A Secondary Phosphorylation of CREB341 at Ser129 Is Required for the cAMP-mediated Control of Gene Expression

A Role for Glycogen Synthase Kinase-3 in the Control of Gene Expression

Carol J. Fiol, John S. Williams, Chin Hua Chou, Q. May Wang, Peter Roach, Ourania M. Andrisani

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Abstract

The cAMP-dependent protein kinase (PKA) phosphorylates CREB327/341 at a single serine residue, Ser119/133, respectively. Phosphorylation at this site creates the sequence motif SXXXS(P), a consensus site of the glycogen synthase kinase-3 (GSK-3) enzyme (Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W., and Roach, P. J. (1987) J. Biol. Chem. 262, 14042-14048). We examined the phosphorylation of CREB at the SXXXS(P) consensus site and its role in CREB transactivation to cAMP induction. Neither isoform of the GSK-3 enzyme (GSK-3 α or β) utilizes CREB as its substrate unless CREB is already phosphorylated at Ser119/133. A 13-amino acid peptide containing the sequence surrounding Ser119/133 was phosphorylated by GSK-3, at Ser115/129, only after the primary phosphorylation of the peptide by PKA (at Ser119/133), suggesting that Ser115/129 is a GSK-3 phosphoacceptor site. Mutant CREB327/341 proteins containing Ser → Ala substitutions confirmed Ser115/129 as the only GSK-3 phosphorylation site. Transfection assays of wild type and mutant Gal4-CREB fusion proteins in PC12 cells demonstrated that Ser → Ala substitution of residue 129 of CREB341 impairs the transcriptional response to cAMP induction. Analogous mutation in CREB327 results in 70% decrease in its transactivation response to cAMP. In undifferentiated F9 cells, which are refractory to cAMP induction, transfected GSK-3β kinase induces a 60-fold increase in cyclic AMP response element-dependent transcription, mediated via the endogenous CREB protein. We propose that the hierarchical phosphorylation at the PKA and GSK-3 sites of CREB are essential for cAMP control of CREB.

Original languageEnglish
Pages (from-to)32187-32193
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number51
StatePublished - Dec 23 1994

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Glycogen Synthase Kinase 3
Phosphorylation
Gene expression
Gene Expression
Cyclic AMP Response Element-Binding Protein
Protein Kinases
Transcriptional Activation
Substitution reactions
Peptides
PC12 Cells
Response Elements
Enzymes
Transcription
Cyclic AMP-Dependent Protein Kinases
Cyclic AMP
Refractory materials
Serine
Transfection
Assays
Protein Isoforms

ASJC Scopus subject areas

  • Biochemistry

Cite this

A Secondary Phosphorylation of CREB341 at Ser129 Is Required for the cAMP-mediated Control of Gene Expression : A Role for Glycogen Synthase Kinase-3 in the Control of Gene Expression. / Fiol, Carol J.; Williams, John S.; Chou, Chin Hua; Wang, Q. May; Roach, Peter; Andrisani, Ourania M.

In: Journal of Biological Chemistry, Vol. 269, No. 51, 23.12.1994, p. 32187-32193.

Research output: Contribution to journalArticle

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title = "A Secondary Phosphorylation of CREB341 at Ser129 Is Required for the cAMP-mediated Control of Gene Expression: A Role for Glycogen Synthase Kinase-3 in the Control of Gene Expression",
abstract = "The cAMP-dependent protein kinase (PKA) phosphorylates CREB327/341 at a single serine residue, Ser119/133, respectively. Phosphorylation at this site creates the sequence motif SXXXS(P), a consensus site of the glycogen synthase kinase-3 (GSK-3) enzyme (Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W., and Roach, P. J. (1987) J. Biol. Chem. 262, 14042-14048). We examined the phosphorylation of CREB at the SXXXS(P) consensus site and its role in CREB transactivation to cAMP induction. Neither isoform of the GSK-3 enzyme (GSK-3 α or β) utilizes CREB as its substrate unless CREB is already phosphorylated at Ser119/133. A 13-amino acid peptide containing the sequence surrounding Ser119/133 was phosphorylated by GSK-3, at Ser115/129, only after the primary phosphorylation of the peptide by PKA (at Ser119/133), suggesting that Ser115/129 is a GSK-3 phosphoacceptor site. Mutant CREB327/341 proteins containing Ser → Ala substitutions confirmed Ser115/129 as the only GSK-3 phosphorylation site. Transfection assays of wild type and mutant Gal4-CREB fusion proteins in PC12 cells demonstrated that Ser → Ala substitution of residue 129 of CREB341 impairs the transcriptional response to cAMP induction. Analogous mutation in CREB327 results in 70{\%} decrease in its transactivation response to cAMP. In undifferentiated F9 cells, which are refractory to cAMP induction, transfected GSK-3β kinase induces a 60-fold increase in cyclic AMP response element-dependent transcription, mediated via the endogenous CREB protein. We propose that the hierarchical phosphorylation at the PKA and GSK-3 sites of CREB are essential for cAMP control of CREB.",
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