scholarly journals Esterase 1 is a Novel Transcriptional Repressor of Growth Hormone Receptor Gene Expression: A Unique Noncatalytic Role for a Carboxyesterase Protein

2011 ◽  
Vol 25 (8) ◽  
pp. 1351-1363
Author(s):  
Jinhong Sun ◽  
P. Anil Kumar ◽  
Jamuna Thimmarayappa ◽  
Natinder Saini ◽  
Pooja Goel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Growth Hormone Receptor ◽  
Chromosome Region ◽  
Receptor Gene ◽  
Nuclear Export Signal ◽  
Subcellular Fractionation ◽  
Phage Clone ◽  
Expression Library

The pleiotropic actions of GH result from its engagement with the GH receptor (GHR). GHR expression is regulated by free fatty acids (FFA). A cDNA phage expression library was screened to identify a phage clone expressing esterase 1 (ES1) binding to the FFA-response element (FARE), L2-D1, in the murine GHR promoter. Ectopically expressed ES1 inhibited GHR promoter activity via effects at two FARE, L2-D1 and L2-A2. Chromatin immunoprecipitation experiments demonstrated specific association of ES1 with the FARE. Catalytically inactive ES1 retained inhibitory activity on the GHR promoter and excluded the possibility that the effect on the GHR promoter was an indirect effect secondary to ES1's actions on the intracellular metabolism of FFA. Ectopically expressed ES1 inhibited the endogenous GHR mRNA and protein expression in 3T3-F442A preadipocytes. Subcellular fractionation and confocal microscopy established that ES1 localizes both to the cytoplasm and the nucleus. Experiments demonstrated chromosome region maintenance 1-dependent nuclear export and the presence of a functional nuclear export signal in ES1. The domain of ES1 responsible for the effect on the GHR promoter was localized to the C-terminal portion of the protein. The in vivo significance of ES1's effect on GHR expression was suggested by decreased liver GHR mRNA expression in mice on a high-fat diet correlating with increased steady-state abundance of liver ES1 mRNA. Our results identify and characterize ES1 as a novel transcriptional regulator of GHR gene expression, thereby establishing a unique nonenzymatic role for a carboxyesterase and expanding the potential biological roles of this protein superfamily.

scholarly journals Exportin Crm1 is important for Swi6 nucleocytoplasmic shuttling and MBF transcription activation in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Kenneth D. Belanger ◽  
William T. Yewdell ◽  
Matthew F. Barber ◽  
Amy N. Russo ◽  
Mark A. Pettit ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Transcription Factor ◽  
Nuclear Export ◽  
Intracellular Localization ◽  
Nuclear Export Signal ◽  
Transcription Activation ◽  
Cell Phenotype ◽  
Export Signal

AbstractThe Swi6 protein acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. While the process of Swi6 nuclear import is well understood, mechanisms underlying its nuclear export remain unclear. Here we investigate Swi6 nuclear export and its impact on Swi6 function. We show that the exportin Crm1, in addition to three other karyopherins previously shown to affect Swi6 localization, is important for Swi6 nuclear export and activity. A truncation of Swi6 that removes a putative Crm1 nuclear export signal results in the loss of changes in nucleocytoplasmic Swi6 localization that normally occur during progression through the cell cycle. Mutagenesis of the NES-like sequence or removal of Crm1 activity using leptomycin B results in a similar decrease in nuclear export as cells enter S-phase. Using two-hybrid analysis, we also show that Swi6 associates with Crm1 in vivo. Alteration of the Crm1 NES in Swi6 results in a decrease in MBF-mediated gene expression, but does not affect expression of an SBF reporter, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the Crm1 NES in Swi6 results in cells that are larger than wild type, but not to the extent of those with a complete Swi6 deletion. Expressing a Swi6 NES mutant in combination with a deletion of Msn5, an exportin involved in Swi6 nuclear export and specifically affecting SBF activation, further increases the large cell phenotype, but still not to the extent observed in a Swi6 deletion mutant. These data suggest that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal and influences expression of a different subset of Swi6-controlled genes.Summary StatementPrecise intracellular localization is important for the proper activity of proteins. Here we provide evidence that the Swi6 transcription factor important for cell cycle progression shuttles between the cell nucleus and cytoplasm, its nuclear export is important for its activity, and that it contains a nuclear export signal (NES) recognized by the Crm1 nuclear transport factor.

scholarly journals c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in αT3-1 Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 839-849 ◽  
Author(s):  
Buffy S. Ellsworth ◽  
Brett R. White ◽  
Ann T. Burns ◽  
Brian D. Cherrington ◽  
Annette M. Otis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Model ◽  
Receptor Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in αT3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in αT3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

scholarly journals Structural determinants of nuclear export signal orientation in binding to exportin CRM1

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Ho Yee Joyce Fung ◽  
Szu-Chin Fu ◽  
Chad A Brautigam ◽  
Yuh Min Chook
Keyword(s):  
Crystal Structures ◽  
Nuclear Export ◽  
Chromosome Region ◽  
Nuclear Export Signal ◽  
Opposite Orientation ◽  
Structural Determinants ◽  
Large Expansion ◽  
Export Signal

The Chromosome Region of Maintenance 1 (CRM1) protein mediates nuclear export of hundreds of proteins through recognition of their nuclear export signals (NESs), which are highly variable in sequence and structure. The plasticity of the CRM1-NES interaction is not well understood, as there are many NES sequences that seem incompatible with structures of the NES-bound CRM1 groove. Crystal structures of CRM1 bound to two different NESs with unusual sequences showed the NES peptides binding the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). Comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome.

Abstract P191: Calpain-Generated Free PKCα Catalytic Domains Induce HDAC5 Nuclear Export and Regulate Cardiac Gene Transcription

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Yan Zhang ◽  
Scot Matkovich ◽  
Abhinav Diwan ◽  
Min-Young Kang ◽  
Gerald W Dorn
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Nuclear Export ◽  
Kinase Inhibitor ◽  
Proteolytic Processing ◽  
Full Length ◽  
Independent Effect ◽  
Independent Manner ◽  
Cardiac Gene

Receptor-mediated activation of protein kinase (PK) C is a central pathway regulating cell growth, homeostasis, and programmed death. Recently, we showed that calpain-mediated proteolytic processing of PKC in ischemic myocardium activates PKC signaling in a receptor-independent manner by releasing a persistent and constitutively active free catalytic C-terminal fragment, PKCα-CT. This unregulated kinase provokes cardiomyopathy, but the mechanisms remain unclear. We examined hypothesis that PKCα-CT has transcriptional activity. Using immunoblot analysis and confocal microscopy, we found that PKCα-CT localized in part to nuclei and spontaneously induced cytosolic relocalization HDAC5 of the transcriptional regulator. Co- expression of calpain 1 with full length PKCα can generate PKCα-CT and produced the same HDAC5 cytosolic relocalization, whereas full length PKCα alone had no such effect. HDAC5 cytosolic relocalization induced by PKCα-CT was abolished by the protein kinase inhibitor GO6976, but not by PKD inhibitor CID 755673. The in vivo relevance of these findings was examined in transgenic mice expressing PKCα and PKCα-CT. To assess the consequence on gene expression, we performed global transcriptome profiling by Affymetrix microarrays and mRNA sequencing. The two techniques substantially agreed. Compared to control hearts, 621 mRNAs were regulated at least 1.3 fold in PKCα-CT hearts (P< 0.001), only 59 in full-length PKCα hearts. MEF2-dependent inflammatory pathway genes which are putative HDAC targets were upregulated in PKCα-CT heart: 15 MEF2 target mRNAs were upregulated in PKCα-CT hearts (p<0.001), only one in PKCα hearts. These results reveal that PKCα-CT is a potent regulator of pathological cardiac gene expression by localizing to nuclei and directly promoting nuclei-cytoplasmic shuttling of HDAC5. Receptor-independent effect of PKCα-CT and HDAC phosphorylation in ischemic hearts has broad ramifications for understanding and preventing the pathological transcriptional stress response.

scholarly journals ADAR1 Interacts with NF90 through Double-Stranded RNA and Regulates NF90-Mediated Gene Expression Independently of RNA Editing

2005 ◽  
Vol 25 (16) ◽  
pp. 6956-6963 ◽  
Author(s):  
Yongzhan Nie ◽  
Li Ding ◽  
Peter N. Kao ◽  
Robert Braun ◽  
Jing-Hua Yang
Keyword(s):  
Gene Expression ◽  
Rna Editing ◽  
Nuclear Export ◽  
Fetal Liver ◽  
Regulation Of Gene Expression ◽  
Nuclear Export Signal ◽  
Binding Domain ◽  
Double Stranded Rna ◽  
Recognition Element ◽  
Dsrna Binding

ABSTRACT The RNA-editing enzyme ADAR1 modifies adenosines by deamination and produces A-to-I mutations in mRNA. ADAR1 was recently demonstrated to function in host defense and in embryonic erythropoiesis during fetal liver development. The mechanisms for these phenotypic effects are not yet known. Here we report a novel function of ADAR1 in the regulation of gene expression by interacting with the nuclear factor 90 (NF90) proteins, known regulators that bind the antigen response recognition element (ARRE-2) and have been demonstrated to stimulate transcription and translation. ADAR1 upregulates NF90-mediated gene expression by interacting with the NF90 proteins, including NF110, NF90, and NF45. A knockdown of NF90 with small interfering RNA suppresses this function of ADAR1. Coimmunoprecipitation and double-stranded RNA (dsRNA) digestion demonstrate that ADAR1 is associated with NF110, NF90, and NF45 through the bridge of cellular dsRNA. Studies with ADAR1 deletions demonstrate that the dsRNA binding domain and a region covering the Z-DNA binding domain and the nuclear export signal comprise the complete function of ADAR1 in upregulating NF90-mediated gene expression. These data suggest that ADAR1 has the potential both to change information content through editing of mRNA and to regulate gene expression through interacting with the NF90 family proteins.

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