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.
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 alpha or beta) 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-3 beta 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.
Fiol
C J
CJ
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202.
Williams
J S
JS
Chou
C H
CH
Wang
Q M
QM
Roach
P J
PJ
Andrisani
O M
OM
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D008969:Molecular Sequence Data; D009154:Mutation; D016716:PC12 Cells; D010449:Peptide Mapping; D010766:Phosphorylation; D010957:Plasmids; D011817:Rabbits; D051381:Rats; D012694:Serine / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D012694:Serine; D000242:Cyclic AMP; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3