Fiol C J, Williams J S, Chou C H, Wang Q M, Roach P J, Andrisani O M
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202.
J Biol Chem. 1994 Dec 23;269(51):32187-93.
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.
环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）分别在单个丝氨酸残基Ser119/133处使CREB327/341磷酸化。该位点的磷酸化产生序列基序SXXXS(P)，这是糖原合酶激酶-3（GSK-3）的共有位点（菲奥尔，C.J.，马伦霍尔茨，A.M.，王，Y.，罗斯克，R.W.，和罗奇，P.J.（1987年）《生物化学杂志》262卷，14042 - 14048页）。我们研究了CREB在SXXXS(P)共有位点的磷酸化及其在CREB对cAMP诱导的反式激活中的作用。GSK-3酶的两种同工型（GSK-3α或β）都不会将CREB用作其底物，除非CREB已经在Ser119/133处被磷酸化。一个包含Ser119/133周围序列的13个氨基酸的肽，只有在被PKA（在Ser119/133处）初步磷酸化后，才会被GSK-3在Ser115/129处磷酸化，这表明Ser115/129是GSK-3的磷酸接受位点。含有Ser→Ala替代的突变型CREB327/341蛋白证实Ser115/129是唯一的GSK-3磷酸化位点。在PC12细胞中对野生型和突变型Gal4-CREB融合蛋白进行转染分析表明，CREB341第129位残基的Ser→Ala替代会损害对cAMP诱导的转录反应。CREB327中的类似突变导致其对cAMP的反式激活反应降低70%。在对cAMP诱导无反应的未分化F9细胞中，转染的GSK-3β激酶通过内源性CREB蛋白介导，使环磷酸腺苷反应元件依赖性转录增加60倍。我们提出，CREB在PKA和GSK-3位点的分级磷酸化对于cAMP对CREB的调控至关重要。
