A perinuclear calcium compartment regulates cardiac myocyte hypertrophy.

The pleiotropic Ca/calmodulin-dependent phosphatase calcineurin is a key regulator of pathological cardiac myocyte hypertrophy. The selective activation of hypertrophic calcineurin signaling under stress conditions has been attributed to compartmentation of Ca signaling in cardiac myocytes. Here, perinuclear signalosomes organized by the scaffold protein muscle A-Kinase Anchoring Protein β (mAKAPβ/AKAP6β) are shown to orchestrate local Ca transients, inducing calcineurin-dependent NFATc nuclear localization and myocyte hypertrophy in response to β-adrenergic receptor activation.

Subcellular Propagation of Cardiomyocyte β-Adrenergic Activation of Calcium Uptake Involves Internal β-Receptors and AKAP7.

β-adrenergic receptor (β-AR) signaling in cardiac myocytes is central to cardiac function, but spatiotemporal activation within myocytes is unresolved. In rabbit ventricular myocytes, β-AR agonists or high extracellular [Ca] were applied locally at one end, to measure β-AR signal propagation as Ca-transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca uptake. High local [Ca], increased CaT amplitude under the pipette faster than did ISO, but was also more spatially restricted.

cAMP at Perinuclear mAKAPα Signalosomes Is Regulated by Local Ca Signaling in Primary Hippocampal Neurons.

The second messenger cyclic adenosine monophosphate (cAMP) is important for the regulation of neuronal structure and function, including neurite extension. A perinuclear cAMP compartment organized by the scaffold protein muscle A-kinase anchoring protein α (mAKAPα/AKAP6α) is sufficient and necessary for axon growth by rat hippocampal neurons Here, we report that cAMP at mAKAPα signalosomes is regulated by local Ca signaling that mediates activity-dependent cAMP elevation within that compartment. Simultaneous Forster resonance energy transfer (FRET) imaging using the protein kinase A (PKA) activity reporter AKAR4 and intensiometric imaging using the RCaMP1h fluorescent Ca sensor revealed that membrane depolarization by KCl selectively induced activation of perinuclear PKA activity.

Muscle A-kinase-anchoring protein-β-bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D.

Myocyte enhancer factor 2 (MEF2) transcription factors are key regulators of the development and adult phenotype of diverse tissues, including skeletal and cardiac muscles. Controlled by multiple post-translational modifications, MEF2D is an effector for the Ca/calmodulin-dependent protein phosphatase calcineurin (CaN, PP2B, and PPP3). CaN-catalyzed dephosphorylation promotes the desumoylation and acetylation of MEF2D, increasing its transcriptional activity.

Calcineurin-AKAP interactions: therapeutic targeting of a pleiotropic enzyme with a little help from its friends.

The ubiquitous Ca /calmodulin-dependent phosphatase calcineurin is a key regulator of pathological cardiac hypertrophy whose therapeutic targeting in heart disease has been elusive due to its role in other essential biological processes. Calcineurin is targeted to diverse intracellular compartments by association with scaffold proteins, including by multivalent A-kinase anchoring proteins (AKAPs) that bind protein kinase A and other important signalling enzymes determining cardiac myocyte function and phenotype. Calcineurin anchoring by AKAPs confers specificity to calcineurin function in the cardiac myocyte.

Bidirectional regulation of HDAC5 by mAKAPβ signalosomes in cardiac myocytes.

Class IIa histone deacetylases (HDACs) are transcriptional repressors whose nuclear export in the cardiac myocyte is associated with the induction of pathological gene expression and cardiac remodeling. Class IIa HDACs are regulated by multiple, functionally opposing post-translational modifications, including phosphorylation by protein kinase D (PKD) that promotes nuclear export and phosphorylation by protein kinase A (PKA) that promotes nuclear import. We have previously shown that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) orchestrates signaling in the cardiac myocyte required for pathological cardiac remodeling, including serving as a scaffold for both PKD and PKA.

Cushing's syndrome mutant PKA exhibits altered substrate specificity.

The PKA hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA and PKA substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions.

Analysis of AKAP7γ Dimerization.

A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that contribute to spatiotemporal regulation of PKA-mediated phosphorylation events. In particular, AKAP7 is a family of alternatively spliced proteins that participates in cardiac calcium dynamics. Here, we demonstrate via pull-down from transfected cells and by direct protein-protein association that AKAP7γ self-associates.

Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation.

Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca(2+) cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca(2+) re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3].

Scaffold state switching amplifies, accelerates, and insulates protein kinase C signaling.

Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes.

Regulation of MEF2 transcriptional activity by calcineurin/mAKAP complexes.

The calcium/calmodulin-dependent protein phosphatase calcineurin is required for the induction of transcriptional events that initiate and promote myogenic differentiation. An important effector for calcineurin in striated muscle is the transcription factor myocyte enhancer factor 2 (MEF2). The targeting of the enzyme and substrate to specific intracellular compartments by scaffold proteins often confers specificity in phosphatase activity.

Spatiotemporal regulation of PKC via interactions with AKAP7 isoforms.

The regulation of kinases by scaffolding proteins greatly contributes to the fidelity of signal transduction. In the present study, we explored an interaction between the ubiquitous enzyme PKC (protein kinase C) and the scaffolding protein AKAP7 (A-kinase-anchoring protein 7). Using protein biochemistry and surface plasmon resonance approaches, we demonstrate that both AKAP7γ and AKAP7α are capable of high-affinity interactions with multiple isoenzymes of PKC.

Myocyte enhancer factor 2 (MEF2) tethering to muscle selective A-kinase anchoring protein (mAKAP) is necessary for myogenic differentiation.

Differentiation of skeletal myoblast cells to functional myotubes involves highly regulated transcriptional dynamics. The myocyte enhancer factor 2 (MEF2) transcription factors are critical to this process, synergizing with the master regulator MyoD to promote muscle specific gene transcription. MEF2 is extensively regulated by myogenic stimuli, both transcriptionally and post-translationally, but to date there has been little progress in understanding how signals upstream of MEF2 are coordinated to produce a coherent response.

A-kinase anchoring proteins: scaffolding proteins in the heart.

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network.

AKAP phosphatase complexes in the heart.

Directed protein phosphorylation is indisputably critical for a multitude of cellular processes. A growing body of research demonstrates A kinase anchoring proteins (AKAPs) to mediate a significant number of phosphorylation events in the heart. By acting as molecular tethers for the regulatory subunit of protein kinase A, AKAPs focus kinase activity onto specific substrate.

Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein.

The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit.

The large isoforms of A-kinase anchoring protein 18 mediate the phosphorylation of inhibitor-1 by protein kinase A and the inhibition of protein phosphatase 1 activity.

Inhibitor-1 (I-1) is phosphorylated on threonine residue 35 (Thr35) by the cAMP-dependent protein kinase (PKA), inducing the potent inhibition of the serine-threonine-specific protein phosphatase 1 (PP1). We now report that the formation of a signaling complex containing PKA and I-1 by the A-kinase anchoring protein 18 (AKAP18) facilitates this regulation in cells. AKAP18 directly bound I-1, and AKAP18/I-1 complexes were isolated from both rat heart extract and transfected heterologous cells.

cAMP-stimulated protein phosphatase 2A activity associated with muscle A kinase-anchoring protein (mAKAP) signaling complexes inhibits the phosphorylation and activity of the cAMP-specific phosphodiesterase PDE4D3.

The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels.

The mAKAP signalosome and cardiac myocyte hypertrophy.

Cardiac hypertrophy is regulated by a large intracellular signal transduction network. Each of the many signaling pathways in this network contributes uniquely to the control of cell growth. In the last few years, it has become apparent that multimolecular signaling complexes or 'signalosomes' are important for mediating crosstalk between different signaling pathways.

Compartmentation of cyclic nucleotide signaling in the heart: the role of A-kinase anchoring proteins.

The activation of the cyclic nucleotide protein kinase A (PKA) and PKG by their respective second messengers is responsible for the modulation of many cellular functions in the heart including cardiac hypertrophy, strength of contraction, and ion flux. However, several studies have revealed that a general increase in cyclic nucleotide concentration in the cell is not sufficient for the specific regulation of target proteins. These studies found that PKA and PKG must be colocalized with their targets to ensure spatial-temporal control of substrate phosphorylation.

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways.

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation.

Spatial restriction of PDK1 activation cascades by anchoring to mAKAPalpha.

The muscle A-kinase anchoring protein (mAKAP) tethers cAMP-dependent enzymes to perinuclear membranes of cardiomyocytes. We now demonstrate that two alternatively spliced forms of mAKAP are expressed: mAKAPalpha and mAKAPbeta. The longer form, mAKAPalpha, is preferentially expressed in the brain.

The mAKAP complex participates in the induction of cardiac myocyte hypertrophy by adrenergic receptor signaling.

Maladaptive cardiac hypertrophy can progress to congestive heart failure, a leading cause of morbidity and mortality in the United States. A better understanding of the intracellular signal transduction network that controls myocyte cell growth may suggest new therapeutic directions. mAKAP is a scaffold protein that has recently been shown to coordinate signal transduction enzymes important for cytokine-induced cardiac hypertrophy.

The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways.

Cyclic adenosine 3', 5'-monophosphate (cAMP) is a ubiquitous mediator of intracellular signalling events. It acts principally through stimulation of cAMP-dependent protein kinases (PKAs) but also activates certain ion channels and guanine nucleotide exchange factors (Epacs). Metabolism of cAMP is catalysed by phosphodiesterases (PDEs).