The mAKAPβ scaffold regulates cardiac myocyte hypertrophy via recruitment of activated calcineurin

Abstract

mAKAPβ is the scaffold for a multimolecular signaling complex in cardiac myocytes that is required for the induction of neonatal myocyte hypertrophy. We now show that the pro-hypertrophic phosphatase calcineurin binds directly to a single site on mAKAPβ that does not conform to any of the previously reported consensus binding sites. Calcineurin–mAKAPβ complex formation is increased in the presence of Ca²⁺/calmodulin and in norepinephrine-stimulated primary cardiac myocytes. This binding is of functional significance because myocytes exhibit diminished norepinephrine-stimulated hypertrophy when expressing a mAKAPβ mutant incapable of binding calcineurin. In addition to calcineurin, the transcription factor NFATc3 also associates with the mAKAPβ scaffold in myocytes. Calcineurin bound to mAKAPβ can dephosphorylate NFATc3 in myocytes, and expression of mAKAPβ is required for NFAT transcriptional activity. Taken together, our results reveal the importance of regulated calcineurin binding to mAKAPβ for the induction of cardiac myocyte hypertrophy. Furthermore, these data illustrate how scaffold proteins organizing localized signaling complexes provide the molecular architecture for signal transduction networks regulating key cellular processes.

Introduction

Cardiac myocyte hypertrophy is the major intrinsic mechanism by which the heart may counterbalance chronically elevated demands for pumping power. Myocyte hypertrophy is controlled by a network of intracellular signaling pathways that are activated by G-protein coupled, growth factor and cytokine receptors and by mechanical and oxidative stress [1]. These signals are transduced by MAPK, cyclic nucleotide, Ca²⁺ and phosphoinositide-dependent pathways. Although much progress has been made over the last 20 years to define this network, it is still unclear how the various constituent pathways act in concert to regulate the overall cellular phenotype [2]. Moreover, while individual signaling pathways may regulate specific cellular functions, the molecules that comprise these signaling pathways often serve multiple functions in the same cells. Therefore, an important question in the field of signal transduction has been how pleiotropic signaling molecules such as protein kinases and phosphatases can specifically regulate individual downstream effectors in response to different upstream stimuli. One mechanism by which specificity in signal transduction is conferred is the formation of multimolecular signaling complexes by scaffold proteins of different combinations of common signaling enzymes [3].

While signaling enzymes may be broadly distributed within the cell, scaffold proteins, such as A-kinase anchoring proteins (AKAPs), recruit small pools of these enzymes to discrete multimolecular complexes that are sequestered in distinct intracellular compartments and that serve different cellular functions [4]. mAKAP (muscle AKAP) was initially identified in a screen for protein kinase A (PKA) binding proteins. mAKAPα and mAKAPβ are the two known isoforms encoded by the single mAKAP (AKAP6) gene and are expressed in neurons and striated myocytes, respectively [5]. As a consequence of alternative mRNA splicing, mAKAPβ is identical to residues 245–2314 (the C-terminus) of mAKAPα. In adult and neonatal cardiac myocytes, mAKAPβ is primarily localized to the outer nuclear membrane through its association with nesprin-1α [6], [7]. In addition to PKA, proteins that have been shown to associate with the mAKAPβ scaffold in myocytes include adenylyl cyclase type 5 [8], the cAMP-specific phosphodiesterase PDE4D3 [9], the cAMP-activated guanine nucleotide exchange factor Epac1 [10], ERK5 and MEK5 mitogen-activated protein kinases (MAPK) [10], the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin Aβ (CaN, PP2B) [11], protein phosphatase 2A [12], hypoxia-inducible factor 1α (HIF1α) and ubiquitin E3-ligases involved in HIF1α regulation [13], myopodin [14], the ryanodine receptor Ca²⁺-release channel (RyR2) [12], [15] and the sodium/calcium exchanger NCX1 [16]. Due to the association of these various enzymes and ion channels with mAKAPβ in the cardiac myocyte, we have proposed that mAKAPβ complexes are important for the regulation of pathologic myocyte remodeling in response to upstream cAMP, calcium, and MAPK signals and hypoxic stress [13], [17]. In support of this hypothesis, mAKAPβ expression in myocytes is required for the full induction of neonatal myocyte hypertrophy in vitro by adrenergic and cytokine agonists [10], [11].

CaN is a pleiotropic Ca²⁺/calmodulin-dependent serine/threonine phosphatase composed of a catalytic A-subunit and a regulatory B-subunit [18]. There are three mammalian A-subunits, of which Aα and Aβ are expressed ubiquitously and Aγ is restricted to testes. Aα and Aβ have been studied by genetic deletion and are not functionally redundant. For example, only the CaNAβ isoform is important for the induction of pathologic cardiac hypertrophy and the survival of myocytes after ischemia [19], [20]. Important calcineurin substrates in vivo include four of the five members of the nuclear factor of activated T-cell transcription factor family (NFATc 1–4). In addition to forming heterodimers with other transcription factors, NFATc can bind directly to CaN through conserved PxIxIT and LxVP motifs [21]. CaN binding facilitates dephosphorylation of the N-terminal NFATc regulatory domain, inducing NFATc nuclear translocation from the cytoplasm. Accordingly, NFATc isoforms serve important roles in cardiac development and myocyte hypertrophy [22].

Previously, we showed that CaNAβ is associated with mAKAPβ in cardiac myocytes [11]. However, it remains unclear how scaffolding by this relatively low abundant protein contributes to CaN signaling. In this study, we characterize the direct binding of CaNAβ to mAKAPβ. Moreover, we provide evidence that recruitment of CaNAβ and NFATc3 to mAKAPβ complexes is important for the transduction of hypertrophic signaling.