein that will be useful in studying the role of specific signal transduction cascades in development.

Animals↗
PubMed1997

The multifunctional role of the co-activator CBP in transcriptional regulation.

One of the most studied and best-understood examples of second messenger-regulated gene transcription involves the activation of genes by the cyclic AMP pathway: stimulation of several hormone, growth factor, and neurotransmitter receptors activates adenylyl cyclase, generating cyclic AMP that, by binding to the regulatory subunit of protein kinase A (PKA), dissociates the PKA catalytic subunit. The free catalytic subunit is transported to the nucleus where it phosphorylates and consequently activates the transcription factor CREB. This phosphorylation of CREB allows interaction with the co-activator CBP, which binds to components of the basal transcriptional machinery. CBP and its homologue p300 are targets for several viral-transforming proteins, implying that these co-activators have a more extensive role in cellular function. Indeed, recent studies have demonstrated that multiple transcription factors bind to CBP, including c-jun, c-myb, MyoD, E2F1, YY1, and members of the steroid hormone receptor superfamily, although it is not yet clear which of these transcription factors depend upon CBP for function. Determining exactly which transcriptional pathways require CBP in vivo and which genes are activated by CBP will provide an important clue in developmental regulation and cell cycle control, since mutations in the human CBP gene have been found to cause developmental abnormalities and a predisposition for some types of cancer. In this review, we will discuss the mechanisms involved in the PKA-dependent activation of CREB and describe how the co-activator CBP and its homologue are involved in this process. In addition, we will outline the various transcription factor pathways that CBP has been proposed to activate. Finally, we will discuss the possible role of CBP in cellular transformation and differentiation.

Animals↗
PubMed1996

A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP.

The Escherichia coli tet-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein-protein interactions can be identified by a positive selection. This assay, designated the "split-hybrid system," contains a two-component reporter. The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator binding sites upstream of HIS3. Interaction of one protein fused to the LexA DNA binding domain with a second protein fused to the VP16 activation domain results in TetR expression. TetR subsequently binds to the tet operators, blocking the expression of HIS3 and preventing yeast growth in media lacking histidine. The utility of the split-hybrid system was analyzed by examining the phosphorylation-dependent interaction of CREB and its coactivator CREB binding protein (CBP). CREB and CBP associate through an interaction that depends upon CREB phosphorylation at Ser-133. Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybrid strains. The split-hybrid system was used to identify other CREB mutations that disrupt its association with CBP. These mutations localized around the site of CREB phosphorylation, indicating that only a small portion of the CREB activation domain is required for CBP interaction. The yeast split-hybrid system should be useful in identifying mutations, proteins, peptides, and drugs that disrupt protein-protein interactions.

Amino Acid Sequence↗
PubMed1996

Analysis of the structural properties of cAMP-responsive element-binding protein (CREB) and phosphorylated CREB.

The transcription factor CREB (cAMP responsive element binding protein) is activated by protein kinase A (PKA) phosphorylation of a single serine residue. To investigate possible mechanisms of CREB regulation by phosphorylation, we initiated a structural and biophysical characterization of the full-length, wild-type CREB protein, an altered CREB protein (CREB/SER) in which the three cysteine residues in the DNA-binding domain were replaced with serine residues and a truncated protein (ACT265) which encompasses the entire activation domain of CREB. Circular dichroism (CD) reveals that CREB and CREB/SER have identical secondary structures and contain approximately 20% alpha-helix, 9% beta-strand, 34% beta-turn, and 37% random coil structures. PKA phosphorylation does not alter the CD spectra, and therefore the secondary structure, of CREB or of CREB bound to DNA. Protease cleavage patterns indicate that PKA phosphorylation does not induce a global conformational change in CREB. Furthermore, PKA phosphorylation does not change the DNA binding affinity of CREB for either canonical or non-canonical CRE sequences as measured by a fluorescence anisotropy DNA binding assay. Since PKA phosphorylation of CREB results in its specific binding to the transcriptional co-activators CREB-binding protein and p300, we suggest that the PKA activation of CREB occurs by the production of specific, complementary interactions with these proteins, rather than through the previously proposed mechanisms of a phosphorylation-dependent conformational change or increased DNA binding affinity.

Amino Acid Sequence↗
PubMed1996

Control of cAMP-regulated enhancers by the viral transactivator Tax through CREB and the co-activator CBP.

The Tax protein of human T-lymphotropic virus (HTLV)-1 activates expression of the HTLV-1 long terminal repeat through a DNA element that resembles the cellular cyclic AMP-regulated enhancer (CRE). Tax contains a transcriptional activation domain, but its ability to activate gene expression depends on interactions with cellular CRE-binding proteins such as CREB. Whether Tax can activate the expression of cellular CRE-containing genes has been controversial. Here we show that Tax can activate both the HTLV-1 and consensus cellular CREs, and propose that this activation may occur through mechanisms that are differentially dependent on CREB phosphorylation. Tax not only increases the binding of CREB to the viral CRE but also recruits the transcriptional co-activator CBP in a manner independent of CREB phosphorylation. In contrast, association of Tax with the cellular CRE occurs through CBP which, in turn, is recruited only in the presence of phosphorylated CREB.

Base Sequence↗
PubMed1996

Fluorescence polarization analysis of protein-DNA and protein-protein interactions.

Fluorescence polarization is a powerful technique for characterizing macromolecular associations and can provide equilibrium determinations of protein-DNA and protein-protein interactions. This technique is particularly useful (and better suited than electrophoretic methods) to study low affinity protein-protein interactions. In this review, we have outlined the principles underlying the use of fluorescence polarization to study the assembly of higher order complexes that bind to the CRE. The availability of simple, relatively inexpensive instrumentation means that this technology is no longer only within the realm of the physical biochemist.

Carrier Proteins↗
PubMed1995

Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP.

The Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome with facial abnormalities, broad thumbs, broad big toes and mental retardation as the main clinical features. Many patients with RTS have been shown to have breakpoints in, and microdeletions of, chromosome 16p13.3 (refs 4-8). Here we report that all these breakpoints are restricted to a region that contains the gene for the human CREB binding protein (CBP), a nuclear protein participating as a co-activator in cyclic-AMP-regulated gene expression. We show that RTS results not only from gross chromosomal rearrangements of chromosome 16p, but also from point mutations in the CBP gene itself. Because the patients are heterozygous for the mutations, we propose that the loss of one functional copy of the CBP gene underlies the developmental abnormalities in RTS and possibly the propensity for malignancy.

Amino Acid Sequence↗
PubMed1995

Adenoviral E1A-associated protein p300 as a functional homologue of the transcriptional co-activator CBP.

The 265K nuclear protein CBP was initially identified as a co-activator for the protein kinase A (PKA)-phosphorylated form of the transcription factor CREB. The domains in CBP that are involved in CREB binding and transcriptional activation are highly related to the adenoviral E1A-associated cellular protein p300 (refs 2, 3), and to two hypothetical proteins from Caenorhabditis elegans, R10E11.1 and K03H1.10 (refs 4 and 5, respectively), whose functions are unknown. Here, we show that CBP and p300 have similar binding affinity for the PKA-phosphorylated form of CREB, and that p300 can substitute for CBP in potentiating CREB-activated gene expression. We find that E1A binds to CBP through a domain conserved with p300 and represses the CREB-dependent co-activator functions of both CBP and p300. Our results indicate that the gene repression and cell immortalization functions associated with E1A involve the inactivation of a family of related proteins that normally participate in second-messenger-regulated gene expression.

Adenovirus E1A Proteins↗