Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.Adipocytes are derived from multipotent mesenchymal stem cells in a process involving commitment to the adipocyte lineage followed by terminal differentiation of the committed preadipocytes. The process is regulated via complex interaction of external and internal clues, where cell shape and cytoskeletal tension converging on regulation of Rho and Rhokinase activity have been demonstrated to play pivotal roles (48,63). Whereas our understanding of the early steps of lineage determination still is limited, regulatory cascades controlling terminal adipocyte differentiation have been elucidated in great detail, particularly the sequential action of different transcription factors culminating in the expression of adipocyte-specific genes (25,30,58). Much information on terminal adipocyte differentiation has been obtained using model cell lines such as 3T3-L1 and 3T3-F442A or mouse embryo fibroblasts (MEFs). In both MEFs and 3T3-L1 preadipocytes, terminal differentiation is initiated upon treatment with fetal calf serum, glucocorticoids, and high levels of insulin or physiological concentrations of insulin-like growth factor 1 (IGF-1). Factors that increase cellular cyclic AMP (cAMP), such as isobutylmethylxanthine (IBMX) or forskolin, strongly accelerate the initiation of the differentiation program (for review, see references 25 and 45).Elevation of cellular cAMP concentration has been associated with crucial events in the early program of differentiation, such as suppression of Wnt10b (5) and Sp1 (64) and induction of CCAAT/enhancer-binding protein ␤ (C/EBP␤) (10,29,70). Moreover, the transcriptional activity of peroxisome proliferator-activated receptor ␦ (PPAR␦) is regulated synergistically by ligands and cAMP (32). In addition, cAMP has been implicated in the production of endogenous PPAR␥ ligand(s) occurring during the initial stages of differentiation (46, 67). The cAMP-responsive element-binding protein (CREB) is a central transcriptional activator of the adipocyte differentiation program. Activated CREB induces expres...

Under fasting conditions, increases in circulating concentrations of pancreatic glucagon maintain glucose homeostasis through induction of gluconeogenic genes by the CREB coactivator CRTC2. Hepatic CRTC2 activity is elevated in obesity, although the extent to which this cofactor contributes to attendant increases in insulin resistance is unclear. Here we show that mice with a knockout of the CRTC2 gene have decreased circulating glucose concentrations during fasting, due to attenuation of the gluconeogenic program. CRTC2 was found to stimulate hepatic gene expression in part through an N-terminal CREB binding domain that enhanced CREB occupancy over relevant promoters in response to glucagon. Deletion of sequences encoding the CREB binding domain in CRTC2 −/− mice lowered circulating blood glucose concentrations and improved insulin sensitivity in the context of diet-induced obesity. Our results suggest that small molecules that attenuate the CREB-CRTC2 pathway may provide therapeutic benefit to individuals with type 2 diabetes.D uring fasting, increases in hepatic gluconeogenesis ensure energy balance for glucose-dependent tissues such as brain and the red blood cell compartment. Hepatic glucose production is elevated in type 2 diabetes, reflecting decreases in insulin signaling that otherwise inhibit the gluconeogenic program (1-3).Increases in circulating glucagon are thought to trigger gluconeogenic gene expression in part through the cAMP-dependent phosphorylation of the transcription factor CREB and through the dephosphorylation of its cognate coactivator CRTC2 (4-6). In parallel, decreases in circulating insulin concentrations during fasting also stimulate gluconeogenic genes by the dephosphorylation of the forkhead activator FOXO1 (7).Localized in the cytoplasm under basal conditions through a phosphorylation-dependent association with 14-3-3 proteins, CRTC2 shuttles to the nucleus following its dephosphorylation at Ser171, where it mediates induction of cellular genes by binding to the bZIP domain of CREB over relevant promoters (8, 9). The importance of CRTC2 for gluconeogenic gene expression is supported by RNAi-mediated knockdown studies, where acute depletion of CRTC2 lowered hepatic glucose production in fasted mice and by overexpression studies in which phosphorylation-defective, active CRTC2 increases circulating glucose levels under both fasting and feeding conditions (4, 6, 10).To determine the importance of the CRTC2-CREB association for induction of the gluconeogenic program, we characterized mice lacking the conserved N-terminal CREB binding domain of CRTC2. We found that gluconeogenic gene expression and hepatic glucose production were reduced in CRTC2-deficient mice during fasting and in the setting of insulin resistance. Our results support an important role for CRTC2 in mediating effects of fasting signals on hepatic gluconeogenesis.

The complex of the subunits (RI␣, C␣) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K d ‫؍‬ 0.25 M) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RI␣ or RII␣ was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RI␣ or RII␣ showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.The cAMP-dependent protein kinase (cA-PK) 1 differs from other kinases in having the catalytic site and the autoinhibitory site on two different subunits. The inactive cA-PK holoenzyme, when studied at nanomolar concentrations, dissociates into catalytic (C) and regulatory (R) subunits in the presence of cAMP (1). There is sparse evidence about the behavior of cA-PK at higher, more physiologically relevant, concentrations. Apparently, it is tacitly assumed that both isozymes (cA-PKI and cA-PKII) are completely dissociated by cAMP in the intact cell. The cAMP-induced decrease of fluorescent resonance transfer between microinjected C␣-FITC and RI␣-TRITC (2), and between genetically encoded fluorescent C␣ and RII␤ (3) has reinforced this notion, although such studies are not designed to tell whether the dissociation of cA-PK is complete or not (4). Recently, C/EBP␤ null mice were shown to have increased liver RI and RII, and attenuated cAMP-stimulated hepatic gene induction (5). Protein kinase inhibitor null mice, having 50% increased muscle RI␣, showed deficient cAMP-stimulated CREB phosphorylation and CRE-dependent gene expression in muscle (6). We have previously observed relatively more holoenzyme-associated kinase than expected from the tissue cAMP content during the pre-replicative cAMP surge in the regenerating liver, in which both RI and RII were up-regulated (7). These observations suggest the possibility that RI or RII subunits may have a negative effect on cA-PK dissociation even at high cAMP concentrations. We used the CRE-luciferase reporter gene to probe for dissociation of cA-PKI and cA-PKII in intact cells. Nuclear translocation of the C subunit requires cA-PK dissociation and is considered a prerequisite for phosphorylation of the CREB/CREM family of nuclear transcription factors, and hence for cAMP stimulation of CRE-governed reporter gene expression (8 -10). We show that cells overexpressing either hRI␣ or hRII␣, even when maximally cAMP challenged, had decreased cAMP responsive gene induction, suggesting that cAMP produced incomplete dissociation of either isozyme in intact cells. We will also present evidence that...

Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.transcription regulation | cellular signaling | protein-protein interaction

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RI␣ and C␣ subunits of PKA type I, but not the RII␣ and C␣ subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.The cAMP-dependent protein kinase (PKA) 1 is a model for protein kinases because of its universal distribution and its relative simplicity because the catalytic moiety and the autoinhibitory moiety are on different subunits. The mammalian tetrameric PKA consists of two catalytic (C) subunit monomers and a regulatory (R) subunit dimmer. The binding of two molecules of cAMP to each R subunit favors dissociation of the C and R subunits. The kinase exists in two major isoforms with different R subunits, RI and RII, each with ␣-and ␤-subforms. The biological significance of the presence of two major isozymes is still uncertain. It was early noticed that RI predominated in many cells with rapid proliferation, rapid growth in cell size, or malignantly transformed cells (1-4). More recently it has been shown by homologous recombination that RI␣Ϫ/Ϫ mice die in embryonic life, whereas RII␤Ϫ/Ϫ, RII␣Ϫ/Ϫ, and RI␤Ϫ/Ϫ mice have less obvious defects, mainly in differentiation of adipose tissue (RII␤) and neural functions (RI␤) (5). An important difference between RI and RII is their relative affinity for anchoring proteins that confine PKA type II, and in some cases PKA type I, to subcellular compartments (see Refs. 6 and 7 for recent reviews). Another striking difference is the ability of PKA type II, but not type I, to become autophosphorylated (8 -10). The interface between the R and C subunits of PKA is complex. The RI and RII subunits bind in part to non-overlapping areas of the C subunit (11). The R subunits contact the substrate binding site of the C subunit as well as areas distant from this site (12) and are, through their (pseudo)substrate moiety, believed to displace substrate from the C subunit and thereby inhibit the kinase activity (10, 12). The subs...