scholarly journals Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines.

1993 ◽  
Vol 13 (11) ◽  
pp. 7180-7190 ◽  
Author(s):  
W Kaszubska ◽  
R Hooft van Huijsduijnen ◽  
P Ghersa ◽  
A M DeRaemy-Schenk ◽  
B P Chen ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Consensus Sequence ◽  
Direct Interaction ◽  
Nf Kappa B ◽  
Independent Manner ◽  
Protein Protein Interaction ◽  
Responsive Element ◽  
And Function

We previously reported that NF-kappa B and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-kappa B in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-kappa B to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-kappa B is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines

1993 ◽  
Vol 13 (11) ◽  
pp. 7180-7190 ◽  
Author(s):  
W Kaszubska ◽  
R Hooft van Huijsduijnen ◽  
P Ghersa ◽  
A M DeRaemy-Schenk ◽  
B P Chen ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Consensus Sequence ◽  
Direct Interaction ◽  
Nf Kappa B ◽  
Independent Manner ◽  
Protein Protein Interaction ◽  
Responsive Element ◽  
And Function

We previously reported that NF-kappa B and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-kappa B in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-kappa B to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-kappa B is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

scholarly journals Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

2002 ◽  
Vol 364 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Gareth J.O. EVANS ◽  
Alan MORGAN
Keyword(s):  
Rat Brain ◽  
Direct Interaction ◽  
Secretory Vesicle ◽  
Brain Membrane ◽  
Synaptotagmin I ◽  
Phosphorylated Form ◽  
Order Of Magnitude ◽  
And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

scholarly journals Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485 ◽  
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

scholarly journals Molecular Characterization of the Mg2+-Responsive PhoP-PhoQ Regulon in Salmonella enterica

2003 ◽  
Vol 185 (21) ◽  
pp. 6287-6294 ◽  
Author(s):  
Sergio Lejona ◽  
Andrés Aguirre ◽  
María Laura Cabeza ◽  
Eleonora García Véscovi ◽  
Fernando C. Soncini
Keyword(s):  
Transcriptional Regulation ◽  
Salmonella Enterica ◽  
Response Regulator ◽  
Consensus Sequence ◽  
Direct Interaction ◽  
Direct Repeat ◽  
Infection Process ◽  
Promoter Regions

ABSTRACT The PhoP/PhoQ two-component system controls the extracellular magnesium deprivation response in Salmonella enterica. In addition, several virulence-associated genes that are mainly required for intramacrophage survival during the infection process are under the control of its transcriptional regulation. Despite shared Mg2+ modulation of the expression of the PhoP-activated genes, no consensus sequence common to all of them could be detected in their promoter regions. We have investigated the transcriptional regulation and the interaction of the response regulator PhoP with the promoter regions of the PhoP-activated loci phoPQ, mgtA, slyB, pmrD, pcgL, phoN, pagC, and mgtCB. A direct repeat of the heptanucleotide sequence (G/T)GTTTA(A/T) was identified as the conserved motif recognized by PhoP to directly control the gene expression of the first five loci, among which the first four are ancestral to enterobacteria. On the other hand, no direct interaction of the response regulator with the promoter of phoN, pagC, or mgtCB was apparent by either in vitro or in vivo assays. These loci are Salmonella specific and were probably acquired by horizontal DNA transfer. Besides, sequence analysis of pag promoters revealed the presence of a conserved PhoP box in 6 out of the 12 genes analyzed. Our results strongly suggest that the expression of a set of Mg2+-controlled genes is driven by PhoP via unknown intermediate regulatory mechanisms that could also involve ancillary factors.

scholarly journals Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta.

1995 ◽  
Vol 15 (9) ◽  
pp. 4971-4979 ◽  
Author(s):  
B Stein ◽  
M X Yang
Keyword(s):  
Estrogen Receptor ◽  
Bone Resorption ◽  
Interleukin 6 ◽  
Binding Sites ◽  
Hormone Replacement ◽  
Direct Interaction ◽  
Nf Kappa B ◽  
Two Factors

Bone metabolism is regulated by a balance between bone resorption caused by osteoclasts and bone formation caused by osteoblasts. This balance is disturbed in postmenopausal women as a result of lower serum estrogen levels. Estrogen, which is used in hormone replacement therapy to prevent postmenopausal osteoporosis, downregulates expression of the interleukin 6 (IL-6) gene in osteoblasts and bone marrow stromal cells. IL-6 is directly involved in bone resorption by activating immature osteoclasts. We show here that NF-kappa B and C/EBP beta are important regulators of IL-6 gene expression in human osteoblasts. Importantly, the IL-6 promoter is inhibited by estrogen in the absence of a functional estrogen receptor (ER) binding site. This inhibition is mediated by the transcription factors NF-kappa B and C/EBP beta. Evidence is presented for a direct interaction between these two factors and ER. We characterized the protein sequence requirements for this association in vitro and in vivo. The physical and functional interaction depends in part on the DNA binding domain and region D of ER and on the Rel homology domain of NF-kappa B and the bZIP region of C/EBP beta. The cross-coupling between ER, NF-kappa B, and C/EBP beta also results in reduced activity of promoters with ER binding sites. We further show that the mechanism of IL-6 gene repression by estrogen is clearly different from that of activation of promoters with ER binding sites. Therefore, drugs that separate the transactivation and transrepression functions of ER will be very helpful for treatment of osteoporosis without causing undesirable side effects.

scholarly journals The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers.

1995 ◽  
Vol 15 (2) ◽  
pp. 872-882 ◽  
Author(s):  
M K Ernst ◽  
L L Dunn ◽  
N R Rice
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Interaction ◽  
Terminal Region ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Functional Capabilities ◽  
C Terminus

In most cells, proteins belonging to the Rel/NF-kappa B family of transcription factors are held in inactive form in the cytoplasm by an inhibitor protein, I kappa B alpha. Stimulation of the cells leads to degradation of the inhibitor and transit of active DNA-binding Rel/NF-kappa B dimers to the nucleus. I kappa B alpha is also able to inhibit DNA binding by Rel/NF-kappa B dimers in vitro, suggesting that it may perform the same function in cells when the activating signal is no longer present. Structurally, the human I kappa B alpha molecule can be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of six ankyrin-like repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. In this study we examined how the structural elements of the I kappa B alpha protein correlate with its functional capabilities both in vitro and in vivo. Using a battery of I kappa B alpha mutants, we show that (i) a dimer binds a single I kappa B alpha molecule, (ii) the acidic C-terminal region of I kappa B alpha is not required for protein-protein binding and does not mask the nuclear localization signal of the dimer, (iii) the same C-terminal region is required for inhibition of DNA binding, and (iv) this inhibition may be accomplished by direct interaction between the PEST-like region and the DNA-binding region of one of the subunits of the dimer.

The RARa-PLZF Oncogenic Protein Inhibits C/EBPa Function in Myeloid Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1825-1825
Author(s):  
Mathieu Tremblay ◽  
Nathalie Girard ◽  
Andre Haman ◽  
Benoit Grondin ◽  
Nathalie Bouchard ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Fusion Gene ◽  
Direct Interaction ◽  
Histone Deacetylation ◽  
Leukemic Cells ◽  
Protein Protein Interaction ◽  
Primary Bone ◽  
Oncogenic Protein

Abstract In acute promyelocytic leukemia (APL), the variant t(15;17) translocation is responsive to differentiation therapy with retinoic acid (RA) while the t(11;17) APL is a more aggressive disease with poor prognosis. The latter produces two fusion proteins, PLZF-RARa and RARa-PLZF, and both proteins are required for leukemogenesis. To define the role of RARa-PLZF, we ectopically expressed the fusion gene in 32D cells and in primary bone marrow cells. First, our results show that RARa-PLZF inhibits myeloid gene expression, specifically CEBPa targets, which fulfill important function in cell survival and differentiation along the granulocytic lineage. Second, we found that repression by RARa-PLZF is dependent on the binding of C/EBPa to its cognate sequence in the promoter of CEBPa target gene, GCSFR. Third, we confirmed by chromatin immuprecipitation that RARa-PLZF associate with C/EBPa on DNA. Fourth, we showed that as PLZF, RARa-PLZF interact directly with HDAC1 and that this interaction causes a deacetylation of histone H3 at the promoter. This inhibition is reversed by treatment with histone deacetylase inhibitor (TSA) both in vitro and in vivo. Thus, this repression is dependent on direct interaction of RP with C/EBPa and recruitment of HDAC1, causing histone deacetylation at C/EBPa target loci. Finally, our data indicate that C/EBPa activity is severely impaired in leukemic cells from patients with t(11;17) APL, as compared to the t(15;17) APL, which is more amenable to therapy. In summary, our study indicates that the oncogene RARa-PLZF inhibits C/EBPa function through direct protein-protein interaction, and thus contributes to leukemogenesis in t(11;17) APL.

scholarly journals Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor

2002 ◽  
Vol 157 (2) ◽  
pp. 303-314 ◽  
Author(s):  
Masaki Kato ◽  
Millan S. Patel ◽  
Regis Levasseur ◽  
Ivan Lobov ◽  
Benny H.-J. Chang ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Osteoblast Proliferation ◽  
Targeted Disruption ◽  
Independent Manner ◽  
Wnt Proteins ◽  
Density Lipoprotein Receptor ◽  
And Function

The low-density lipoprotein receptor–related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.

scholarly journals Cardioprotective actions of the sodium-activated potassium channel Slack (aka Slo2.2, KNa1.1 or KCNT1)

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Kuret ◽  
R Ehinger ◽  
H Bischof ◽  
A Luczak ◽  
A Bausch ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Research Unit ◽  
In Vivo Model ◽  
Extracellular Potassium ◽  
Funding Source ◽  
Cardiac Damage ◽  
Independent Manner ◽  
And Function

Abstract Background Sodium-activated potassium channels (KNa) Slack (sequence like a calcium-activated potassium channel, aka Slo 2.2, KNa1.1 or KCNT1) are widely expressed throughout neuronal tissue, whereas their presence and function in the cardiovascular system is not well understood. Due to high intracellular sodium concentrations ([Na+]i) necessary to induce half-maximal channel activation, we hypothesized that Slack function is attributed to pathophysiological conditions such as myocardial ischemia. Purpose To elucidate the putative functions of Slack in the murine heart and in cardiomyocytes (CMs) and to explore whether the ischemia and reperfusion (I/R)-induced cardiac damage is affected by endogenous Slack channel activity. Methods I/R injury was evaluated in global and CM-specific Slack knockout mice (Slack gKO, CM Slack KO) and compared to litter-matched controls (Slack gWT, CM Slack CTR) by applying an in vivo model of acute myocardial infarction (MI). Infarct size (IS) was assessed at baseline, after ischemic pre- (iPre) and postconditioning (iPost) and in response to cinaciguat (CIN), a cGMP-elevating agent. Moreover, Slack expression and function in CMs was studied by biochemical and electrophysiological means and by utilizing the newly developed FRET-based K+ probe GEPII 1.0. Results IS in Slack gKO mice was increased in comparison to gWT littermates. In addition, the cardioprotection afforded by iPost was attenuated in the absence of Slack. To test if the increased vulnerability to I/R injury of the Slack gKO mouse model was originating from Slack activity in CMs, we subjected CM-specific Slack CTR and KO mutants to an identical MI procedure. IS measurements confirmed increased cardiac damage at baseline and reduced cardioprotective effects afforded by iPre and iPost in CM Slack KO mice. Interestingly, CIN (i.p., 30 min prior to I/R) reduced IS to a similar extent in both genotypes, suggesting that Slack functions in a cGMP-independent manner. Whole-cell patch clamp experiments on CMs demonstrated a reduction of the KNa-inhibitors clofilium- and chinidine-sensitive K+ outward currents in Slack gKO CM. Extracellular potassium ([K+]ex) accumulation measured with GEPII 1.0 was lower in Slack gKO versus gWT CM pools exposed to membrane permeabilizing agent digitonin. Accordingly, [K+]ex evoked by the Slack activators niclosamide and bithionol was lower in the absence of functional Slack in CMs. Conclusion The presented findings establish an important role of Slack channels for cardioprotective signalling mechanisms during I/R in vivo and for mediating beneficial effects of mechanical conditioning on IS. Corroborating in vitro studies on adult CMs exhibit an impaired [K+]ex dynamic in response to genetic or pharmacological modulation of Slack activity. Thus, we conclude that Slack-dependent K+ signalling pathways in CMs may represent a promising drug target that renders the heart muscle less vulnerable to the I/R-induced damage. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Work in the authors' laboratories is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (to R.L.) and the DFG Research Unit 2060, “cGMP Signaling in Cell Growth and Survival” (to R.L. and P.R.).

Sign in / Sign up

Export Citation Format

Share Document