Cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein β functions through inhibition of glycogen syntase kinase-3

Abstract

The CCAAT enhancer-binding protein (C/EBPβ) plays an important role in the regulation of gene expression during cell proliferation, differentiation, and apoptosis. We previously showed that C/EBPβ 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/EBPβ by inhibiting glycogen synthase kinase-3β (GSK-3β). Phosphorylation of GSK-3β 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/EBPβ dephosphorylation. Previous studies with GSK-3 inhibitors suggest that GSK-3β is a C/EBPβ kinase in resting cells. We determined that GSK-3β phosphorylated C/EBPβ in vitro on Thr189, Ser185, Ser181, and Ser177; C/EBPβ 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/EBPβ prevented C/EBPβ phosphorylation in resting cells, enhanced C/EBPβ DNA binding, and led to increased target gene transactivation, mimicking the stimulatory effects of cGMP on C/EBPβ. cGMP regulation of C/EBPβ was disrupted by a mutant GSK-3β(Ala 9) resistant to cGMP/PKG phosphorylation and inhibition. We conclude that cGMP increases the DNA binding potential of C/EBPβ by preventing the negative effects of GSK-3 phosphorylation. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.