AKAP12 Signaling Complex: Impacts of Compartmentalizing cAMP‐Dependent Signaling Pathways in the Heart and Various Signaling Systems

Abstract

Heart failure is a complex clinical syndrome, represented as an impairment in ventricular filling and myocardial blood ejection. As such, heart failure is one of the leading causes of death in the United States. With a mortality rate of 1 per 8 individuals and a prevalence of 6.2 million Americans, it has been projected that heart failure prevalence will increase by 46% by 2030. Cardiac remodeling (a general determinant of heart failure) is regulated by an extensive network of intertwined intracellular signaling pathways. The ability of signalosomes (molecular signaling complexes) to compartmentalize several cellular pathways has been recently established. These signalosome signaling complexes provide an additional level of specificity to general signaling pathways by regulating the association of upstream signals with downstream effector molecules. In cardiac myocytes, the AKAP12 (A‐kinase anchoring protein 12) scaffolds a large signalosome that orchestrates spatiotemporal signaling through stabilizing pools of phosphatases and kinases. Predominantly upon β‐AR (β₂‐adrenergic‐receptor) stimulation, the AKAP12 signalosome is recruited near the plasma membrane and binds tightly to β‐AR. Thus, one major function of AKAP12 is compartmentalizing PKA (protein kinase A) signaling near the plasma membrane. In addition, it is involved in regulating desensitization, downregulation, and recycling of β‐AR. In this review, the critical roles of AKAP12 as a scaffold protein in mediating signaling downstream GPCRs (G protein–coupled receptor) are discussed with an emphasis on its reported and potential roles in cardiovascular disease initiation and progression.

Nonstandard Abbreviations and Acronyms

AKAP

A‐kinase‐anchoring protein

β‐AR

β‐adrenergic receptor

ERK

extracellular signal–regulated kinase

GPCR

G protein–coupled receptor

PKA

protein kinase A

PKC

protein kinase C

PLN

phospholamban

SERCA

sarco/endoplasmic reticulum Ca²⁺‐ATPase

SR

sarcoplasmic reticulum

VIP

vasoactive intestinal peptide

AKAPs (A‐kinase anchoring proteins) (AKAPs) belong to a family of scaffolding proteins that organize complex signal transduction events descending from cell surface–stimulated receptors.1 AKAPs mediate sequestering of protein kinases, phosphatases, and signal termination molecules, with their target substrates.1 Thus, AKAPs coordinate phosphorylation and dephosphorylation states within the cell to confer the specificity of intracellular events. When first identified, regardless of the general structural diversity,2 they were functionally characterized on the basis of their ability to bind to PKA (protein kinase A).3 This binding capacity is mainly attributed to their highly conserved amphipathic helix that anchors to PKA regulatory subunits.4 In addition, AKAPs localize to diverse subcellular locations, including, but not limited to, the plasma membrane, mitochondria, nuclear envelope, and cytoskeleton.5, 6, 7

Briefly, on their binding to the regulatory subunit of PKA, AKAPs localize the activation of PKA catalytic subunits, utilizing the specific targeting sequences located near their N terminus.8 This explains how such a common signaling pathway (PKA signaling) can serve many different functions with high selectivity. However, later studies have shown that these scaffold proteins further can bind to other substrates and even homo‐ or heterodimerize/oligomerize,9 indicating a more complex spatial and temporal signaling profile.10, 11, 12

Kinases and phosphatases are key regulators of enzymatic activities and protein interactions. AKAPs colocalize these enzymes by playing a major role in influencing cellular activities, where any deviation from their normal function may induce states of imbalance and disease. Several AKAPs have been identified within the past decade. However, their full function/mechanism of action in different systems under physiologic conditions and possible involvement in pathophysiologic states is not fully understood. Thus, in this review we covers some of the major AKAPs, with a stronger emphasis on AKAP12 and its possible associated cardiovascular pathways/effects.

AKAP–PKA Communication Platform

Recent studies have shown that distinct AKAPs bind to different protein kinases/phosphatases, uniquely colocalizing them to their targets. An exception is PKA, which is the common protein kinase that all AKAPs share the capacity to bind.3 Considering the diverse network through which PKA signals, it is peculiar how it can still be highly specific to its targets. Further understanding of binding/signaling mechanisms through PKA gives insights into the general AKAP signalosome.

Briefly, PKA holoenzyme (a heterotetramer) contains 2 possible regulatory subunits, either RI or RII.13 The regulatory subunits keep the enzyme in a dormant state at low levels of cAMP through inhibiting the 2 catalytic subunits (PKAc).13 When PKAc subunits are activated, they phosphorylate serine/threonine residues on their multiple target substrates, triggering downstream signaling pathways.14

To manage a more distinct signal specificity of such diverse pathways, adjustments take place within the cell. Such adjustments include sequestration of AKAP complexes near their targets, a local increase in cAMP levels, and the appearance of termination mechanisms.13, 15 Although the local increase of cAMP levels postactivation of the upstream regulators of PKA may assure some level of specificity, yet another signal termination mechanism must exist to maintain homeostasis. AKAPs assure this through not only linking PKA to its upstream modulators but also to signal termination molecules such as phosphodiesterase through scaffolding to them as well.16

So far, all studied AKAPs contain the highly conserved sequence of 14 to 18 amino acids, usually Gln4–Lys21, which forms a 5‐turn amphipathic (alpha) helix.17 This helix is important because of its ability to slide into the binding pocket near the N terminus of PKA.17, 18 The majority of AKAPs preferentially anchor the RII dimer of the PKA holoenzyme.19 However, D‐AKAP1 and D‐AKAP2 are capable of binding to either subtype of PKA‐regulatory subunits.19 Furthermore, recent studies have indicated that AKAP11 and sphingosine kinase–interacting protein selectively bind to the RIα subunit of PKA, forming a dynamic cytoplasmic signaling complex.14, 20 However, it remains unknown whether AKAP anchoring to either RI or RII subunits of PKA has differential effects on its signaling outcomes. Nonetheless, it is established that the binding of AKAPs to the RI or RII subunits neither affected the tertiary nor the quaternary conformations of the dimer.18 On the contrary, exposure to elevated levels of cAMP induces such change.21 Thus, AKAPs are not essential for the phosphorylation/activation of PKA. Rather they are crucial to spatially restrict the activation of PKA to areas where cAMP levels are high with the stimulation of upstream proteins/receptors.21

As mentioned previously, AKAPs can directly bind and sequester various signaling proteins. AKAPs can bind protein kinases (PKC [protein kinase C] and mitogen‐activated protein kinase), phosphatases, cAMP phosphodiesterase (PDE), guanosine triphosphate–binding proteins, and adaptor proteins. PDEs reduce the local levels of cAMP available for triggering downstream signaling pathways.22 Considering that PKA is mainly affected by cAMP levels, crosstalk between PKA and PDE is crucial to forming a feedback loop, which is believed to be mainly controlled through their colocalization by AKAPs at the same subcellular compartments.23

Another key signaling transduction mechanism that ultimately activates nuclear transcription factors is regulated by the calcium–calmodulin‐dependent serine–threonine protein phosphatase 2B/calcineurin. Certain AKAPs, such as AKAP79/150, anchor protein phosphatase 2B/calcineurin to facilitate the downstream signaling. Ultimately, this signalosome complex activates effector molecules within the nucleus, including the nuclear factor of activated T cells.24 This diversity of AKAPs and their complexes indicates their ability to control a vast array of opposing signaling pathways within the cells to maintain homeostasis. Regardless, the exact signaling mechanism driving their ability to compartmentalize the complexes near their targets is still speculative because of the high structural diversity of AKAPs and their anchoring domains. It is worthy of mention that the presence of adenylyl cyclase subtypes reported in some AKAP (AKAP79, Yotiao, and mAKAP) complexes25 demonstrates a possible constitutional adenylyl cyclase–AKAP binding that maintains these unique clusters near their targets at basal levels where cAMP will be increased once the upstream receptors are stimulated. However, this scheme cannot be generalized for all AKAPs. For instance, myristylation of N‐terminal sequences, along with the presence of basic amino acid–rich regions (polybasic domains) within the N terminus on AKAP12 is sufficient to mediate this localization near the β₂‐AR.26, 27

Notably, the AKAP12 association with PDE4D3 and β₂‐AR (β₂‐adrenergic receptor) recruitment was enhanced with activation of PKA.23, 28 PDE4 is exceptionally important in cardiovascular signaling, which is delineated by its signaling under cardiac diseases. For instance, under cardiac hypertrophy, PDE4 activity is decreased29 and associated with propagation of heart failure.30 Furthermore, acute PDE inhibition enhances cardiomyocyte function, whereas chronic inhibition results in increased mortality.31 This complexity may be explained by the compartmentalized controls of cAMP levels by PDE4, in an agonist‐dependent manner downstream of β₂‐AR. Such properties make PDE4 a favorable partner for many AKAPs.

AKAP Functional Roles

Generally, signaling interactions are governed by the global abundance of proteins in addition to their specific interactions. The specificity of such interactions is fundamental to maintain homeostasis and is controlled by the organized subcellular microdomains, coordinated by several scaffolding proteins, some of which are AKAPs. AKAPs dictate how, when, and where each member of their complexes will initiate a certain action. Usually, under pathophysiologic conditions, the global balance of signaling molecules, such as cAMP and PKA, is not affected. Rather imbalances within certain AKAP complexes are evident, thus making them valuable therapeutic targets. In what follows we briefly cover the involvement of different AKAPs in variable systems (Table 1),32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 followed by a more detailed discussion of their functions in the cardiovascular system.

Table 1.

AKAP‐Reported Functions in Various Signaling Systems

System	AKAP	Effect	Mechanism
Nervous32, 33, 34	AKAP5 (AKAP79/150)	Promotes synaptic plasticity; essential for NMDA receptor–mediated LTD and normal motor coordination	Scaffolding of PKA, PKC, and PP2B to phosphorylate glutamate receptors
Immune35, 36	Ezrin	Suppression of T‐cell replication	Scaffolding of PKA to phosphorylate C‐terminal Src kinase
	D‐AKAP1	HIV progression	Cofactor of HIV reverse transcriptase
Reproductive37, 38, 39	AKAP1	Defective maturation of ovaries	Disrupted association of PKA to AKAP1
	AKAP3	Impaired sperm motility	PI3 interference with AKAP3–PKA binding
	AKAP4	Impaired sperm motility	Failure of association of glycolytic enzymes to the fibrous sheath
Endocrine40, 41	AKAP79/150	Regulation of insulin secretion	Reduction of PP2B activity
	AKAP18α/γ	Enhance/reduction of insulin secretion respectively	Controlling glucagon‐like peptide‐1–mediated insulin secretion
Renal42, 43	AKAP18δ	Trafficking of aquaporins AQP2 toward plasma membrane	PKA Phosphorylation at serine (S)256, S261, S264, and S269 at AQP2
	AKAP‐Lbc	Redistribution of AQP2 from intracellular vesicles to the periphery of medullary collecting duct principal (IMCD) cells	PKA phosphorylation of RhoA at S188 inducing its inhibition
Respiratory44	AKAP1	Worsening of hyperoxia‐induced acute lung injury	Inhibition of lung NF‐κB p65 activity
Gastrointestinal45, 46	AKAP150	Regulation of pepsinogen secretion	Unclear
	Ezrin	Parietal cell activation	Scaffolding of ezrin with Stx3
Liver47	Ezrin	Gap junction modulation	PKA phosphorylation of connexin 43

AKAP indicates A‐kinase anchoring protein; AQP2 aquaporins; LTD, long‐term depression; NMDA, N‐methyl‐D‐aspartate receptor; PKA, protein kinase A; PKC, protein kinase C; PP2B, protein phosphatase 2B; Stx3, syntaxin3; NF‐κB, nuclear factor‐kappa light‐chain enhancer of activated B cells; PI3; phosphoinositide 3; SRC, proto‐oncogene tyrosine–protein kinase; Stx3, syntaxin 3; and RhoA, Ras homolog family member A.

AKAPs and Cardiovascular System

Cardiac function is strongly impacted by the adrenergic system, mainly through the interaction of catecholamines, with β‐ARs controlling both contractility and heart rate.48 Such distinct cellular events are mainly controlled via PKA followed by the secondary messenger cAMP, on stimulation of GPCRs (G protein–coupled receptors).49 Importantly, several reports showed that cAMP levels increase within specific cellular compartments, as a “local increase” in a stimulus‐induced manner, assuring some level of specificity.50, 51, 52 Also, multiple AKAPs have been associated with the compartmentalization of cardiac signaling pathways. Of the >50 AKAPs characterized so far, several have been associated with cardiac development, contractility, cardiac morphology, and rhythm (Table 2).53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64

Table 2.

Cardiac AKAP‐Reported Functions

AKAP	Alternative Names	Effect	Mechanism
AKAP153	D‐AKAP, AKAP121	Reduction of ROS in the heart	Increase SOD2 mitochondrial expression
AKAP553	AKAP79, AKAP75, AKAP150	Increase ROS, and inflammatory responses	Activation of PKC
AKAP654	AKAP100, mAKAP	Regulation of cardiomyocyte oxygen homeostasis	Enhances the transcriptional activation of HIF‐1α–regulated genes
AKAP755	AKAP15, AKAP18	Regulation of calcium handling	Coordinates PKA phosphorylation of PLN
AKAP956	Yotiao	Increases the slow outward potassium ion current	Targeting PKA and PP1 to hKCNQ1
AKAP1057	D‐AKAP2	Regulation of cardiac rhythm	Unknown
AKAP1258,60,61	Gravin, SSeCKS	Cardiac pathophysiology (to be defined)	Unknown
AKAP1353, 62	AKAP Lbc	Mediates metabolic switch during the development of compensatory hypertrophy	Activation of PKD1
Synemin63	SYNM	Maintenance of normal ventricular function	Unknown
Myosprin64	CMYA5	Modulates the clustering of RyR	Unknown

AKAP indicates A‐kinase anchoring protein, HIF‐1α, hypoxia‐inducible factor‐1α; hKCNQ1, human potassium voltage‐gated channel subfamily Q member 1; PKA, protein kinase A; PKC, protein kinase C; PKD1, polycystin 1; PLN, phospholamban; PP1, protein phosphatase 1; ROS, reactive oxygen species; RyR, ryanodine receptor; and SOD2, superoxide dismutase 2.

Cardiac Development

During cardiac development (Figure 1),62, 65 AKAP‐LBC (AKAP13) is postulated as a key regulator of cardiomyocyte differentiation and morphogenesis.65 The mechanism of this regulation of differentiation and morphogenesis is believed to be through producing a platform that links Gα12 and Rho signaling to an essential transcriptional program, as confirmed by the observed embryonic lethality upon deletion of AKAP13 in mice.66

Figure 1.

Role of AKAP13 in cardiac function. GPCR signaling is directed by the type of ligand binding to this receptor. The nature of the ligand would favorably shift the equilibrium to a specific conformation of the receptor, which signals through a distinct G protein. For AKAP13 and GPCR signaling in the heart, it has been associated with 2 distinct G protein–signaling pathways (Gα12 and Gαs). A, When stimulation of β‐AR stabilizes its conformation to signal through Gα12, AKAP13 signalosome is attracted near the receptor. AKAP13 interacts with Gα12 allowing Rho‐GEF stimulation. Upon Rho‐GEF stimulation, both SRF (Rho‐GEF guanosine triphosphatase) and myocyte enhancer factor 2C (transcription factor) activities are increased. However, the mechanism of their activation depends on different regions of AKAP13. Activation of myocyte enhancer factor 2C depends on the carboxyl region of AKAP13 (BRX), whereas SRF activation depends on the GEF region of AKAP13. Collectively, this suggests that increased gene expression of factors associated with cardiac development may be partially dependent on AKAP13.65 B, AKAP13 has been reported in the cardiac hypertrophic signaling. Under states of increased hemodynamic load, it is usually accompanied by a favorable state of Gαs pathway signaling within the cardiomyocytes. AKAP13 signalosome releases PKD to phosphorylate HDAC5. Phosphorylation of HDAC5 activates MEF2, which drives the transcription of genes associated with cell growth, Ca²⁺ handling, and contraction. The aforementioned multistep process of PKD release starts with PKA activation as a response to the Gαs pathway. PKA further phosphorylates PKC. Phosphorylation of PKC activates PKD, leading to its dissociation from the signalosome.62 α12 indicates α subunit of Gα12 protein; αs, α subunit of Gαs protein; β‐AR, β‐adrenergic receptor; AKAP, A‐kinase anchoring protein; GPCR, G protein–coupled receptor; HDAC5, histone deacetylase 5; MEF2C, myocyte enhancer factor 2C; PKA, protein kinase A; PKC, protein kinase C; PKD, protein kinase D; PM, plasma membrane; Rho‐GEF, Rho guanine nucleotide exchange factor; and SRF, serum response factor.

Cardiac Contractility

Regarding cardiac contractility (Figure 2), normal contraction of the heart is controlled by a delicate regulation of cytosolic Ca²⁺ levels through a complex interplay between the ryanodine receptor, L‐type calcium channels, SR (sarcoplasmic reticulum), and SERCA (sarco/endoplasmic reticulum Ca²⁺‐adenosine triphosphatase). Several AKAPs have been involved in facilitating the proper signal transduction within these complexes, such as AKAP79/150, AKAP12, AKAP15/18, mAKAP, and AKAP18δ. For instance, Ca²⁺ signaling on the surface of the cell was linked to AKAP15/18 ability to target the PKA to the C terminus of L‐type voltage‐gated calcium channel channels. Nuclear Ca²⁺ signaling is regulated through mAKAP acting on the nuclear membrane, whereas AKAP18δ affects SR levels of Ca²⁺.66 mAKAP forms macromolecular complexes, composed of PKA, PDE4D3, adenylyl cyclase 5, protein phosphatase 2A, mitogen‐activated protein kinase 5, ERK5 (extracellular signal–regulated kinase 5) Rap1, and Epac1.67, 68, 69, 70 The strategic location of this complex on the nuclear membrane allows mAKAP to promote the opening of ryanodine receptor channels located on the nuclear envelope (through targeting PKA to these channels using leucine/isoleucine zipper motifs within the mAKAP), impacting both Ca²⁺ rise and perinuclear flux to further increase cardiac function.66, 71 Historically, mAKAP was thought to play a crucial role in cardiac contractility on the basis of studies identifying its localization on the SR.72, 73 However, later studies alternatively have established that mAKAP is located on the nuclear membrane rather than SR and it acts on the nuclear ryanodine receptor.74, 75 Considering the differences in the signaling between cytoplasmic and nuclear Ca²⁺ pools, mAKAP is primarily involved in gene expression regulation rather than cardiac contractility.76

Figure 2.

Role of AKAP complexes in cardiac contractility. Cardiac contractility is controlled mainly by the delicate interplay between RyR, calcium channels (Ca²⁺ _v1.2), SR, and SERCA. On electrical stimulation, the voltage‐gated Ca²⁺ channels increase Ca²⁺ influx, leading to local areas of high Ca²⁺ (1). The local increase of Ca²⁺ activates the RyR channels located on the SR (2). The RyR channels activation increases Ca²⁺ efflux from the SR, causing a cytosolic (global) increased Ca²⁺ (3). The global increase in Ca²⁺ triggers muscle contraction cycle, as a result of Ca²⁺ binding to troponin (4). SERCA plays a major role in maintaining consistency in the contractile force between cardiac beats. As a response to high cytosolic levels of Ca²⁺, SERCA reuptakes Ca²⁺ back into the SR to return to baseline levels in the cytoplasm. SERCA activity is controlled by PLN, which, when phosphorylated by PKA, allows SERCA to reuptake Ca²⁺. AKAPs play a crucial role in each of these phases; AKAP15/18 facilitates Ca²⁺ influx through targeting PKA to the C terminus of Ca²⁺ _v1.2 channels, AKAP79/150 binds PKA and PKCα to Ca_v1.2 channels to facilitate their normal gating, and AKAP18δ mediates PKA phosphorylation of PLN causing it to detach from SERCA, thus allowing the reuptake of Ca²⁺. AKAP indicates A‐kinase anchoring protein; Ca²⁺ _v1.2, calcium channel; Na⁺/Ca²⁺ exchanger, sodium–calcium exchanger; PKA, protein kinase A; PKC, protein kinase C; PLN, phospholamban; PM, plasma membrane; RyR, ryanodine receptor; SERCA, sarco/endoplasmic reticulum Ca²⁺‐adenosine triphosphatase; and SR, sarcoplasmic reticulum.

AKAP18δ influences SERCA2 needed for reuptake of Ca²⁺ from the cytoplasm to assure consistency of cardiac contractile force within each beat. Briefly, SERCA2 activity is affected by calcium gradients, level of SERCA2 expression, and the levels of the SERCA2 suppressor PLN (phospholamban). In this manner, AKAP18δ mediates PKA phosphorylation of serine 16 on PLN, dissociating it from SERCA2, which subsequently increases the uptake of Ca²⁺ into the SR and enhances cardiac function.55 Other AKAPs, such as synemin77 and myomegalin, have also been proposed to play a role in cardiac contractility because of their sarcomere intracellular localization.77 ^, 78

In addition to the aforementioned function of AKAP79/150 in cardiac contractility, it has been also linked to cardiac rhythm regulation.79 This regulation of cardiac rhythm is potentially achieved through binding PKA and PKCα to L‐type voltage‐gated calcium channels to facilitate their normal gating, thus protecting against arrhythmias.79 Moreover, Yotiao (AKAP9) regulates normal cardiac rhythm by recruiting PKA and protein phosphatase‐1 to the C‐terminal subunit of the slowly activating delayed rectifier potassium channels, where these channels are responsible for conducting ionic currents.80 Finally, D‐AKAP2 (AKAP10) was proposed to control pacemaker sensitivity to cholinergic stimulation.57 It is has been proposed that a coordinated interplay between these 3 AKAPs (AKAP79/150, Yotiao, and D‐AKAP2) regulates heart rhythm in distinct yet interconnected pathways.

Cardiac Hypertrophy

Cardiac hypertrophy, defined as an increase in cardiomyocyte size (adaptive remodeling of the heart) under stress or continuous stimulation, can develop into further pathophysiologic states, such as heart failure. Thus, the involvement of mAKAP, AKAP79/150, and AKAP‐LBC was examined in mediating the pathologic state of cardiac hypertrophy. So far, mAKAP and AKAP79/150 activation has been associated with nuclear translocation of nuclear factor of activated cells, promoting the transcription of hypertrophic genes.81 In addition, mAKAP activates the mitogen‐activated protein kinase pathway in a cAMP‐dependent manner, which entails further activation of the prohypertrophic factor myocyte enhancer factor 2C.69 Alternatively, AKAP‐LBC (AKAP13) facilitates PKC/PKD coupling to inactivate histone deacetylase to induce cardiac hypertrophy (Figure 1).82

Together, it is evident from the accumulated studies that AKAPs play a major role in controlling many aspects of cardiac function, and that any alteration within their complexes may contribute to the development of cardiac disease. This notion is supported by the important roles reported on other AKAP signalosomes in developing cardiac hypertrophy. For instance, in heart failure there is upregulated gene expression of AKAP‐LBC, AKAP18, and AKAP2, whereas the AKAP121 gene was downregulated.83 Interestingly, AKAP12 (detailed in what follows) knockout ameliorated cardiac function in an animal heart failure model58; however, the exact mechanism is not yet fully understood, and more research is needed to elucidate this observation.

Importantly, AKAPs may be involved in alterations of spatial localization of β‐AR in heart failure. In failing hearts, β₂‐AR signaling is redistributed from T tubules to cell crest. As a result, β₂‐AR uncouples localized PKA pools, mediating a cellwide rather than localized cAMP propagation.84 Such alteration in signaling further propagates the heart failure phenotype. As of now, roles of AKAPs in mediating such responses in cardiomyocytes are remain unknown.85

β₂‐AR dysregulation is associated with heart failure.86, 87 Stimulated β₂‐ARs couples with Gαs and can subsequently switch to Gαi, regulating cAMP levels, PKA activity, and phosphorylation of several downstream pathways.88 Alterations in β₂‐AR desensitization and resensitization play a major role in development of heart failure. Mechanisms of β₂‐AR desensitization are well characterized,89, 90, 91, 92 but few studies have investigated β₂‐AR resensitization schemes.92, 93, 94 Notably, one study reported the importance of Ser‐355/356 phosphorylation in regulating physiologic resensitization in neonatal cardiomyocytes.93 Another study using an integrated whole‐cell response microfluidic system suggested the need for Src‐regulated processes and dynamin (cytoskeleton‐associated protein) for β₂‐AR de/resensitization.92 Interestingly, elevated AKAP121 levels reduced apoptosis of cardiomyocytes understimulated ischemia, through its action on dynamin‐like receptor‐1.95 Accordingly, reports of failing human hearts showed diminished levels of AKAP121.83, 96

Collectively, AKAP complexes contain multiple proteins that play a significant role in regulating β‐AR signaling and can be a valuable therapeutic target, as they contribute to mediating several cardiac functions. Thus, future studies should focus on the biochemical details of these protein networks and their involvement in both physiologic and pathophysiologic aspects.

AKAP12 is an A‐Kinase Anchoring Protein

AKAP12 was first recognized as an autoantigen in serum from myasthenia gravis patients,97 hence the alias name (Gravin). It is also known as the Src‐suppressed C kinase substrate, which is considered the rodent ortholog of the human identified Gravin98; however, these terms are used interchangeably. This 250‐kd multivalent AKAP interacts with PKA, along with several other molecules and signaling proteins such as PKC, β₂‐AR, PDE4D, β‐1,4‐galactosyltransferase, non‐receptor tyrosine kinase Src, and Ca²⁺/calmodulin. The AKAP12 signalosome plays a central role in organizing (GPCRs) to protein kinases and phosphatases. In particular, it targets PKA and other signaling molecules to the cell periphery near β₂‐ARs, to regulate its resensitization and recycling.99, 100, 101

It has been proposed that AKAP12 subcellular distribution is dynamic and affected by intracellular Ca²⁺ concentration, in addition to the level of PKC activation. This introduces the possibility of crosstalk between PKA, PKC, Ca²⁺/calmodulin,99, 100 and potentially other additional molecules.

Upon evaluating AKAP12's structure (Figure 3),102, 103, 104 domains responsible for its targeting to the cell periphery are believed to be the N terminal's myristoylation sites along with its 3 polybasic domains located near the N terminus.26 However, the exact mechanism is still unclear. In particular, one study reported that the presence of the polybasic subunits was sufficient to only dissociate AKAP12 from the cell periphery in response to phorbol ester treatment, whereas the putative N‐terminal myristoylation was required for AKAP12's redistribution to intracellular vesicular compartments.26 Yet another study demonstrated that the presence of any 2 polybasic domains, as described in their constructs lacking the myristoylation sites, was sufficient for targeting AKAP12 to the cell periphery and subsequent regulation of β₂‐AR resensitization.100

Figure 3. AKAP12 schematic topological map shows the known and potential protein–protein binding and phosphorylation domains.

2–7 coiled coils indicates 2–7 alpha‐helices usually involved in gene expression102, 103, 104β2‐AR indicates β₂‐adrenergic receptor; BD, binding domain; EGF, epidermal growth factor; GalTase, β‐1,4‐galactosyltransferase; PKA, protein kinase A; PKC, protein kinase C; SRC, non‐receptor tyrosine kinase; and WSK, short motifs composed of 3 conserved residues found in a WXSXK motif.

Furthermore, 3 distinct isoforms of AKAP12 (α, β, or γ) have been identified so far and they share >95% amino acid sequence identity, with variability between the isoforms only shown to be within their N terminus. Thus, having 3 AKAP12 isoforms can potentially explain the distinct spatial distribution isoform profiles such that the myristoylation of an AKAP12α is necessary for targeting it to the endoplasmic reticulum.105 Notably, these isoforms are independent transcripts under the control of separate promoters, showing distinct spatiotemporal mRNA expression in different organs.105

In summary, these 2 features of AKAP12‐‐‐(1) the presence of many docking/binding domains for kinases/phosphatases and (2) its broad expression‐‐‐drives its ability to influence cellular signaling pathways in many systems. Thus, these cellular signaling roles of AKAP12 are discussed in more detail, along with an emphasis on its cardiac capacities, in what follows.

AKAP12 Signaling in Cellular Systems

AKAP12 is probably one of the most studied AKAPs in cell cycle regulation. It is known as a negative regulator of the G1/S‐phase transition and important for the completion of cytokinesis through altering expression levels of cyclin D1 by either blocking its synthesis106 or scaffolding PKC to control the formation of actin–myosin rings.107 Accordingly, AKAP12 was intensively studied in the cancer field and reported as a metastasis suppressor in a variety of cancers,102 such as prostate and skin cancers.108 Metastasis suppression has been attributed to attenuating tumor‐intrinsic PKC and Src pathways108 or reducing the secretion of tumor chemoattractants in the peritoneum.106 In contrast, a study examining the mechanism underlying glioblastoma multiform reported that patients with higher AKAP12 mRNA levels had overall worse survival rates.109 Although AKAP12 expression eas shown to be downregulated in several cancers, including prostate, breast, gastric, skin, and colon cancers, in addition to hepatocellular carcinoma and melanoma, its upregulation may still be a risk factor in other types of cancer.109

When examining the roles of AKAP12 in endothelial tissues, it has been proposed that vascular endothelial dysfunction, generated by lipopolysaccharide treatment, induces upregulation of AKAP12. Such upregulation of AKAP12 may improve endothelium‐dependent relaxation through its binding to the atypical isoform of PKC (PKCζ), a well‐known pathologic mediator of endothelial dysfunction.110 Upon binding, AKAP12 inhibits PKCζ‐mediated reduction of the ERK5 pathway.110 Furthermore, depletion of AKAP12 was shown to increase pro‐inflammatory proteins (tumor necrosis factor‐α, interleukin‐1β, and interleukin‐6) and reactive oxygen species formation while reducing both endothelial nitric oxide synthase expression and ERK5 transcription.110 This suggests the potential participation of AKAP12 in altering cytoskeletal morphology during inflammation.

In addition, lipopolysaccharide‐induced inflammatory response in astrocytes has revealed that the β‐1,4‐galactosyltransferase‐I–AKAP12 complex colocalizes in the perinuclear region at either basal levels or in the presence of lipopolysaccharide.111 The complex was directed to the trans‐Golgi network, where astrocyte migration was inhibited during lipopolysaccharide treatment.111 This may be explained by inhibition of β‐1,4‐galactosylation of integrin‐β1, suggesting that AKAP12 integrates the various functions attributed to the galactosyltransferase I cytoplasmic domain.111

In line with these findings, AKAP12 has been associated with macrophage regulation during the convalescent stage of inflammation. In particular, this was shown by AKAP12 activating the M2 macrophages (anti‐inflammatory type), which then induced a more compact extracellular matrix. This was further confirmed by the positive correlation between both CD206 (M2 marker) and AKAP12 expression in humans and animal models.112 Interestingly, a recent study investigating GPCR‐mediated transactivation of epidermal growth factor receptor in epithelial cells showed that AKAP12 stabilized naked cuticle homolog 2 by facilitating PKA phosphorylation of Ser‐223 on naked cuticle homolog 2. This further ensured efficient cell‐surface delivery of transforming growth factor‐α to increase epidermal growth factor receptor activation. Furthermore, AKAP12 knockout canceled baseline phosphorylation of the naked cuticle homolog 2 domain and reduced it in response to forskolin, VIP (vasoactive intestinal peptide), and prostaglandin estradiol,113 stipulating a potential constitutive phosphorylating capacity of AKAP12 within its domains. Considering the homeostatic capacity of epithelial cells within multiple organs, further studies are crucial to examine such constitutive activity.

Within the pulmonary smooth muscles, the main signaling pathways are governed by coupling of the G protein, either Gαs or Gαq, to β‐ARs. The Gαq pathway controls smooth muscle contraction and tone through activating phospholipase C and diacylglycerol, whereas the Gαs pathway causes local elevation of cAMP, which further causes activation of PKA and Epac in airway smooth muscles to influence contraction/relaxation of these muscles.114 AKAP12 and AKAP5 are the main mediators of compartmentalizing phospholipase C, diacylglycerol, and PKA near the β₂‐ARs and muscarinic receptor 3. AKAP5 increases G protein–coupled receptor kinase 2 recruitment to the cell surface, causing β₂‐AR desensitization and internalization. AKAP12, however, is not involved in agonist‐specific desensitization or the internalization of GPCRs.115, 116, 117 Instead, it is responsible for the dephosphorylation, resensitization, and recycling of these receptors from intracellular vesicles back to the periphery. Thus, a balance between these AKAPs in addition to AKAP78 (involved in β₂‐AR internalization) is needed for proper function of the airways.118, 119

In addition, both AKAP12 and AKAP5 have been proposed to be regulators of pulmonary endothelial barrier function, and AKAP12 is required for this cAMP‐mediated barrier stabilization.120, 121 However, a study examining the mechanism of cigarette smoke extract deterioration on airway endothelial function showed that mRNA expression levels of AKAP12 and AKAP5 were not affected, whereas a reduction in expression levels of another AKAP member (AKAP9) was noted and associated with reduced E‐cadherin levels.122 Interestingly, another study linking hypoxia in endothelial cells, widely implicated in many pathologic conditions, has also been linked to elevated AKAP12 gene expression, which is in contrast to other members of the AKAP family (AKAP1, AKAP17A, AKAP79, and AKAP100). Moreover, an analysis of the cloned promoter of AKAP12 revealed a functional hypoxia‐responsive domain with 2 binding sites for hypoxia‐inducible factor.123

Furthermore, AKAP12 gene expression has also been inversely associated with estrogen receptor gene expression, particularly Erβ.124 In Erβ‐null granulosa cells, upregulation of AKAP12 genes was identified as a mechanism that reduced cAMP levels through combating the sequestration of PKA regulatory subunits.124 This was further supported in a study examining the agonist‐dependent AKAP12 role in regulating cAMP levels. The study demonstrated that AKAP12 expression is crucial to allow (human embryonic kidney 293 cell line and human epidermoid carcinoma cell line) cells to recover their cAMP response to the agonist.118

Overall, it is apparent that AKAP12 plays a critical role in regulating multiple signaling pathways, but further studies are necessary to elucidate its role under different conditions.

AKAP12 Role in Cardiovascular System

In contrast to the extensive studies involving AKAP12's role in cancer research, there is little knowledge of its role in the cardiovascular system. AKAP12 is highly expressed in the heart103 and functions to scaffold its signalosome complex near β₂‐AR to influence resensitization and sequestration events.116 Usually, scaffold proteins physically and transiently tether their complexes. However, AKAP12/β₂‐AR interaction is unique as it was found to be strengthened upon stimulation of β₂‐AR and remains intact even during the internalization of these receptors.59 Considering that cardiac function is mainly influenced by β‐Ars, and that AKAP12 directly binds to β₂‐AR, it is logical to assume that AKAP12 plays a role in regulating cardiac function.

To date, few studies have investigated AKAP12's involvement in cardiac pathophysiology, with the majority of studies indicating that AKAP12 downregulation improves cardiac function. For example, downregulation of AKAP12 reduced ventricular remodeling after chronic myocardial infarction (MI) surgery in transgenic mice overexpressing thioredoxin‐1. This is believed to be associated with AKAP12's regulation of redox‐sensitive transcription factor hypoxia‐inducible factor‐α.125 Hypoxia‐inducible factor‐α is a critical expression regulator of both vascular endothelial growth factor and endothelial nitric oxide synthase, where AKAP12 destabilizes hypoxia‐inducible factor‐α by enhancing its proteosomal degredation.126 Thus, the thioredoxin‐1 cardioprotective effects of overexpression are partially attributed to reduced oxidative stress–mediated stabilization of hypoxia‐inducible factor‐α, which thereby increases vascular endothelial growth factor and endothelial nitric oxide levels.126

Knowing that cardiac hypertrophy and fibrosis are strongly associated with increased mortality in diabetic patients, the role of vascular endothelial growth factor, endothelial nitric oxide, and heat shock proteins in relation to AKAP12 were studied in the diabetic milieu of post‐myocardial infarction (MI) surgery. Surprisingly, the study confirmed that heat shock protein A12B attenuated AKAP12 action, mediated cardioprotection through upregulating (thioredoxin‐1), and suggested AKAP12 involvement in the downregulation of thioredoxin‐interacting protein. In addition, on the basis of previous reports of heat shock protein A12B binding directly to a specific domain within AKAP12 (829–860 amino acid residues) located between the N and C terminus,101 cardioprotection may be induced through genetic modifications causing AKAP12 downregulation. Taking into account that AKAP12 downregulation affected the cycling of the β₂‐AR, this observation likely occurs through reduced recruitment of proteins involved in receptor desensitization (G protein–coupled receptor kinase 2 and β‐arrestin).28

In efforts to better understand the mechanism of AKAP12 in β₂‐AR signaling pathways, AKAP12‐t/t mice were examined after acute infusion with the nonspecific β‐AR agonist isoproterenol.58, 59 In contrast to a study fully ablated AKAP12,60 AKAP12‐t/t only lack the critical region required for β₂‐AR, PKA, or PKC binding.58, 59 This reduces the probability of influence of the upregulated compensatory mechanisms, as in full‐knockout AKAP12 gene models. Acute β‐AR stimulation of AKAP12‐t/t delineated enhanced contractility and basal activity. The proposed mechanism of increased basal activity was because of increased cMyBPC Ser‐273 phosphorylation by heat shock protein 20, although increased cardiac contractility could be associated with altering PKA phosphorylating capacity as well as activity of PDE4D.58 Also, it was suggested that AKAP12‐t/t muscle exhibited increased myofilament responsiveness to Ca²⁺,59 supporting the concept of AKAP12 being more than just a scaffold protein.

An additional study speculated that AKAP12 ablation aggravated heart failure induced post–angiotensin II infusion by promoting oxidative stress, apoptosis, cardiac fibrosis, and inflammatory response.60 In that study, the authors suggested that the mechanism of increased cardiac fibrosis was mainly through increased levels of transforming growth factor‐β1 and collagen, as well as through activation of the SMAD2/3 pathway.60

In addition to these findings, it has been reported recently that AKAP12‐t/t mice exhibit a delayed high‐fat‐diet–induced hyperlipidemia and atherosclerosis.61 It is believed that such resistance to hyperlipidemia and atherosclerosis development is associated with reduced gene expression of sterol regulatory element‐binding protein 2. Further reducing gene expression of liver 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase and low‐density lipoprotein receptor.61 Consistent with these findings, Choi et al reported that overexpression of AKAP12 is involved in sterol regulatory element‐binding protein 2 activation in a SCAP‐dependent manner,59, 127 probably through enhancing [3H]‐cholesterol efflux to extracellular acceptors. These findings further argue in favor of the AKAP12‐t/t cardioprotective phenotype from a pathophysiologic risk perspective (heart failure development as a result of hyperlipidemia and atherosclerosis).

Finally, it is noteworthy that none of these studies addresses the expression levels of other AKAPs. This is important because several AKAPs are present in the heart. Thus, observed alterations that were accredited to AKAP12 may have been influenced by other AKAPs, especially AKAP5, as AKAP5 also binds to β₂‐AR and is capable of switching the coupling of β₂‐AR from the Gαs to the Gαi pathway.128 In addition, AKAP5 can form not only homodimers but also a higher order supermolecular homo‐oligomeric complex, thus influencing the stability of protein–protein interactions and signaling loops affecting the AKAP12 signalosome.9 Therefore, these aspects stress the importance of taking into consideration the AKAP5 expression levels in future studies. Together, on the basis of the aforementioned data, AKAP12's role in the modulating cardiac function seems clearer. Thus, ongoing research of AKAP12's overexpression and how this overexpression impacts the cardiovascular system may resolve some of these issues.

In conclusion, AKAPs mediate the kinase signaling pathways needed for optimal cellular responses and alterations in their levels; either upregulation or downregulation explains the development or mitigation of different pathologic states. AKAP12 cellular signaling roles are established clearly in some systems (Figure 4).99, 102, 129 However, the implication of this finding in cardiovascular diseases is still speculative, especially in the area of heart failure progression. Further understanding of AKAP12 binding domains and signaling pathways may be the initial step in developing specific peptide‐targeting drugs.

Figure 4.

Proposed AKAP12 signaling downstream of β2‐AR. AKAP12 scaffolds multiple kinases, in addition to polybasic domains, which control its interactions and signaling. Under the unstimulated β₂‐AR state, AKAP12 is found mainly within the cytoplasm. However, when β2‐AR is stimulated, the polybasic domains found within the AKAP12 signalosome targets the complex to the cell membrane. This colocalization of the signalosome facilitates a stable binding of β₂‐AR and AKAP12. Activation of PKA is associated with increased levels of cAMP in its proximity. β₂‐AR stimulation induces local increases of cAMP. AKAP12 is important for PKA activation through scaffolding and collocating PKA near areas of high cAMP levels. The active form of PKA phosphorylates multiple downstream molecules, such as CREB (cellular transcription factor), SRC (tyrosine kinase), and GRK (G protein kinases), all playing a role in downstream signaling of β₂‐AR. Importantly, PKA phosphorylates AKAP12, which stabilizes the interaction between AKAP12 and the β₂‐AR, even during post‐internalization of the receptor.102 On the other hand, phosphorylation of AKAP12 by PKC decreases AKAP12–PKC scaffolding (1),99 inhibits the PKC‐Raf/MEK/ERK pathway (2),129 and induces translocation of the AKAP12/PKA/β2‐AR complex to the perinuclear space (3).99 AKAP indicates A‐kinase anchoring protein; β2‐AR, β₂‐adrenergic receptor; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CREB, cellular transcription factor; EPAC, exchange protein directly activated by cAMP; ERK, extracellular signal–regulated kinase; GRK, G protein kinase; MEK, mitogen‐activated protein kinase; PDE4, phosphodiesterase‐4; PKA, protein kinase A; PKC, protein kinase C; PM, plasma membrane; Raf, rapidly accelerated fibrosarcoma; and SRC, tyrosine kinase.

Sources of Funding

This study was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (award number R15 HL141963 to B.K.M.); the American Heart Association (award number 18AIREA 33960175 to B.K.M.); and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (to B.K.M.). The funders had no role in the preparation or decision to publish this work.

Disclosures

None.

(J Am Heart Assoc. 2020;9:e016615 DOI: 10.1161/JAHA.120.016615.)