Scaffold Proteins: From Coordinating Signaling Pathways to Metabolic Regulation

Abstract

Among their pleiotropic functions, scaffold proteins are required for the accurate coordination of signaling pathways. It has only been within the past 10 years that their roles in glucose homeostasis and metabolism have emerged. It is well appreciated that changes in the expression or function of signaling effectors, such as receptors or kinases, can influence the development of chronic diseases such as diabetes and obesity. However, little is known regarding whether scaffolds have similar roles in the pathogenesis of metabolic diseases. In general, scaffolds are often underappreciated in the context of metabolism or metabolic diseases. In the present review, we discuss various scaffold proteins and their involvement in signaling pathways related to metabolism and metabolic diseases. The aims of the present review were to highlight the importance of scaffold proteins and to raise awareness of their physiological contributions. A thorough understanding of how scaffolds influence metabolism could aid in the discovery of novel therapeutic approaches to treat chronic conditions, such as diabetes, obesity, and cardiovascular disease, for which the incidence of all continue to increase at alarming rates.

Transduction of receptor-mediated signaling pathways requires upstream receptors at the cell membrane and their downstream effector molecules, such as kinases or transcription factors. Precise coordination of signaling events is necessary to ensure that stimulus-dependent effects are accurately evoked in a particular cell type, and dysregulation of signaling events can lead to the development of diseases, including cancer (1–3). Molecular scaffolds or adaptor proteins (herein referred to as scaffolds) have been proposed to be the principal regulators that ensure such coordination (4, 5). Scaffolds can bind to one or more signaling effectors to facilitate their spatial or temporal localization within a cell and also regulate the cellular functions of various effectors, including those involved in metabolic signaling pathways (4–6). For example, scaffolds belonging to the 14-3-3 protein family interact with the phosphorylated form of the transcription factor, FOXO1, leading to it sequestration in the cytoplasm and inhibition of its transcriptional activity (7). Furthermore, they can regulate the enzymatic activity of kinases, such as hormone-sensitive lipase (HSL) (8).

Despite the knowledge that scaffolds are needed to accurately coordinate signaling pathways, it is only within the past 10 years that their roles in metabolism have emerged. For decades, receptors and kinases have been considered to be the key signaling effectors responsible for metabolic processes, such as glucose uptake, lipolysis, and insulin secretion (9, 10). However, little is known whether scaffolds have similar importance in metabolic processes or in the pathogenesis of metabolic diseases, such as diabetes and obesity. Thus, scaffolds have often been underappreciated in the context of metabolism and metabolic diseases.

In this mini-review, we have summarized the contributions of various scaffold proteins to metabolism and cardiometabolic diseases. We describe their participation in processes underlying whole-body metabolism and whether alterations in their function might contribute to disease development. Overall, we sought to highlight the importance of scaffolds in metabolism and metabolic diseases and to raise awareness of the need to consider their functions when developing new therapeutic targets for the treatment of obesity, diabetes, and cardiovascular disease.

Scaffold Proteins and Their Importance to Cellular Signaling/Processes

The role of scaffold proteins in coordinating cellular signaling events have been reviewed in several recent publications (11–15). We briefly discuss their importance in signal transduction pathways related to metabolic regulation.

Many hormones, such as incretins, catecholamines, and glucagon, exert their cellular effects through the activation of 7-transmembrane G protein–coupled receptors (GPCRs) (13, 16–20). Scaffolds of GPCRs might directly regulate receptor signaling by acting as allosteric modulators of receptor conformation (21). For example, after agonist binding, changes in receptor conformation can lead to enhanced interaction of scaffold proteins with G proteins and GPCRs (13, 21, 22). Most scaffolds that interact with GPCRs contain PDZ (PSD-95/Discs-large/ZO-1 homology) domains, which facilitate their interactions with the carboxyl-termini of other proteins (23). One of the best characterized scaffold families that interacts with GPCRs and G proteins are the β-arrestins, and their metabolic functions have been discussed in the subsequent sections.

Scaffolds proteins are also involved in promoting the association of ion channels with other signaling molecules in specific microdomains. For example, the activity and localization of transient receptor potential (TRP) channels, which are involved in intracellular Ca²⁺ homeostasis (24), are determined by their interactions with various scaffolds. Homer proteins, including Homer1, Homer2, and Homer3, are scaffolds that were first identified in the brain and have been shown to interact with TRP channels (25, 26). Enhanced spontaneous Ca²⁺ influx in cells from Homer1 knockout mice has been observed due to increased TRP channel activity (26). Impairment of Homer function might also attenuate thrombin-evoked Ca²⁺ entry and the maintenance of store-operated Ca²⁺ entry in human platelets, which demonstrates cell type-dependent functions (27). In addition to the Homer protein family, a number of additional scaffold proteins are associated with TRP proteins. TRPC4 contains a PDZ-interacting domain at the C-terminus, which is an interaction site with scaffolds such as EBP50/NHERF. A TRPC4 mutant lacking the ability to interact with EBP50/NHERF leads to intracellular accumulation. In contrast, wild-type channels that can bind to this scaffold will be uniformly distributed in the plasma membrane in HEK-293 cells (28).

The activity of the heat-activated ion channel TRPV1 is modulated by protein kinase A (PKA) and protein kinase C (PKC), in addition to the phosphatase calcineurin. The interactions between TRPV1 and PKA, PKC, or calcineurin are dependent on the ability of AKAP150 to organize and form a signaling complex (29). RACK-1 (receptor for activated C kinase 1) is another scaffold protein involved in the regulation of TRP channels and has been reported to inhibit TRPP3 channel activity (30). It also facilitates the association of TRPC3 with a number of proteins, including type I IP3R, STIM1, and Orai1 (31, 32). Finally, caveolins also display scaffold functions because they promote the localization TRPC1 channels within lipid raft domains and aid in the internalization of insulin receptors (33–35). Caveolin-1 has also been found to have a predominant role in insulin granule exocytosis and insulin signaling in metabolic target tissues (34, 36).

Receptor tyrosine kinases (RTKs) and their downstream signaling pathways have important roles in development and metabolic homeostasis. RTKs can include, but are not limited to, the epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and the insulin receptor families (37–40). In the case of RTKs, autophosphorylation at NXXY motifs recruit scaffolds that contain phosphotyrosine-binding domains. These scaffolds include members of the insulin-receptor substrate (IRS), Dok, FGF-receptor substrate 2, and Shc families (37–41). Once associated with an RTK, the scaffold protein is phosphorylated at tyrosine motifs, resulting in the recruitment of Src homology (SH) 2 domain proteins and the subsequent activation of intracellular pathways (42). This series of events has been well-described with respect to the scaffold Shc1 (43, 44). The Shc1 gene encodes three proteins of 46, 52, and 66 kDa that share an N-terminal phosphotyrosine-binding domain and a C-terminal SH2 domain, flanking a central region (CH1) containing key phosphorylation sites (Tyr239/240 and Tyr313) (44). Modification of these phosphorylation sites creates pYXN binding motifs for the SH2 domain of the Grb2 adaptor. Through its SH3 domains, Grb2 recruits proteins, such as Sos1 and Gab1, and cause downstream activation of ERK and phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (Akt) pathways (45, 46). Collectively, the pleiotropic roles of scaffolds in various signaling pathways demonstrate their importance in cell biology; however, whether scaffolds are required for metabolic signaling pathways is unclear.

Scaffolds With Known Metabolic Roles

Although many scaffold proteins have been identified, it is not clear whether they are all involved in the regulation of metabolic homeostasis. Some scaffolds have multiple isoforms, suggesting that they might have redundant cellular functions in metabolic signaling pathways. However, those with well-established roles in metabolism have been discussed in subsequent paragraphs (Table 1).

Table 1.

Involvement of Scaffolds in Cardiometabolic Functions

Scaffold Protein	Model	Modifications	Metabolic Outcome	Reference
β-Arrestin 1	INS1 cells	siRNA knockdown	Impaired GSIS	Sonoda et al. (47)
		Overexpression	Protection from glucose-apoptosis	Quoyer et al. (48)
	Mouse	Systemic knockout	Increased adiposity	Ruderman et al. (49)
			Decreased insulin sensitivity
			Potentiated HFD-induced obesity
	Mouse cardiomyocytes	Overexpression	Resistance to apoptosis	Song et al. (50), Eguchi et al. (51), and Nackiewicz et al. (52)
β-Arrestin-2	Mouse	Liver-specific knockout	Increased gluconeogenesis	Zhu et al. (53)
	Hepatocytes	Overexpression	Decreased gluconeogenesis	Zhu et al. (53)
	Mouse cardiomyocytes	Overexpression	Stress-induced cardiac damage	Eguchi et al. (51)
	Mouse	β-Cell specific knockout	Worsened glucose tolerance after HFD	Zhu et al. (54)
			GSIS- and KCl-induced insulin secretion reduced
		β-Cell overexpression	Enhanced GSIS	Zhu et al. (54)
	Human EndoC-βH1 cells	siRNA knockdown	Impaired GSIS	Zhu et al. (54)
AKAPs	β-Cell	AKAP150 systemic knockout	Impaired insulin secretion	Hinke et al. (55)
			Impacts l-type Ca2+currents
			Attenuates cytoplasmic accumulation of Ca2+ and cAMP
	Muscle	AKAP150 systemic knockout	Increased insulin sensitivity	Hinke et al. (55)
	Mouse cardiomyocytes	AKAP18α overexpression	Regulate Ca2+ influx via PKA in response to β-adrenergic stimulation	Gray et al. (56) and Fraser et al. (57)
		AKAP5 systemic knockout	Impaired trafficking and signaling of β1AR	Klauck et al. (58)
KSR2	C2C12 cells	Expression of KSR2 mutants with human mutations	Reduced glucose oxidation	Pearce et al. (59)
			Increased fatty acid oxidation
	Mouse	Systemic knockout	Develops obesity	Liu et al. (60)
			Hyperinsulinemia
			Glucose intolerance
			Increased AMPK-dependent glucose uptake
			Hypophagic
NCK1/2	MIN6 cells	Nck1 knockdown by siRNA	Improves β-cell function	Yamani et al. (61)
			Resistance to ER stress
	Mouse	Systemic Nck1 knockout	Improves glucose homeostasis	Latreille and Larose (62)
			Enhanced hepatic insulin signaling
			Protects against β-cell apoptosis
	Mouse	Systemic Nck2 knockout	Increased adiposity	Dusseault et al. (63)
14-3-3 Proteins	MIN6 cells	Pharmacological inhibition	Apoptosis	Lim et al. (4)
	Mouse islets	Pharmacological inhibition	Apoptosis	Lim et al. (4)
14-3-3ζ	MIN6 cells	siRNA knockdown	Apoptosis	Lim et al. (4)
	MIN6 cells	Overexpression	Protection from apoptosis	Lim et al. (4)
	3T3-L1 preadipocyte	siRNA knockdown	Inhibition of adipogenesis	Lim et al. (64)
	Mouse	Systemic knockout	Impaired visceral adipogenesis	Lim et al. (64, 65)
			Insulin resistance
			Improved oral glucose tolerance via GLP-1
	Mouse	Systemic overexpression	Enhanced age- and HFD-induced weight gain	Lim et al. (64)
Homer1	Human platelets	Knockdown	Increases TRP channel activity	Jardin et al. (27)
			Enhanced spontaneous Ca2+ influx
Caveolin-1	Mouse	Systemic knockout	HFD-induced postprandial hyperinsulinemia	Cohen et al. (33) and Razani et al. (34)
			Decreases insulin sensitivity
			Alterations in lipid homeostasis
			Resistant to HFD-induced obesity
	MIN6 cells	siRNA knockdown	Increases basal insulin secretion	Boothe et al. (35)
	MIN6 cells	siRNA knockdown	Impaired insulin receptor internalization and ERK activation	Nevins et al. (36)
JIP1	INS1 cells	siRNA knockdown	Increases susceptibility to apoptosis	Morel et al. (66)
	Mouse	Systemic knockout	Insulin resistance	Jaeschke et al. (67)

Abbreviations: AMPK, AMP-activated protein kinase; ER, endoplasmic reticulum; GSIS, glucose-induced insulin secretion; HFD, high-fat diet; KSR, kinase suppressor of RAS; Nck, noncatalytic region of tyrosine kinase; siRNA, small interfering RNA.

β-Arrestins

Receptors belonging to the β-adrenergic receptor (βAR; βAR1, βAR2, and βAR3) are GPCRs that have been studied extensively in terms of their structure, regulation, and downstream signaling pathways (13, 68, 69). Scaffold proteins have been found to be necessary for efficient internalization of GPCRs and the initiation of downstream signaling events (68, 70–72). For example, β-arrestin 1 and 2 are prototypical GPCR scaffolds that translocate to the plasma membrane after agonist stimulation, where they are recruited to βARs and phosphorylated by GPCR kinases. This results in agonist-induced desensitization of βARs, as a result of their internalization or degradation and subsequent recycling to the plasma membrane (Fig. 1) (73–75). Although this has demonstrated that β-arrestins can have negative effects on GPCR signaling (76, 77), recent studies have also revealed that β-arrestins are required for GPCR-dependent metabolic signaling pathways (78–82). Activation of the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) involves Gs-proteins for cAMP production, and β-arrestin 1 was shown to be required for GLP-1–induced cAMP synthesis and insulin secretion from INS-1 β-cells (47). GLP-1 potentiates insulin secretion from β-cells in a glucose-dependent manner, and β-arrestin 1 knockdown in INS1 cells impaired GLP-1–induced insulin release due to reduced cAMP production and decreased activation of PKA and PKA-independent pathways (47, 83–85). Also, β-arrestin 1 was found to mediate the antiapoptotic effect of GLP-1 in β-cells by influencing GLP-1–induced ERK activation and promoting the phosphorylation of Bad on Ser112, an established inhibitory phosphorylation site (48). In pancreatic β-cells, GLP-1R signaling events associated with insulin secretion or β-cell survival have been well-characterized. However, when considering the role of scaffolds such as β-arrestin 1, this has demonstrated the increased complexity of GLP-1 actions. Despite a high degree of homology, β-arrestin 2 and its functions in β-cells differ. Systemic deletion in mice has been associated with impaired β-cell mass expansion in response to a high-fat diet (86). Furthermore, systemic β-arrestin 2 knockout mice do not display defects in insulin secretion, and Zhu et al. (54) reported that β-cell–specific deletion or overexpression of β-arrestin 2 impaired or potentiated insulin secretion, respectively. Moreover, depletion of β-arrestin 2 in the human β-cell line, EndoC-βH1, was also associated with decreased insulin secretory function (54).

Figure 1.

A model for β-arrestin–mediated regulation of metabolic processes. Ligand binding stimulates the activation of a GPCR, leading to the dissociation of the G protein heterotrimeric complex and subsequent initiation of G protein-dependent signaling. The ligand-occupied GPCR is phosphorylated by G protein–related kinases (GRKs), followed by the recruitment of β-arrestin 1/2 to the phosphorylated GPCR. β-Arrestin 1/2 subsequently interacts with proteins belonging to cAMP–PKA or ERK1/2 pathways to promote metabolic responses.

β-Arrestins were also found to be involved in liver metabolism. Selective inactivation of β-arrestin 2 in hepatocytes of adult mice results in glucose intolerance and enhanced hepatic glucagon receptor signaling and glucagon-induced glucose mobilization (53). Hepatic insulin sensitivity and β-adrenergic signaling were not affected. Hepatocyte-specific overexpression of β-arrestin 2 greatly reduced hepatic glucagon receptor signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Control mice displayed glucose intolerance, increased gluconeogenesis, and enhanced glucagon sensitivity compared with mutant mice (68).

cAMP-dependent kinase–anchoring proteins

cAMP-dependent kinase–anchoring proteins (AKAPs) are scaffolds involved in GPCR signaling and play central roles in facilitating GPCR association with protein kinases and phosphatases (Fig. 2) (6, 87, 88). AKAPs can also associate with phosphodiesterases that degrade the second messenger, cAMP (89). Multiple AKAP proteins have been identified, and one, AKAP150 (encoded by AKAP5), has been shown to coordinate key aspects of adrenergic signaling, such as Ca²⁺ cycling and excitation–contraction in cardiomyocytes (90). In addition to binding to PKA, AKAP150 can also bind to the phosphatase calcineurin (also known as PP2B), which has been identified as a central regulator of cardiac hypertrophy (91). Specific to glucose homeostasis, AKAP150-null mice secrete less insulin from β-cells but display improved glucose handling owing to increased insulin sensitivity in skeletal muscle (55). This metabolic phenotype is retained in AKAP150ΔPIX mice that lack a seven amino acid sequence required for interactions with calcineurin (55). Hence, AKAP150 has pleiotropic functions in the regulation of cardiomyocyte function and metabolic homeostasis.

Figure 2.

Basic scheme of AKAP–PKA signaling. Ligand-activated GPCRs that transduce via the Gα subunit of the heterotrimeric G protein complex cause the activation of adenylyl cyclase (AC), which catalyzes the formation of cAMP. Binding of cAMP to the PKA complex leads to dissociation of the catalytic subunits from the holoenzyme to activate PKA. Attenuation of cAMP-dependent signaling is facilitated by phosphodiesterases (PDEs), which hydrolyze cAMP to modulate the duration and extent of cAMP-dependent signaling. AKAP family members function as scaffolds to bring together PKA and PDEs in close proximity to ensure precise cAMP-dependent PKA signaling. Reductions in AKAP expression or function can lead to deleterious effects on metabolic and cardiovascular physiology.

Insulin receptor substrates

Although not often thought of as scaffolds, IRS proteins represent a critical group of proteins required for downstream transduction of insulin and IGF-1 receptor signaling events, including activation of PI3K. Among its pleiotropic metabolic effects, insulin is essential for stimulating glucose uptake in peripheral tissues, and defects at various steps of the insulin-signaling pathway promote insulin resistance, which is one of the earliest metabolic abnormalities detected in individuals with the metabolic syndrome (92, 93). Tyrosine phosphorylation of IRS molecules is normally required for transducing the signals from activated receptors; however, phosphorylation of serine/threonine residues on IRS proteins has negative effects on their function (94, 95). For example, c-Jun NH2-terminal kinase (JNK) phosphorylates IRS1 at serine and threonine residues, which prevents the recruitment of IRS1 to the insulin receptor and inhibits insulin signaling (96).

JNK-interacting proteins (JIPs) are scaffold proteins involved in coordinating JNK signaling (97, 98), and they have also been found to interact with the mixed-lineage protein kinase group of MAPKs, MKK7, and AKT (97). IRS and JIP scaffold proteins might function independently to regulate insulin- and JNK-dependent signal transduction, respectively; however, these scaffolds can exhibit cross-talk. This was exemplified by a study using Jip1 knockout mice, which exhibit decreased high-fat diet-induced JNK activation in white adipose tissue, phosphorylation of IRS1 on Ser-307, and insulin resistance (67).

Kinase suppressor of RAS

Several scaffold proteins have been identified in different MAPK pathways; however, it is unclear whether they all have roles in metabolic signaling. One of best characterized MAPK scaffolds is the kinase suppressor of RAS (KSR) 2, which is required for ERK1/2 signaling (Fig. 3) (99). KSR was originally identified in RAS-dependent genetic screens in Drosophila melanogaster and Caenorhabditis elegans (100–102). Variants in KSR2 disrupt Raf-MEK-ERK signaling and can impair cellular fatty acid oxidation and glucose oxidation (59). In C2C12 cells overexpressing KSR2 variants with identified human mutations markedly reduced glucose oxidation was observed. In contrast, overexpression of wild-type KSR2 increased palmitate-stimulated oxygen consumption, and this effect could be attenuated by cotransfection of KSR2 mutants (59). Preincubation of cells with metformin increased palmitate-stimulated oxygen consumption in nontransfected cells, and the increase in basal fatty acid oxidation due to wild-type KSR2 overexpression could be further increased with metformin. The reduced basal level of fatty acid oxidation seen with the KSR2 mutations was completely rescued in all cases by the addition of metformin (59).

Figure 3.

The role of KSR in ERK-dependent metabolic signaling. Following receptor stimulation, Grb2 recruits SOS to activate Ras. This is necessary for the recruitment of Raf to the membrane. Translocation of kinase suppressor of ras 2 (KSR2) to the cell membrane leads to the formation of a protein complex with activated Raf-1, MAPK kinase (MEK1/2), and ERK1/2. This complex amplifies ERK1/2-dependent signaling to facilitate various metabolic outcomes.

Noncatalytic region of tyrosine kinase proteins

Noncatalytic region of tyrosine kinase (Nck) scaffold proteins include Nck1 and Nck2 (103, 104). Nck1 and Nck2 are involved in critical biological processes, including embryonic development, actin cytoskeletal reorganization, axonal guidance, proliferation, and the unfolded protein response (105–112). In MIN6 insulinoma cells, Nck1 enhances protein kinase R–like endoplasmic reticulum kinase (PERK) basal activity, leading to PERK-dependent insulin biosynthesis (61). In addition, Yamani et al. (61) reported that silencing Nck1 in MIN6 cells is protective against cell death induced by chemical inducers of endoplasmic reticulum stress. Depletion of Nck1 in MIN6 cells upregulates the unfolded protein response, as demonstrated by enhanced basal PERK activity, phosphorylation of eIF2α, and increased ATF4 mRNA and nuclear protein levels (61). Thus, silencing Nck1 improves β-cell function and resistance to endoplasmic reticulum stress. Whole body Nck1 knockout mice display improved glucose homeostasis and enhanced hepatic insulin signaling compared with obese littermate controls (62). Nck1 also has regulatory roles in the activation of the PI3K/Akt pathway via a protein tyrosine phosphatase 1B–dependent mechanism. Primary hepatocytes isolated from Nck1 knockout mice display enhanced insulin-mediated Akt activation due to Nck1-dependent regulation of protein tyrosine phosphatase 1B expression (113).

In contrast, Nck2 has been found to regulate adipogenesis and glucose homeostasis, because Nck2 deletion in mice increases adiposity through adipocyte hypertrophy, promotes glucose intolerance, and decreases insulin sensitivity. In addition, Nck2 knockdown has been found to enhance adipocyte differentiation in vitro (63). Recent work has provided pharmacological evidence of abrogation of the interaction between Nck1 and PERK to improve β-cell function and survival (114). Through the use of the TAT-pY561 phosphopeptide, increases in basal PERK activation was observed, resulting in protection against glucolipotoxicity-induced apoptosis and enhanced insulin production and secretion from pancreatic β-cells.

14-3-3 Proteins

14-3-3 Proteins are a ubiquitously expressed family of scaffolds consisting of seven isoforms (β, ε, γ, η, θ, σ, and ζ). They bind to phosphorylated proteins containing specific phosphoserine or phosphothreonine motifs (RSxpS/TxP or R'pS/TxP, respectively) (115–117). Different roles of 14-3-3 proteins in cell cycle progression, development, and neurite growth have been described (115, 118, 119); however, their roles in energy metabolism are still poorly understood. In vitro studies have documented the abilities of 14-3-3 proteins to regulate enzyme activity, protein phosphorylation status, protein stability, and granule transport and exocytosis (115, 120–124). Regarding metabolism and glucose homeostasis, 14-3-3 isoforms have demonstrated to interact with effectors in the insulin signaling pathway, such as IRS-1, Raf-1, AS160/TBD1C4, and FOXO1 (Fig. 4) (4, 64, 120, 121, 125–129). Collectively, these data suggest that 14-3-3 proteins could have important metabolic roles in vivo. In the context of lipid and glucose metabolism, 14-3-3 proteins interact with enzymes involved in fatty acid synthesis and glycolysis, such as fatty acid synthase and fructose-2,6-bisphosphate kinase/phosphatase, respectively (130–132). 14-3-3 Proteins might also regulate PKA-dependent HSL activity. It has been suggested that the phosphorylation of HSL by PKA during lipolysis leads to the creation of phosphorylation-binding sites for 14-3-3 proteins (133). Site-directed mutagenesis of these phosphorylation-binding sites is associated with decreased HSL activity (8, 134). Further studies using in vivo models are required to assess when 14-3-3 proteins can have such pleiotropic effects in glucose homeostasis and metabolism.

Figure 4.

Metabolic roles of 14-3-3 proteins via their interactions with client proteins. Serine or threonine phosphorylation of client proteins by various kinases generates recognition sites for dimeric 14-3-3 proteins. The interactomes of 14-3-3 proteins are large and diverse, and the interactions of 14-3-3 proteins with various metabolic effectors have been identified. This suggests regulatory roles for 14-3-3 proteins in processes such as glucose uptake, pancreatic β-cell survival, and adipogenesis.

Of the seven isoforms, we determined that 14-3-3ζ was essential for adipocyte differentiation in vitro and in vivo (64). 14-3-3ζ knockout mice were markedly lean from birth with specific reductions in visceral fat depots. In contrast, transgenic overexpression of 14-3-3ζ in mice potentiated obesity without metabolic complications (64). Through the use of isoform-specific small interfering RNAs (siRNAs), we found that only the 14-3-3ζ isoform was essential for 3T3-L1 adipogenesis, and 14-3-3ζ depletion promoted autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α (64). Given their ability to bind to proteins phosphorylated by multiple kinases, including PKA, Akt, and Raf-1, the interactomes of 14-3-3 proteins are large and diverse (122, 133, 135–138) and suggest that novel regulators of adipocyte differentiation could be identified within the 14-3-3ζ interactome. Thus, we used proteomics to elucidate the 14-3-3ζ interactome during adipogenesis and observed >100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners (138). siRNA-mediated depletion of RNA splicing factors, found to be highly abundant within the 14-3-3ζ interactome during adipocyte differentiation, revealed that some factors such as Hnrpf have essential roles in adipogenesis and in the alternative splicing of Pparg (138).

With respect to glucose homeostasis, we demonstrated that systemic 14-3-3ζ deletion improves glucose tolerance through a GLP-1–dependent mechanism (65). 14-3-3ζ Knockout mice displayed improved glucose tolerance after an oral glucose gavage compared with an intraperitoneal glucose bolus. This improvement was associated with significantly elevated fasting GLP-1 levels, and this improvement in oral glucose tolerance could be abrogated through the use of a GLP-1 receptor antagonist (65). Collectively, these findings suggest that 14-3-3ζ and perhaps its related isoforms have critical roles in the regulation of metabolism, glucose homeostasis, and adipogenesis.

Contributions of Scaffold Proteins to Cardiometabolic Diseases

Alterations in the expression of scaffolds, such as 14-3-3 proteins, have been suggested to lead to detrimental outcomes, notably carcinogenesis and neurologic disorders (115, 118, 139, 140). Also, it is not well-appreciated whether changes in the function or expression of scaffolds can have detrimental effects on metabolic health. We have provided an overview of notable examples of scaffold proteins linked to metabolic diseases.

Obesity

Obesity is a major risk for insulin resistance, type 2 diabetes, and cardiovascular disease and is associated with significantly increased adipose tissue mass (141). Multiple, complex pathways facilitate adipogenesis, which demonstrates the requirement of scaffold proteins to coordinate signaling events for accuracy. This raises the question of whether alterations in their expression or function could potentiate the development of obesity and other metabolic diseases.

Recently, we identified critical roles for the 14-3-3 protein family and, in particular, 14-3-3ζ in the regulation of visceral adipogenesis and adipocyte differentiation (64). Mice lacking 14-3-3ζ were smaller and had substantially reduced fat mass compared with their wild-type littermates. However, transgenic overexpression of 14-3-3ζ exacerbated age-onset weight gain and potentiated high-fat diet–induced obesity (64). To the best of our knowledge, this was the first study to report how changes in 14-3-3ζ levels could affect metabolic health. It was previously reported that adipose tissue from obese individuals had increased levels of 14-3-3ζ and other isoforms (142, 143), and a single nucleotide protein close to YWHAZ (gene encoding 14-3-3ζ) was significantly associated with weight gain (144). Taken together, these lines of evidence suggest an intimate role of 14-3-3ζ and perhaps it related isoforms in the pathogenesis of obesity.

Rare variants in KSR2 have been associated with the development of early-onset obesity and severe insulin resistance in humans (59). These KSR2 variants promote changes in food intake, basal metabolic rate, fatty acids, and glucose oxidation in humans. When expressed in C2C12 cells, KSR2 mutations cause impaired fatty acid and glucose oxidation (59). Through the use of proteomics, KSR2 has been found to interact with multiple proteins, including AMP-activated protein kinase (AMPK) (60, 145). This interaction is thought to be essential for metabolism, because Ksr2 knockout mice develop obesity, hyperinsulinemia, and impaired glucose tolerance (145–147). Moreover, Ksr2 knockout mice are hypophagic and expend less energy than wild-type mice (145). However, deletion of Ksr2 also impaired the thermogenic actions of AMPK activation, which suggests that Ksr2 might bridge the activity of both ERK1/2 and AMPK signaling pathways for metabolic homeostasis (145).

SH2 B adaptor protein 1 (SH2B1) is a scaffold protein involved in different RTK and cytokine receptor signaling pathways that require Janus kinases. Mice lacking the Sh2b1 gene display leptin resistance, increased food intake, severe obesity, and insulin resistance (148, 149). Four SH2B1 gene variants in humans (P90H, T175N, P322S, and F344Lfs*20) identified via the Genetics of Obesity Study cohort are associated with early-onset obesity and insulin resistance (150).

Although no gene variants or mutations for the β-arrestins in humans have been reported, β-arrestin 1 knockout mice are susceptible to diet-induced obesity, and they exhibit increased fat mass accumulation and decreased whole body insulin sensitivity when fed a high-fat diet (151). Collectively, these observations indicate that scaffold proteins themselves could influence the development of obesity and demonstrate the need for further detailed study into their contributions to obesity.

Type 2 diabetes and insulin resistance

In addition to the progressive decline in pancreatic β-cell function and mass, insulin resistance is known to contribute to the development of type 2 diabetes (49, 152). Despite the different causal factors and pathogenic mechanisms, alterations in the activity of scaffold proteins themselves could be a causal factor that promotes the development of type 2 diabetes.

It has been reported that the gene encoding the scaffold protein JIP1, MAPK8IP1, is a candidate type 2 diabetes gene in humans (153). Knockdown by siRNA or expression of a protein variant harboring a missense mutation (559N) in an insulinoma cell line were also associated with decreased INS transcriptional activity and increased susceptibility to apoptosis (153). Mice that express a point mutation in Jip1 (Thr103Ala) displayed decreased Jnk activation, which results in a protection against high-fat diet–associated insulin resistance (66).

Luan et al. (154) have proposed a relationship between β-arrestin 2 and insulin resistance. They reported that β-arrestin 2 expression in muscle and liver is severely downregulated in insulin-resistant animal models (154), and a similar relationship was observed in liver samples from a small cohort of patients with type 2 diabetes. In wild-type mice, deletion of β-arrestin 2 caused postprandial hyperglycemia and glucose intolerance, both hallmarks of insulin resistance. In contrast, administration of β-arrestin 2 in db/db mice via adenoviral delivery improved insulin sensitivity. These studies also revealed that insulin stimulates the formation of a β-arrestin 2 signaling complex, in which β-arrestin 2 scaffolds Akt and Src to the insulin receptor (154).

It has been proposed that 14-3-3 proteins might bind to various components of the insulin signaling network (4, 64, 120, 121, 125–129), suggesting that these proteins could have important roles in various insulin-induced metabolic effects in different tissues. For example, decreased levels of 14-3-3ζ are associated with decreased insulin sensitivity in humans (155). We found that systemic 14-3-3ζ knockout mice displayed decreased insulin sensitivity after peripheral administration of exogenous insulin (64). Collectively, these lines of evidence suggest that changes in 14-3-3ζ expression might cause defects in glucose transporter type 4–mediated glucose uptake in skeletal muscle or fat. The Rab GTPase-activating protein AS160/TBC1D4 (TBC1 domain family member 4) controls a distal signaling event that regulates translocation of glucose transporter type 4–containing vesicles to the plasma membrane, and its inhibitory activity is attenuated through interactions with 14-3-3 proteins (127, 156). Transgenic mice overexpressing an AS160/TBC1D4 mutant that is unable to bind to 14-3-3 proteins leads to decreased insulin sensitivity, presumably owing to the inability of 14-3-3 proteins to inhibit AS160 activity (127).

The regulatory roles of the immune system on metabolism has become an emerging area of research of importance (157), and it is possible that the scaffolds required for coordinating immune signaling could also have metabolic roles. Toll-like receptor 4 (TLR4) represents one of several immune receptors that link immune system function to metabolic health, and it has been found to be involved in the development of obesity, insulin resistance, and pancreatic islet inflammation and dysfunction (50–52, 158, 159). SAM and SH3 domain-containing protein 1 (SASH1) is a scaffold that coordinates TLR4 signaling (160), and interactions of SASH1 with different downstream effectors of TLR4, including nuclear factor κ-light chain enhancer of activated B, JNK, and p38 MAPK, is required for production of proinflammatory cytokines (160). These observations suggest a possible role for SASH1 in the development of these metabolic disorders through the production of proinflammatory cytokines and warrants further investigation. With numerous proinflammatory factors having effects on metabolic regulation (157, 161), identifying the scaffolds that coordinate the downstream pathways of their respective receptors represents an unexplored and necessary area of study.

Cardiovascular diseases

Cardiovascular diseases are the most common cause of mortality in developed countries. Chronic Gs protein activity due to prolonged β-adrenergic signaling can lead to increased myocyte size, apoptosis, and contractile failure (162–164). To date, one of the most effective treatments of heart failure is to target the βARs (β1AR, β2AR). Antagonists for these receptors function by attenuating G-protein–mediated signaling and G-protein–independent mechanisms, such as those involving receptor phosphorylation by G-protein receptor kinases and the recruitment of β-arrestins (Fig. 1). Transactivation of the EGFR has been shown to have cardioprotective effects in response to chronic catecholamine stress, and this is thought to be through a β-arrestin 1–mediated mechanism (165). Comparisons of transgenic mice overexpressing wild-type β1AR or a mutant β1AR lacking G-protein receptor kinase phosphorylation sites under conditions of chronic catecholamine stress revealed that mutant mice failed to exhibit β-arrestin–mediated EGFR transactivation and had pronounced myocyte apoptosis and left ventricular dilatation (165). Taken together, these findings demonstrate the importance of β-arrestin 1 in cardiovascular function.

In contrast, β-arrestin 2 has been identified as a potential pathogenic factor in cardiac ischemia-reperfusion (I/R) injury (166). Three major findings were observed. First, β-arrestin 2 in the heart is upregulated in response to cardiac oxidative stress and I/R injury. Second, upregulation of β-arrestin 2 triggers cardiomyocyte death and exaggerates oxidative stress-induced cardiac damage and cardiomyopathy. Finally, β-arrestin 2 binds to the p85 subunit of PI3K and subsequently prevents its interaction with CaV3, resulting in impaired activation of the PI3K-Akt-GSK3β prosurvival pathway. Moreover, downregulation of β-arrestin 2 was found to have cardioprotective effects against I/R-induced myocardial injury.

Angiotensin II type IA receptors are another family of GPCRs that require β-arrestins and are implicated in heart failure. Mechanical stretch of heart tissue can trigger a conformational change in angiotensin II type IA receptors, leading to β-arrestin recruitment in the absence of ligand (167). Hearts from mice lacking β-arrestin or angiotensin II type IA receptors failed to induce a cardioprotective response to mechanical stretch. Also, the same heart displayed altered ERK and Akt activation, impaired transactivation of the EGFR, and enhanced myocyte apoptosis. Collectively, these findings support differential roles for β-arrestins in the context of cardiovascular disease.

Another scaffold protein family implicated in βARs signaling in the heart is the AKAP family. AKAP members have differential roles in cardiac physiology. For example, AKAP18α (AKAP7α) targets l-type calcium channels to regulate Ca²⁺ influx in response to β-adrenergic stimulation (56, 57). Murine AKAP150 and human AKAP79 have been shown to interact with different enzymes such as PKA, PKC, and calcineurin and their potential substrates, including l-type calcium channels, β-ARs, caveolin-3, and adenylyl cyclase (58, 73, 168–174). Moreover, loss of AKAP150 disrupts intracellular trafficking of β-ARs in cardiomyocytes (175). Defects in agonist-internalized β1-AR recycling have been observed in AKAP5 knockout myocytes, and this could be rescued when full-length AKAP5 was reintroduced (175). This suggested that AKAP5 exerted specific and profound effects on β1-AR recycling in mammalian cells.

Some observations have proposed a link between MAPK/ERK signaling and cardiac hypertrophy. For example, the four and a half LIM domain protein-1 (FHL1) is a novel MAPK scaffold protein that mediates MAPK signaling responses at the sarcomere during stress-induced cardiac hypertrophy (176). FHL1 is ubiquitously expressed in mice and humans and increase in the heart during hypertrophic conditions (177–181). In vivo studies using transgenic mice overexpressing a constitutively active form of Gq selectively in the heart demonstrated that FHL1 influences Gαq signaling, as Fhl1 deficiency prevented Gq-dependent cardiomyopathies (176).

Taken together, the differential roles of scaffold proteins with respect to cardiomyopathies have been identified, demonstrating their importance to cardiovascular health and disease.

Concluding Remarks

Scaffold proteins have emerged as multifunctional molecules with essential roles in cell signaling. Thus, a greater understanding of the contributions of scaffolds to metabolism and cardiometabolic diseases should provide a framework for the identification of new, potential targets for the treatment of these diseases. For example, increasing our current understanding of how scaffolds regulate and interact with their target proteins could provide additional information on the pathogenic mechanisms of potential therapeutic targets. One method to understand in greater detail how a scaffold regulates multiple pathways within metabolic tissues will be to examine the interactomes of various scaffolds under physiological and pathophysiological conditions. Such studies will expand our understanding of their metabolic functions and potentially open new avenues of research to better metabolism and metabolic disorders.

Glossary

Abbreviations:

AKAP

cAMP-dependent kinase–anchoring protein

Akt

protein kinase B

AMPK

AMP-activated protein kinase

EGFR

epidermal growth factor receptor

FGFR

fibroblast growth factor receptor

FHL1

four and a half LIM domain protein-1

GLP-1

glucagon-like peptide-1

GLP-1R

glucagon-like peptide-1 receptor

GPCR

G protein–coupled receptor

HSL

hormone-sensitive lipase

I/R

ischemia/reperfusion

IRS

insulin-receptor substrate

JNK

c-Jun NH2-terminal kinase

KSR

kinase suppressor of RAS

Nck

noncatalytic region of tyrosine kinase

PERK

protein kinase R–like endoplasmic reticulum kinase

PI3K

phosphatidylinositol 3′-kinase

PKA

protein kinase A

PKC

protein kinase C

RTK

receptor tyrosine kinase

SASH1

SAM and SH3 domain-containing protein 1

SH

Src homology

SH2B1

SH2 B adaptor protein 1

siRNA

small interfering RNA

TLR4

toll-like receptor 4

TRP

transient receptor potential

VEGFR

vascular endothelial growth factor receptor

βAR

β-adrenergic receptor