Targeting CREB's Activity

Abstract

Although growth factors and stress stimulate the phosphorylation of cAMP response element-binding protein (CREB), these signals do not trigger the expression of the same genes as those activated by stimuli that activate cAMP signaling. Two groups (Ravnskjaer et al . and Xu et al .) report that transducers of regulated CREB (TORCs) are responsible for cAMP-selective CREB-mediated gene expression. Ravnskjaer et al . found that exposure of HEK293T cells to forskolin (to increase intacellular concentrations of cAMP) or a phorbol ester (to mimic stress signaling) activated different sets of CREB target genes. Modification of CREB proteins with hydrophobic substitutions at Ser 142 (S142L or Ser142F), which increased the affinity of CREB for histone acetylases p300 or CBP (CBP/p300), resulted in increased recruitment of p300/CBP to cAMP-responsive genes and activation of those genes when phorbol ester was the stimulus. Dephosphorylation and nuclear localization of TORC2, which is the predominant form present in the HEK293T cells, occurred only in response to forskolin and not phorbol ester. Kinetic chromatin immunoprecipitation analysis showed that TORC2 was the first protein recruited to the cAMP-responsive promoter, followed by phosphorylated CREB, and finally CBP. TORC2 and CBP appeared to directly interact based on several assays, and RNAi-mediated knockdown of TORC2 decreased the recruitment of CBP to cAMP-responsive promoters. Thus, the authors proposed that the activation of TORC2 by cAMP signaling promotes the formation of a complex containing TORC2, CREB, and CBP/p300 and that the direct interaction between TORC2 and CBP/p300 increases the stability of the histone acetylases at the CREB-responsive gene promoters, allowing efficient gene activation. Xu et al . created mouse embryo fibroblasts (MEFs) in which the interaction between CREB and CBP/p300 was disrupted by mutating the KIX domains in CPB and p300, which are responsible for mediating the CREB-CBP/p300 interaction. Although recruitment of CBP/p300 to CREB target genes was reduced in cells in which the KIX domains were mutated, binding was not eliminated, which suggests that there is another interaction allowing CBP/p300 to bind. Overexpression of TORC2 restored cAMP-gene expression in MEFs in which the KIX domains were mutated. If the interaction between TORC2 and CREB was disrupted by mutating Arg 314 in CREB and the KIX domains in CBP/p300 were also mutated, then all cAMP-inducible gene expression was lost. Sequential chromatin immunoprecipitation experiments indicated that CBP/p300 was recruited to cAMP-responsive promoters both through an interaction with phosphorylated CREB and through an interaction with TORC2. A dominant-negative TORC2 construct inhibited recruitment of CBP/p300 to CREB target genes and inhibited cAMP-responsive gene expression. Thus, together these two studies show that TORC2 is a specific component of cAMP-inducible transcriptional activation complex. K. Ravnskjaer, H. Kester, Y. Liu, X. Zhang, D. Lee, J. R. Yates III, M. Montminy, Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression. EMBO J. 26 , 2880-2889 (2007). [PubMed] W. Xu, L. H. Kasper, S. Lerach, T. Jeevan, P. K. Brindle, Individual CREB-target genes dictate usage of distinct cAMP-responsive coactivation mechanisms. EMBO J. 26 , 2890-2903 (2007). [PubMed]