scholarly journals Protein Kinase A Regulates Cholinergic Gene Expression in PC12 Cells: REST4 Silences the Silencing Activity of Neuron-Restrictive Silencer Factor/REST

1999 ◽  
Vol 19 (10) ◽  
pp. 6788-6795 ◽  
Author(s):  
Masahito Shimojo ◽  
Alice J. Paquette ◽  
David J. Anderson ◽  
Louis B. Hersh
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Pc12 Cells ◽  
Gene Locus ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Mobility Shift ◽  
Kinase A

ABSTRACT The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A-deficient PC12 mutant, A126.1B2, in which transcription of the gene is reduced. The site of action of protein kinase A was localized to a neuron-restrictive silencer element/repressor element 1 (NRSE/RE-1) sequence within the cholinergic gene. Neuron-restrictive silencer factor (NRSF)/RE-1-silencing transcription factor (REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2 cells. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform REST4 was expressed in PC12 cells but not in A126.1B2. REST4 inhibited binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Coimmunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in A126.1B2 was sufficient to transcriptionally activate the cholinergic gene locus. Thus, in PC12 cells, protein kinase A promotes the production of REST4, which inhibits repression of the cholinergic gene locus by NRSF/REST.

scholarly journals The Cholinergic Gene Locus Is Coordinately Regulated by Protein Kinase A II in PC12 Cells

2002 ◽  
Vol 71 (3) ◽  
pp. 1118-1126 ◽  
Author(s):  
Masahito Shimojo ◽  
Donghai Wu ◽  
Louis B. Hersh
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Pc12 Cells ◽  
Gene Locus ◽  
Kinase A

scholarly journals Physiological Phosphorylation of Protein Kinase A at Thr-197 Is by a Protein Kinase A Kinase

1998 ◽  
Vol 18 (3) ◽  
pp. 1416-1423 ◽  
Author(s):  
Robert D. Cauthron ◽  
Karen B. Carter ◽  
Susanne Liauw ◽  
Robert A. Steinberg
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Time Course ◽  
Catalytic Subunit ◽  
Wild Type ◽  
Mobility Shift ◽  
Efficient Catalyst ◽  
Gel Mobility Shift ◽  
Kinase A

ABSTRACT Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coliand in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a lowKm for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [γ-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.

Regulation of arginine vasopressin mRNA in rat fetal hypothalamic cell culture. Role of protein kinases and glucocorticoids

1993 ◽  
Vol 10 (1) ◽  
pp. 51-57 ◽  
Author(s):  
S-B Hu ◽  
L A Tannahill ◽  
S L Lightman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Mrna Expression ◽  
Protein Kinase A ◽  
Arginine Vasopressin ◽  
In Situ Hybridization Histochemistry ◽  
Kinase C ◽  
Kinase A

ABSTRACT Studies have been performed to investigate the regulation of arginine vasopressin (AVP) mRNA expression in fetal hypothalamic cultures. AVP mRNA-positive neurones were identified by in-situ hybridization histochemistry, and changes in mRNA expression were quantitated by nuclease protection assay. Both protein kinase C and protein kinase A activators increased the expression of AVP mRNA, in contrast to dexamethasone, which inhibited the responses to both protein kinase C and protein kinase A activation.

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP

1994 ◽  
Vol 14 (10) ◽  
pp. 6522-6530
Author(s):  
R R Vaillancourt ◽  
A M Gardner ◽  
G L Johnson
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinase A ◽  
Pc12 Cells ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Camp Levels ◽  
Kinase A

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.

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