Glucose homeostasis is regulated systemically by hormones such as insulin and glucagon, and at the cellular level by energy status. Glucagon enhances glucose output from the liver during fasting by stimulating the transcription of gluconeogenic genes via the cyclic AMP-inducible factor CREB (CRE binding protein). When cellular ATP levels are low, however, the energy-sensing kinase AMPK inhibits hepatic gluconeogenesis through an unknown mechanism. Here we show that hormonal and energy-sensing pathways converge on the coactivator TORC2 (transducer of regulated CREB activity 2) to modulate glucose output. Sequestered in the cytoplasm under feeding conditions, TORC2 is dephosphorylated and transported to the nucleus where it enhances CREB-dependent transcription in response to fasting stimuli. Conversely, signals that activate AMPK attenuate the gluconeogenic programme by promoting TORC2 phosphorylation and blocking its nuclear accumulation. Individuals with type 2 diabetes often exhibit fasting hyperglycaemia due to elevated gluconeogenesis; compounds that enhance TORC2 phosphorylation may offer therapeutic benefits in this setting.

Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 in response to hormonal stimuli triggers cellular gene expression via the recruitment of the histone acetylase coactivator paralogs CREB binding protein (CBP) and p300 to the promoter. The NMR structure of the CREB:CBP complex, using relevant interaction domains called KID and KIX, respectively, reveals a shallow hydrophobic groove on the surface of KIX that accommodates an amphipathic helix in phospho (Ser-133) KID. Using an NMR-based screening approach on a preselected smallmolecule library, we identified several compounds that bind to different surfaces on KIX. One of these, KG-501 (2-naphthol-AS-Ephosphate), targeted a surface distal to the CREB binding groove that includes Arg-600, a residue that is required for the CREB:CBP interaction. When added to live cells, KG-501 disrupted the CREB: CBP complex and attenuated target gene induction in response to cAMP agonist. These results demonstrate the ability of small molecules to interfere with second-messenger signaling cascades by inhibiting specific protein-protein interactions in the nucleus.cAMP response element binding protein ͉ transcription P rotein-protein interactions often serve as key regulatory points for signal propagation in response to extracellular stimuli. The formation of protein-protein complexes is of particular interest in the field of transcriptional regulation, where multiple low-affinity interactions appear to contribute to the recruitment of the transcriptional apparatus to the promoter (1). Initially thought to result from low energy interactions between unstructured regions, the recognition of an activator by its coactivator cognate often involves discrete surface contacts between well-folded protein domains. Moreover, the interaction surfaces between such protein pairs often are modulated by covalent modifications at residues near the protein-protein interface.Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 stimulates its association with the coactivator paralogs CREB binding protein (CBP) and p300 via a direct mechanism (2); the Ser-133 phosphate forms a hydrogen bond with Tyr-658 and an ion pair with Lys-662 in the KIX domain (3). Binding of CREB to CBP͞p300 is further stabilized by a random coil-to-helix transition in KID that provides the majority of hydrophobic surface contacts with residues lining a shallow groove in KIX (4, 5). The KIX domain folds into a three-helix structure with helices ␣1 and ␣3 aligned in nearly parallel fashion to form a groove that accommodates hydrophobic residues in KID. Helix ␣2 does not participate directly in surface contacts with KID but appears to stabilize KIX structure.The CREB binding site in KIX also recognizes other activators, most notably the protooncogene c-Myb (6). Similar to phospho (Ser-133) KID, c-Myb binds to KIX via an amphipathic helix that forms numerous surface contacts with residues lining the shallow hydrophobic groove in KIX (7). Notably, surfaces distal to the CREB-bi...

We have employed a hidden Markov model (HMM) based on known cAMP responsive elements to search for putative CREB target genes. The best scoring sites were positionally conserved between mouse and human orthologs, suggesting that this parameter can be used to enrich for true CREB targets. Target validation experiments revealed a core promoter requirement for transcriptional induction via CREB; TATA-less promoters were unresponsive to cAMP compared to TATA-containing genes, despite comparable binding of CREB to both sets of genes in vivo. Indeed, insertion of a TATA box motif rescued cAMP responsiveness on a TATA-less promoter. These results illustrate a mechanism by which subsets of target genes for a transcription factor are differentially regulated depending on core promoter configuration.

stimulate cooperative interactions with TFIID that facilitate recruitment of RNA polymerase II complexes (Horikoshi et al., 1988). In this regard, CREB has been found to interact with the hTAF II 130 component of TFIID via a glutamine-rich Q2 trans-activation domain; and this