Cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein beta functions through inhibition of glycogen synthase kinase-3.
The CCAAT enhancer-binding protein (C/EBPbeta) plays an important role in the regulation of gene expression during cell proliferation, differentiation, and apoptosis. We previously showed that C/EBPbeta participates in cGMP-regulated transcription of c-fos in osteoblasts (Chen, Y., Zhuang, S., Cassenaer, S., Casteel, D. E., Gudi, T., Boss, G. R., and Pilz, R. B. (2003) Mol. Cell. Biol. 23, 4066-4082). In the present work, we show that cGMP/cGMP-dependent protein kinase (PKG) induced dephosphorylation and activation of C/EBPbeta by inhibiting glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of GSK-3beta on Ser9 negatively regulates the enzyme activity, and we found that PKG phosphorylated this site both in vitro and in vivo; the in vivo phosphorylation occurred rapidly and preceded C/EBPbeta dephosphorylation. Previous studies with GSK-3 inhibitors suggest that GSK-3beta is a C/EBPbeta kinase in resting cells. We determined that GSK-3beta phosphorylated C/EBPbeta in vitro on Thr189, Ser185, Ser181, and Ser177; C/EBPbeta was phosphorylated on these same sites in intact, unstimulated osteoblasts, and phosphorylation was decreased in cGMP-treated cells. Mutation of the GSK-3 phosphorylation sites in C/EBPbeta prevented C/EBPbeta phosphorylation in resting cells, enhanced C/EBPbeta DNA binding, and led to increased target gene transactivation, mimicking the stimulatory effects of cGMP on C/EBPbeta. cGMP regulation of C/EBPbeta was disrupted by a mutant GSK-3beta(Ala9) resistant to cGMP/PKG phosphorylation and inhibition. We conclude that cGMP increases the DNA binding potential of C/EBPbeta by preventing the negative effects of GSK-3 phosphorylation.
Zhao
Xin
X
Department of Medicine and Cancer Center, University of California at San Diego, La Jolla, California 92093, USA.
Zhuang
Shunhui
S
Chen
Yongchang
Y
Boss
Gerry R
GR
Pilz
Renate B
RB
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D045744:Cell Line, Tumor; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010006:Osteoblasts / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D015533:Transcriptional Activation
D022782:CCAAT-Enhancer-Binding Protein-beta; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D004247:DNA; D017869:Cyclic GMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D000469:Alkaline Phosphatase; D006152:Cyclic GMP