CREB and the CRTC co-activators: sensors for hormonal and metabolic signals

Abstract

The cyclic AMP-responsive element-binding protein (CREB) is phosphorylated in response to a wide variety of signals, yet target gene transcription is only increased in a subset of cases. Recent studies indicate that CREB functions in concert with a family of latent cytoplasmic co-activators called cAMP-regulated transcriptional co-activators (CRTCs), which are activated through dephosphorylation. A dual requirement for CREB phosphorylation and CRTC dephosphorylation is likely to explain how these activator-co-activator cognates discriminate between different stimuli. Following their activation, CREB and CRTCs mediate the effects of fasting and feeding signals on the expression of metabolic programmes in insulin-sensitive tissues.

Figure 1. cAMP stimulates creB phosphorylation

The binding of ligand to G protein-coupled receptors (GPCR) that are linked to the stimulatory G proteins, which are comprised of α-, β- and γ-subunits, leads to the activation of adenylate cyclase (AC), which catalyses the synthesis of cyclic AMP. Increases in cellular cAMP stimulate protein kinase A (PKA) signalling. cAMP binds to the regulatory (R) subunits of PKA, thereby promoting their dissociation from the catalytic subunits. The liberated catalytic subunits enter the nucleus by passive diffusion and phosphorylate the cAMP-responsive element (CRE)-binding protein (CREB) at Ser133. Phosphorylated CREB promotes target gene expression at promoters containing CREs.

Figure 2. Modular organization of creB and its co-activators

a | Cyclic AMP-responsive element-binding protein (CREB) contains two Glu-rich domains (Q1 and Q2), a central kinase-inducible domain (KID) and a carboxy-terminal basic Leu zipper (bZIP) domain. The KID domain and the Q2 domain make up the amino-terminal transactivation domain (TAD). The Q2 domain binds to TBP-associated factor 4 (TAF4); phosphorylation of the KID domain at Ser133 promotes an interaction with CREB-binding protein (CBP) and its paralogue p300. Ser133 is phosphorylated by a number of basic directed kinases including protein kinase A (PKA) and PKC. Two clusters of phosphorylation sites flanking Ser133 inhibit CBP/p300 binding; these sites are phosphorylated by ataxia-telangiectasia mutated (ATM) and calcium- and calmodulin-dependent kinase II (CaMKII) as indicated. The bZIP domain promotes CREB DNA binding and dimerization; it also mediates CREB binding to cAMP-regulated transcriptional co-activators (CRTCs). Arg314 in the bZIP domain is critical for the CREB–CRTC interaction. b | Domain structure of CBP/p300, showing the nuclear receptor-interaction domain (RID), Cys and His-rich region 1 (CH1) and CH3, CREB-binding KIX domain, bromodomain (BR), plant homeodomain (PHD), histone acetyltransferase (HAT) domain, zinc-binding domain (ZZ) and interferon response factor-binding domain (IBID). Phosphorylation of both CBP and p300 at Ser89 by salt-inducible kinase 2 (SIK2) or AMP-activated protein kinase (AMPK) inhibits CRTC2 binding. Phosphorylation of CBP, but not p300, at Ser436 by atypical PKCι/λ (aPKCι/λ), within the CH1 region, inhibits binding to CREB. c | Domain structure of the CRTC family of CREB co-activators, as exemplified by CRTC2. CRTCs contain an N-terminal CREB binding domain (CBD), a central regulatory region (REG), a splicing domain (SD) and a C-terminal TAD. CRTC phosphorylation at Ser171 (by AMPK and SIK2), Ser275 (by microtubule affinity-regulating kinase 2 (MARK2)), and Ser307 (by SIK2) promotes 14-3-3 protein binding and the cytoplasmic sequestration of CRTC2. In contrast with CREB, CRTC2 phosphorylation in the TAD domain has not been described to date.

Figure 3. crTc nuclear shuttling is regulated by phosphorylation

Cyclic AMP and calcium signals regulate cAMP-responsive element (CRE)-binding protein (CREB) target genes by stimulating the nuclear translocation of cAMP-regulated transcriptional co-activators (CRTCs). Under basal conditions, CRTCs are phosphorylated and sequestered in the cytoplasm through interactions with 14-3-3 proteins. cAMP and calcium signals promote CRTC dephosphorylation through inhibition of the salt-inducible kinases (SIKs), which phosphorylate CRTCs, and through induction of the CRTC phosphatase calcineurin (CN), respectively. Dephosphorylated CRTC translocates to the nucleus where it binds to CREB and stimulates its activity. Image is modified, with permission, from REF. © (2004) Elsevier.

Figure 4. creB stimulates the gluconeogenic programme

Cyclic AMP-responsive element (CRE)-binding protein (CREB) stimulates gluconeogenic gene expression through direct and feedforward mechanisms. During short-term fasting, CREB activity increases in response to glucagon and it directly stimulates the expression of the pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase 1 (PEPCK1) and glucose-6-phosphatase (G6PC) genes following its binding to CREs within their promoters. CREB activation also stimulates expression of peroxisome proliferator-activated receptor-γco-activator 1α (PGC1α) and members of the nuclear receptor subfamily 4 group A (NR4A) family (NR4A1, NR4A2 and NR4A3) of orphan nuclear receptors. PGC1α and NR4A1 further induce the expression of gluconeogenic genes as well as the glucose transporter 2 (GLUT2). PGC1α stimulates hepatic glucose production through its role as a co-activator for glucocorticoid receptor (GR), hepatocyte nuclear factor 4 (HNF4) and/or forkhead box (FOXO) transcription factor. NR4A1 stimulates hepatic fasting gene expression by binding to NGFIB-response elements (NBREs) within the promoters of the G6PC, GLUT2, enolase 3, and fructose-1,6-bisphosphatase 1 (FBP1) and FBP2 genes. Increases in PGC1α and NR4A1 expression increase gluconeogenic gene expression when fasting is prolonged.

Figure 5. Glucagon and insulin antagonism

Opposing effects of glucagon and insulin on the cyclic AMP-responsive element (CRE)-binding protein (CREB) pathway. a | Glucagon stimulates the protein kinase A (PKA)-mediated phosphorylation of CREB at Ser133. PKA also stimulates cAMP-regulated transcriptional co-activator 2 (CRTC2) activity via the phosphorylation and inhibition of salt-inducible kinase 2 (SIK2), leading to the dephosphorylation of CRTC2. Dephosphorylated CRTC2 translocates to the nucleus, where it binds to CREB and promotes the recruitment of TBP-associated factor 4 (TAF4) and CREB-binding protein (CBP) and its paralogue p300. Nuclear CRTC2 is transiently stabilized by CBP/p300-mediated acetylation (Ac) at Lys628. b | Insulin signalling stimulates AKT, which phosphorylates and activates SIK2. Active SIK2 disrupts the CRTC2–CBP/p300 interaction by phosphorylating CBP/p300 at Ser89, leading to the deacetylation and ubiquitin (Ub)-dependent degradation of CRTC2. SIK2 also promotes the cytoplasmic translocation of CRTC2 through phosphorylation at Ser171.

Figure 6. Leptin promotes lipolysis and energy expenditure

a | The adipocyte-derived hormone leptin increases energy expenditure by acting on hypothalamic centres that increase sympathetic outflow. Increased sympathetic tone stimulates lipolysis and the release of fatty acids (FAs) from white adipose stores. Circulating FAs are taken up and oxidized by brown adipose tissue. b | In white adipocytes, catecholamines bind to β-adrenergic receptors and subsequently stimulate adenylate cyclase (AC) and cyclic AMP production. Increased cellular cAMP levels stimulate protein kinase A (PKA), which phosphorylates and activates hormone-sensitive lipase (HSL). During lipolysis, triacylglycerol (TAG) is hydrolysed by adipocyte triglyeride lipase (ATGL). The resultant diacylglycerol (DAG) is subsequently hydrolysed to monoacylglycerol (MAG) by HSL. MAG is further hydrolysed by MAG lipase (MGL) to generate glycerol, which enters the circulation. The FAs generated during the lipolysis of TAG may also enter the circulation. Alternatively, the FAs may undergo β-oxidation or may be re-esterified to TAG. Glycerol-3-phosphate (G-3-P) is utilized as the backbone for TAG synthesis and is generated from glucose, which is transported into the adipocyte via glucose transporters (GLUTs).