This study illuminates an unexpected function of an RGS protein, RGS13, as a direct repressor of CREB-mediated gene expression. Although a large body of evidence supports a biological role for RGS proteins as regulators of GPCR signaling through their GAP function, RGS13 inhibited CREB independently of its classical GAP activity. Rather, RGS13 impaired cAMP-evoked gene transcription by accumulating in the nucleus, binding pCREB in a complex with CBP/p300 in the absence of DNA, and reducing the subsequent interaction of pCREB with CRE promoter sites. In addition, RGS13, in complex with CRE-bound pCREB, attenuated CBP recruitment. The biological relevance of these findings was supported by the enhanced CREB-dependent gene expression in HEK293T cells or primary B lymphocytes deficient in RGS13.
An unresolved issue in gene regulation is how CREB binding to DNA is regulated. Although CREB interaction with consensus CREs may be sensitive to Mg2+
concentration (Craig et al., 2001
) and methylation of a central CpG within the CRE (Martinowich et al., 2003
), the effect of CREB phosphorylation at S133
on its binding to naked DNA templates in vitro or CRE-containing promoters in vivo is controversial. Although it has been assumed that CREB binding to gene promoters was constitutive, ChIP and footprinting studies have shown signal-dependent recruitment of CREB to the CRE and cell-type-specific CREB promoter binding to identical CREs (Hiroi et al., 2004
; Cha-Molstad et al., 2004
). Our results suggest that additional nuclear factors such as RGS13 could contribute to the selective binding of CREB to target CREs.
CREB-coactivator interactions may also be subject to acute regulation. Immunofluorescence experiments with antiserum specific for the KID-KIX complex demonstrated that stimulation of cells with cAMP and non-cAMP signals induced formation of the complex to differing degrees (Wagner et al., 2000
). Potentiation of CREB-CBP complex formation is mediated by the mixed lineage leukemia protein (MLL), which binds to KIX (Ernst et al., 2001
). Recently, a small molecule antagonist of CREB-CBP binding was identified that also interacts with KIX (Best et al., 2004
). By contrast, no endogenous cellular inhibitors of CREB-CBP interactions have been isolated. Our results suggest that RGS13 reduces the affinity of DNA-bound pCREB for CBP. As CBP ChIP would not discriminate between reduced CBP promoter binding due to diminished pCREB association with the CRE or impaired pCREB-CBP interaction, immunostaining of RGS13hi
cells with the KID-KIX antiserum may illuminate how RGS13 affects CREB-coactivator interactions in vivo.
The precise mechanism whereby RGS13 impairs pCREB-DNA binding requires further study. RGS13 did not co-immunoprecipitate with the DNA binding domain of CREB (B-ZIP) when B-ZIP was expressed as a GAL4 fusion protein. It seems unlikely that RGS13’s association with KID blocks CREB-DNA binding competitively as it was recovered in pCREB-CBP-CRE complexes by DNA ‘pulldown’. Rather, since we detected less pCREB at the Nr4a2 promoter in chromatin from RGS13hi cells by ChIP, RGS13 may act allosterically to transmit a conformational change to pCREB that a) reduces its apparent affinity for the CRE and/or b) decreases its association with CBP/p300 at the CRE. Our in vitro analyses suggest that both occur. Further biochemical studies such as crystallization of the pKID-CH/1-KIX-RGS13 complex may resolve the specific details of how RGS13 interferes with CREB induction of the transcriptional machinery.
Most likely because unmodified CREB contains a largely disordered KID, RGS13 did not interact with CREB in the absence of agonist or PKA-induced phosphorylation. Accordingly, we found that KID residues that contribute to the CBP/p300-induced α-helical conformation of pCREB are crucial for RGS13 binding. Regulation of CREB-mediated transcription by RGS13 may also depend on the duration and intensity of agonist stimulation since RGS protein expression is often low in unstimulated cells but is highly transcriptionally regulated (Bansal et al., 2007
). Further control of this process may be achieved by recruitment of RGS13 to the nucleus by certain stimuli.
The physiological impact of increased CREB target gene transcription in B lymphocytes deficient in RGS13 remains to be determined. RGS13 is also concentrated in neuroendocrine cells in the gastrointestinal and respiratory tracts (data not shown
), suggesting possible regulation of CREB-dependent hormone secretion (Gevrey et al., 2004
). Given the ubiquity and importance of CREB as a transcription factor, direct repression of CREB-mediated gene transcription by RGS13 could have an impact on numerous cellular processes for which CREB has an essential function.