Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB

doi:10.1016/0092-8674(92)90537-M

Cell

Volume 70, Issue 1, 10 July 1992, Pages 105-113

https://doi.org/10.1016/0092-8674(92)90537-M Get rights and content

Abstract

We have examined the mechanism by which the transcriptional activity of the cAMP-responsive factor CREB is attenuated following Induction with forskolin. Metabolic labeling studies reveal that, after an initial burst of phosphorylation in response to CAMP, CREB is dephosphorylated and transcription of the cAMP-responsive somatostatin gene is correspondingly reduced. The phosphatase inhibitor 1 protein and okadaic acid both prevented the dephosphorylation of CREB at Ser-133 in PC12 cells and also augmented the transcriptional response to cAMP. Of the four Ser/Thr phosphateses described to date, only PP-1 appears to be similarly inhibited by these agents. As PP-1 specifically dephosphorylates CREB at Ser-133 and inhibits cAMP-dependent transcription, we propose that this phosphatase is the major regulator of CREB activity in cAMP-responsive cells.

References (19)

M. Boshart et al.
The tissue specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase
Cell
(1991)
P.T.W. Cohen
Two isoforms of protein phosphatase 1 may be produced from the same gene
FEBS Lett.
(1988)
A. Elbrecht et al.
Molecular cloning of protein phosphatase inhibitor-1 and its expresssion in rat and rabbit tissues
J. Biol. Chem.
(1990)
N.S. Foulkes et al.
CREM gene: use of alternative DNA-binding domains generates multiple antagonists of CAMP-induced transcription
Cell
(1991)
G.A. Gonzalez et al.
Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133
Cell
(1989)
K.W. Jones et al.
Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A
Cell
(1991)
K. Sasaki et al.
Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription
J. Biol. Chem.
(1984)
F.S. Ambesi-Impiombato et al.
Culture of hormone-dependent functional epithelial cells from rat thyroids
P. Angel et al.
Oncogene jun encodes a sequence specific trans-activator similar to AP-1
Nature
(1988)

There are more references available in the full text version of this article.

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Abstract

References (19)

The tissue specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase

Cell

Two isoforms of protein phosphatase 1 may be produced from the same gene

FEBS Lett.

Molecular cloning of protein phosphatase inhibitor-1 and its expresssion in rat and rabbit tissues

J. Biol. Chem.

CREM gene: use of alternative DNA-binding domains generates multiple antagonists of CAMP-induced transcription

Cell

Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133

Cell

Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A

Cell

Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription

J. Biol. Chem.

Culture of hormone-dependent functional epithelial cells from rat thyroids

Oncogene jun encodes a sequence specific trans-activator similar to AP-1

Nature

Cited by (438)

Mitigating transcriptional bottleneck using a constitutively active transcription factor, VP16-CREB, in mammalian cells

PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells

Regulation of learned fear expression through the MgN-amygdala pathway

Mechanistic insights into the activation of ester prodrugs of 666-15

A suite of bioassays to evaluate CREB inhibitors

Zbtb7c is a critical gluconeogenic transcription factor that induces glucose-6-phosphatase and phosphoenylpyruvate carboxykinase 1 genes expression during mice fasting