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Cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein beta functions through inhibition of
Xin Zhao1, Shunhui Zhuang, Yongchang Chen
1Department of Medicine and Cancer Center, University of California at San Diego, La Jolla, California 92093, USA.
Abstract:
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.
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