Evidence suggests that both α and βγ subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) inhibit adenylyl cyclase. Although type I cyclase is inhibited directly by exogenous βγ, inhibition G iα has not been convincingly demonstrated in vitro. Concentration-dependent cyclases purified described. Activated but oα was effective, myristoylation required. The characteristics the inhibitory effect were dependent on nature activator enzyme. concentrations required...

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a alpha subunit and high-affinity complex beta gamma subunits. There is molecular heterogeneity gamma, but the significance this diversity poorly understood. Different G protein subunits have been expressed both singly in combinations Sf9 cells. Although expression individual achieved all cases, activity (support pertussis toxin-catalyzed ADP-ribosylation rGi 1) detected only when are concurrently. Of six...

DNA regulatory elements frequently harbor multiple recognition sites for several transcriptional activators. The response mounted from such compound is often more pronounced than the simple sum of effects observed at single binding sites. determinants synergy and its control, however, are poorly understood. Through a genetic approach, we have uncovered novel protein motif that limits DNA-binding regulators. Disruption these conserved control motifs (SC motifs) selectively increases activity...

Abstract Lysophosphatidic acid receptors stimulate a Gα12/13/RhoA-dependent gene transcription program involving the serum response factor (SRF) and its coactivator oncogene, megakaryoblastic leukemia 1 (MKL1). Inhibitors of this pathway could serve as useful biological probes potential cancer therapeutic agents. Through transcription-based high-throughput element-luciferase screening assay, we identified two small-molecule inhibitors pathway. Mechanistic studies on more potent CCG-1423 show...

The beta and gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) form tightly associated complexes. To examine functional differences among the large number possible combinations unique subunits, we have synthesized characterized complexes containing 5 7, two widely distributed subunits. When either or 7 is expressed concurrently with 1 2 in a baculovirus/Sf9 cell system, all four subunit support pertussis toxin-catalyzed ADP-ribosylation rGi alpha...

Dynamin-related protein (Drp) 1 is a key regulator of mitochondrial fission and composed GTP-binding, Middle, insert B, C-terminal GTPase effector (GED) domains. Drp1 associates with sites promotes membrane constriction through its intrinsic activity. The mechanisms that regulate activity remain poorly understood but are likely to involve reversible post-translational modifications, such as conjugation small ubiquitin-like modifier (SUMO) proteins. Through detailed analysis, we find...

The voltage-gated potassium (Kv) channel Kv1.5 mediates the I Kur repolarizing current in human atrial myocytes and regulates vascular tone multiple peripheral beds. Understanding complex regulation of function is substantial interest because it represents a promising pharmacological target for treatment fibrillation hypoxic pulmonary hypertension. Herein we demonstrate that posttranslational modification by small ubiquitin-like modifier (SUMO) proteins modulates function. We have identified...

One of the most common forms functional interaction among transcription factors is more than additive effect at promoters harboring multiple copies a response element. The mechanisms that enable or control synergy such compound elements are poorly understood. We recently defined motif within negative regulatory regions operates by regulating their transcriptional synergy. have identified (SC) embedded "attenuator domain" CCAAT/enhancer-binding protein α (C/EBPα), key regulator energy...

Functional interactions between factors bound at multiple sites on DNA often lead to a synergistic or more-than-additive transcriptional response. We previously defined class of peptide sequences termed synergy control motifs (SC motifs) that function in regulators by selectively inhibiting activity driven from but not single response elements. By studying the prototypic SC glucocorticoid receptor, we show inhibit transcription per se both cis and trans, requirement for contacts with renders...

A 210-amino acid region, termed enh2, near the N terminus of rat glucocorticoid receptor, is necessary for both transcriptional activation and repression. The mechanism(s) regulation conferred by this however, are poorly understood. We screened in Saccharomyces cerevisiae a library random mutants enh2 region constitutive receptor derivative isolated series multiply substituted receptors that specifically defective activation. Although many substitutions area were tolerated, three amino...

The number of ion channels expressed on the cell surface shapes complex electrical response excitable cells. Maintaining a balance between anterograde and retrograde trafficking channel proteins is vital in regulating steady-state expression. Kv1.5 an important voltage-gated K+ cardiovascular system underlying ultra-rapid rectifying potassium current (Ikur), major repolarizing atrial myocytes, resting membrane potential excitability smooth muscle Defects expression are associated with...

The neurodegenerative disorder spinal and bulbar muscular atrophy or Kennedy disease is caused by a CAG trinucleotide repeat expansion within the androgen receptor (AR) gene. resulting expanded polyglutamine tract in N-terminal region of renders AR prone to ligand-dependent misfolding formation oligomers aggregates that are linked neuronal toxicity. How influenced post-translational modifications, however, poorly understood. target SUMOylation, this modification inhibits activity promoter...

Keratin polypeptide 8 (K8) associates noncovalently with its partners K18 and/or K19 to form the intermediate filament cytoskeleton of hepatocytes and other simple-type epithelial cells. Human K8, K18, variants predispose liver disease, whereas site-specific keratin phosphorylation confers hepatoprotection. Because stress-induced protein regulates sumoylation, we hypothesized that keratins are sumoylated in an injury-dependent manner sumoylation is important regulatory modification. We...

Expansion of the polyglutamine (polyQ) tract within androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated SBMA; however, extent to which altered contributes pathogenesis remains controversial. Here, we sought dissociate effects from polyQ-mediated proteotoxicity by enhancing activity polyQ AR. To...

Small ubiquitin-like modifier (SUMO) modification of sequence-specific transcription factors has profound regulatory consequences.By providing an intrinsic inhibitory function, SUMO isoforms can suppress transcriptional activation, particularly at promoters harboring multiple response elements.Through a comprehensive structure-function analysis, we have identified single critical sector along the second beta sheet and following alpha helix SUMO2.This distinct surface is defined by four basic...

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor selectively expressed in the adrenal cortex and gonads, where it mediates hormonal stimulation of multiple genes involved steroid hormone biosynthesis. SF-1 target both phosphorylation SUMOylation, but how these modifications interact or contribute to regulation endogenous remains poorly defined. We found that SUMOylated at K194 Y1 adrenocarcinoma cells although SUMOylation does not alter subcellular localization SF-1, modification...

Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) K313 in the N-terminal domain (NTD) K721 within ligand-binding domain. of NTD sites mediates negative effect synergy control motifs on promoters with closely spaced binding sites. There scarce evidence role SUMO conjugation to its impact transcriptional activity. We have previously shown that...

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated substrates, such as the receptor, triggering events leading to desensitization. beta ARK activity is markedly stimulated by isoprenylated gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G gamma), which translocates plasma membrane and thereby targets it substrate. amino-terminal two-thirds ARK1 composes recognition catalytic domains, while carboxyl third contains G binding...

GATA sequences are required for the optimal expression of endothelial cell-specific genes, including endothelin-1 (ET-1). We have identified PIASy in a search new GATA-2 interacting proteins that can regulate GATA-2-mediated gene expression. Notably, among cell populations comprising vascular walls, mRNA is selectively expressed cells, and its be regulated by angiogenic growth factors. show covalently modified small ubiquitin-like modifier (SUMO)-1 -2 PIASy, through E3 SUMO ligase activity,...

Abstract Multiple transcription factors, including members of the nuclear receptor family, harbor one or more copies a short regulatory motif that limits synergistic transactivation in context-dependent manner. These synergy control (SC) motifs exert their effects by serving as sites for posttranslational modification small ubiquitin-like modifier (SUMO) proteins. By analyzing requirements both and SUMOylation glucocorticoid (GR), we find an intact ligand-binding domain engaged DNA- binding...