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Cardiac fibrosis is a major cause of dysfunctions and arrhythmias in failing hearts. At the cellular level fibrosis is mediated by cardiac myofibroblasts, which display an increased migratory capacity and secrete large amounts of extracellular matrix. These properties allow myofibroblasts to invade, remodel and stiffen the myocardium and eventually alter cardiac function. While the enhanced ability of cardiac myofibroblasts to migrate has been proposed to contribute to the initiation of the fibrotic process, the molecular mechanisms controlling their motile function have been poorly defined. In this context, our current findings indicate that A-kinase anchoring protein 2 (AKAP2) associates with actin at the leading edge of migrating cardiac myofibroblasts. Proteomic analysis of the AKAP2 interactome revealed that this anchoring protein assembles a signaling complex composed of the extracellular regulated kinase 1 (ERK1) and its upstream activator Grb2 that mediates the activation of ERK in cardiac myofibroblasts. Silencing AKAP2 expression results in a significant reduction in the phosphorylation of ERK1 and its downstream effector WAVE2, a protein involved in actin polymerization, and impairs the ability of cardiac myofibroblasts to migrate. Importantly, disruption of the interaction between AKAP2 and F-actin using cell-permeant competitor peptides, inhibits the activation of the ERK-WAVE2 signaling axis, resulting in a reduction of the translocation of Arp2 to the leading-edge membrane and in inhibition of cardiac myofibroblast migration. Collectively, these findings suggest that AKAP2 functions as an F-actin bound molecular scaffold mediating the activation of an ERK1-dependent promigratory transduction pathway in cardiac myofibroblasts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Prostate cancer (PC) is the second leading cause of cancer-related death in men. The growth of primary prostate cancer cells relies on circulating androgens and thus the standard therapy for the treatment of localized and advanced PC is the androgen deprivation therapy. Prostatic neuroendocrine carcinoma (PNEC) is an aggressive and highly metastatic subtype of prostate cancer, which displays poor prognosis and high lethality. Most of PNECs develop from prostate adenocarcinoma in response to androgen deprivation therapy, however the mechanisms involved in this transition and in the elevated biological aggressiveness of PNECs are poorly defined. Our current findings indicate that AKAP2 expression is dramatically upregulated in PNECs as compared to non-cancerous prostate tissues. Using a PNEC cell model, we could show that AKAP2 is localized both intracellularly and at the cell periphery where it colocalizes with F-actin. AKAP2 and F-actin interact directly through a newly identified actin-binding domain located on AKAP2. RNAi-mediated silencing of AKAP2 promotes the phosphorylation and deactivation of cofilin, a protein involved in actin turnover. This effect correlates with a significant reduction in cell migration and invasion. Co-immunoprecipitation experiments and proximity ligation assays revealed that AKAP2 forms a complex with the catalytic subunit of protein phosphatase 1 (PP1) in PNECs. Importantly, AKAP2-mediated anchoring of PP1 to the actin cytoskeleton regulates cofilin dephosphorylation and activation, which, in turn, enhances F-actin dynamics and favors migration and invasion. In conclusion, this study identified AKAP2 as an anchoring protein overexpressed in PNECs that controls cancer cell invasive properties by regulating cofilin phosphorylation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Extra-pituitary ACTH secretion is associated with a variety of neoplastic conditions and may cause the so-called ectopic ACTH-dependent Cushing syndrome (CS). The clarification of the mechanisms of extra-pituitary ACTH expression would provide potential therapeutic targets for this complex and severe disease. In the adenohypophysis, the transcription factor TPIT, co-operating with other molecules, induces POMC expression and ACTH production. However, no data are currently available on the presence and role of TPIT expression in extra-pituitary ACTH-producing neoplasms. This study was designed to explore TPIT expression in a series of pulmonary and pancreatic ACTH-producing tumors, either CS-associated or not. Forty-one extra-pituitary ACTH-producing neuroendocrine tumors (NETs) were included in the study, encompassing 32 NETs of the lung (LuNETs), 7 of the pancreas (PanNETs), and 2 pheochromocytomas. Of these, 9 LuNETs, all PanNETs, and the two pheochromocytomas were CS-associated. For comparison, 6 NETs of the pituitary gland (PitNETs; 3 ACTH-secreting and 3 ACTH-negative) and 35 ACTH-negative extra-pituitary NETs (15 Lu-NETs and 20 PanNETs) were analyzed. Immunohistochemistry with specific anti-TPIT antibodies and quantitative real-time PCR (qRT-PCR) were performed using standard protocols. TPIT expression was completely absent (protein and mRNA) in PanNETs, pheochromocytomas, and all ACTH-negative NETs. In contrast, it was expressed in 16/32 LuNETs, although with lower levels than in PitNETs. No definite relationship was found between immunohistochemistry TPIT expression and NET grade or the presence of Cushing syndrome. This study further highlights the clinical and biological heterogeneity of extra-pituitary ACTH secretion and suggests that the differences between ACTH-secreting PanNETs and LuNETs may mirror distinct molecular mechanisms underlying POMC expression. Our results point towards the recognition of a real corticotroph-like phenotype of ACTH-producing LuNETs, that is not a feature of ACTH-producing PanNETs., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nanoparticle-based drug delivery systems have the potential for increasing the efficiency of chemotherapeutics by enhancing the drug accumulation at specific target sites, thereby reducing adverse side effects and mitigating patient acquired resistance. In particular, photo-responsive nanomaterials have attracted much interest due to their ability to release molecular cargos on demand upon light irradiation. In some settings, they can also provide complementary information by optical imaging on the (sub)cellular scale. We herein present a system based on lithium niobate harmonic nanoparticles (LNO HNPs) for the decoupled multi-harmonic cell imaging and near-infrared light-triggered delivery of an erlotinib derivative ( ELA ) for the treatment of epidermal growth factor receptor (EGFR)-overexpressing carcinomas. The ELA cargo was covalently conjugated to the surface of silica-coated LNO HNPs through a coumarinyl photo-cleavable linker, achieving a surface loading of the active molecule of 27 nmol/mg NPs. The resulting nanoconjugates ( LNO-CM-ELA NPs) were successfully imaged upon pulsed laser excitation at 1250 nm in EGFR-overexpressing human prostate cancer cells DU145 by detecting the second harmonic emission at 625 nm, in the tissue transparency window. Tuning the laser at 790 nm resulted in the uncaging of the ELA cargo as a result of the second harmonic emission of the inorganic HNP core at 395 nm. This protocol induced a significant growth inhibition in DU145 cells, which was only observed upon specific irradiation at 790 nm, highlighting the promising capabilities of LNO-CM-ELA NPs for theranostic applications., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Aims: Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis., Methods and Results: EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-β)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts., Conclusion: Ang II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Myocardial infarction (MI) is a leading cause of maladaptive cardiac remodeling and heart failure. In the damaged heart, loss of function is mainly due to cardiomyocyte death and remodeling of the cardiac tissue. The current study shows that A-kinase anchoring protein 2 (AKAP2) orchestrates cellular processes favoring cardioprotection in infarcted hearts. Induction of AKAP2 knockout (KO) in cardiomyocytes of adult mice increases infarct size and exacerbates cardiac dysfunction after MI, as visualized by increased left ventricular dilation and reduced fractional shortening and ejection fraction. In cardiomyocytes, AKAP2 forms a signaling complex with PKA and the steroid receptor co-activator 3 (Src3). Upon activation of cAMP signaling, the AKAP2/PKA/Src3 complex favors PKA-mediated phosphorylation and activation of estrogen receptor α (ERα). This results in the upregulation of ER-dependent genes involved in protection against apoptosis and angiogenesis, including Bcl2 and the vascular endothelial growth factor a (VEGFa). In line with these findings, cardiomyocyte-specific AKAP2 KO reduces Bcl2 and VEGFa expression, increases myocardial apoptosis and impairs the formation of new blood vessels in infarcted hearts. Collectively, our findings suggest that AKAP2 organizes a transcriptional complex that mediates pro-angiogenic and anti-apoptotic responses that protect infarcted hearts.

Whereas most of the reports on the nonlinear properties of micro- and nanostructures address the generation of distinct signals, such as second or third harmonic, here we demonstrate that the novel generation of dual output lasers recently developed for microscopy can readily increase the accessible parameter space and enable the simultaneous excitation and detection of multiple emission orders such as several harmonics and signals stemming from various sum and difference frequency mixing processes. This rich response, which in our case features 10 distinct emissions and encompasses the whole spectral range from the deep ultraviolet to the short-wave infrared region, is demonstrated using various nonlinear oxide nanomaterials while being characterized and simulated temporally and spectrally. Notably, we show that the response is conserved when the particles are embedded in biological media opening the way to novel biolabeling and phototriggering strategies.

Myocardial stress and injury invariably promote remodeling of the cardiac tissue, which is associated with cardiomyocyte death and development of fibrosis. The fibrotic process is initially triggered by the differentiation of resident cardiac fibroblasts into myofibroblasts. These activated fibroblasts display increased proliferative capacity and secrete large amounts of extracellular matrix. Uncontrolled myofibroblast activation can thus promote heart stiffness, cardiac dysfunction, arrhythmias, and progression to heart failure. Despite the well-established role of myofibroblasts in mediating cardiac disease, our current knowledge on how signaling pathways promoting fibrosis are regulated and coordinated in this cell type is largely incomplete. In this respect, cyclic adenosine monophosphate (cAMP) signaling acts as a major modulator of fibrotic responses activated in fibroblasts of injured or stressed hearts. In particular, accumulating evidence now suggests that upstream cAMP modulators including G protein-coupled receptors, adenylyl cyclases (ACs), and phosphodiesterases (PDEs); downstream cAMP effectors such as protein kinase A (PKA) and the guanine nucleotide exchange factor Epac; and cAMP signaling organizers such as A-kinase anchoring proteins (AKAPs) modulate a variety of fundamental cellular processes involved in myocardial fibrosis including myofibroblast differentiation, proliferation, collagen secretion, and invasiveness. The current review will discuss recent advances highlighting the role of cAMP and AKAP-mediated signaling in regulating pathophysiological responses controlling cardiac fibrosis.

Cardiac stress initiates a pathological remodeling process that is associated with cardiomyocyte loss and fibrosis that ultimately leads to heart failure. In the injured heart, a pathologically elevated synthesis of reactive oxygen species (ROS) is the main driver of oxidative stress and consequent cardiomyocyte dysfunction and death. In this context, the cAMP-dependent protein kinase (PKA) plays a central role in regulating signaling pathways that protect the heart against ROS-induced cardiac damage. In cardiac cells, spatiotemporal regulation of PKA activity is controlled by A-kinase anchoring proteins (AKAPs). This family of scaffolding proteins tether PKA and other transduction enzymes at subcellular microdomains where they can co-ordinate cellular responses regulating oxidative stress. In this review, we will discuss recent literature illustrating the role of PKA and AKAPs in modulating the detrimental impact of ROS production on cardiac function., (© 2019 The Author(s).)

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Heart failure is a lethal disease that can develop after myocardial infarction, hypertension, or anticancer therapy. In the damaged heart, loss of function is mainly due to cardiomyocyte death and associated cardiac remodeling and fibrosis. In this context, A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that facilitate the spatiotemporal activation of the cyclic adenosine monophosphate (AMP)-dependent protein kinase (PKA) and other transduction enzymes involved in cardiac remodeling. AKAP-Lbc, a cardiac enriched anchoring protein, has been shown to act as a key coordinator of the activity of signaling pathways involved in cardiac protection and remodeling. This review will summarize and discuss recent advances highlighting the role of the AKAP-Lbc signalosome in orchestrating adaptive responses in the stressed heart., Competing Interests: The authors declare no conflict of interest.

Doxorubicin (DOX) is a chemotherapic agent that is widely used to treat hematological and solid tumors. Despite its efficacy, DOX displays significant cardiac toxicity associated with cardiomyocytes death and heart failure. Cardiac toxicity is mainly associated with the ability of DOX to alter mitochondrial function. The current lack of treatments to efficiently prevent DOX cardiotoxicity underscores the need of new therapeutic approaches. Our current findings show that stimulation of cardiomyocytes with the α1-adrenergic receptor (AR) agonist phenylephrine (PE) significantly inhibits the apoptotic effect of DOX. Importantly, our results indicate that AKAP-Lbc is critical for transducing protective signals downstream of α1-ARs. In particular, we could show that suppression of AKAP-Lbc expression by infecting primary cultures of ventricular myocytes with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly impairs the ability of PE to reduce DOX-induced apoptosis. AKAP-Lbc-mediated cardiomyocyte protection requires the activation of anchored protein kinase D1 (PKD1)-dependent prosurvival pathways that promote the expression of the anti-apoptotic protein Bcl2 and inhibit the translocation of the pro-apoptotic protein Bax to mitochondria. In conclusion, AKAP-Lbc emerges as a coordinator of signals that protect cardiomyocytes against the toxic effects of DOX., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cancer development is a multifactorial process resulting from the aberrant activation of multiple signaling pathways. It has become increasingly clear that the coordination of the signaling events leading to cancer formation and progression is under the control of macromolecular transduction complexes organized by scaffolding proteins. A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins involved in the spatio-temporal activation of pathways controlling cancer cell proliferation, cell survival, and invasion. Mutations or altered expression of AKAP coding genes results in unregulated signaling associated with oncogenesis, cancer maintenance, and metastasis. This review will focus on recent advances illustrating the role of AKAPs in cancer pathophysiology as well as on their potential as therapeutic targets., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Uncontrolled activation of Rho signaling by RhoGEFs, in particular AKAP13 (Lbc) and its close homologs, is implicated in a number of human tumors with poor prognosis and resistance to therapy. Structure predictions and alanine scanning mutagenesis of Lbc identified a circumscribed hot region for RhoA recognition and activation. Virtual screening targeting that region led to the discovery of an inhibitor of Lbc-RhoA interaction inside cells. By interacting with the DH domain, the compound inhibits the catalytic activity of Lbc, halts cellular responses to activation of oncogenic Lbc pathways, and reverses a number of prostate cancer cell phenotypes such as proliferation, migration, and invasiveness. This study provides insights into the structural determinants of Lbc-RhoA recognition. This is a successful example of structure-based discovery of a small protein-protein interaction inhibitor able to halt oncogenic Rho signaling in cancer cells with therapeutic implications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Heart and blood vessels ensure adequate perfusion of peripheral organs with blood and nutrients. Alteration of the homeostatic functions of the cardiovascular system can cause hypertension, atherosclerosis, and coronary artery disease leading to heart injury and failure. A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that are crucially involved in modulating the function of the cardiovascular system both under physiological and pathological conditions. AKAPs assemble multifunctional signaling complexes that ensure correct targeting of the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to precise subcellular compartments. This allows local regulation of specific effector proteins that control the function of vascular and cardiac cells. This review will focus on recent advances illustrating the role of AKAPs in cardiovascular pathophysiology. The accent will be mainly placed on the molecular events linked to the control of vascular integrity and blood pressure as well as on the cardiac remodeling process associated with heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aims: The adult mammalian heart has poor regenerative capacity. In contrast, the zebrafish heart retains a robust capacity for regeneration into adulthood. These distinct responses are consequences of a differential utilization of evolutionary-conserved gene regulatory networks in the damaged heart. To systematically identify miRNA-dependent networks controlling cardiac repair following injury, we performed comparative gene and miRNA profiling of the cardiac transcriptome in adult mice and zebrafish., Methods and Results: Using an integrated approach, we show that 45 miRNA-dependent networks, involved in critical biological pathways, are differentially modulated in the injured zebrafish vs. mouse hearts. We study, more particularly, the miR-26a-dependent response. Therefore, miR-26a is down-regulated in the fish heart after injury, whereas its expression remains constant in the mouse heart. Targets of miR-26a involve activators of the cell cycle and Ezh2, a component of the polycomb repressive complex 2 (PRC2). Importantly, PRC2 exerts repressive functions on negative regulators of the cell cycle. In cultured neonatal cardiomyocytes, inhibition of miR-26a stimulates, therefore, cardiomyocyte proliferation. Accordingly, miR-26a knockdown prolongs the proliferative window of cardiomyocytes in the post-natal mouse heart., Conclusions: This novel strategy identifies a series of miRNAs and associated pathways, in particular miR-26a, which represent attractive therapeutic targets for inducing repair in the injured heart., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or pathological stimuli resulting in increased cardiomyocyte size and myocardial mass. Whereas cardiac hypertrophy can be an adaptive mechanism to stressful conditions of the heart, prolonged hypertrophy can lead to heart failure which represents the primary cause of human morbidity and mortality. Among G protein-coupled receptors, the α1-adrenergic receptors (α1-ARs) play an important role in the development of cardiac hypertrophy as demonstrated by numerous studies in the past decades, both in primary cardiomyocyte cultures and genetically modified mice. The results of these studies have provided evidence of a large variety of α1-AR-induced signaling events contributing to the defining molecular and cellular features of cardiac hypertrophy. Recently, novel signaling mechanisms have been identified and new hypotheses have emerged concerning the functional role of the α1-adrenergic receptors in the heart. This review will summarize the main signaling pathways activated by the α1-AR in the heart and their functional implications in cardiac hypertrophy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In response to stress or injury the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy and fibrosis. Transformation of cardiac fibroblasts to myofibroblasts is a crucial event initiating the fibrotic process. Cardiac myofibroblasts invade the myocardium and secrete excess amounts of extracellular matrix proteins, which cause myocardial stiffening, cardiac dysfunctions and progression to heart failure. While several studies indicate that the small GTPase RhoA can promote profibrotic responses, the exchange factors that modulate its activity in cardiac fibroblasts are yet to be identified. In the present study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor (GEF) activity, is critical for activating RhoA and transducing profibrotic signals downstream of type I angiotensin II receptors (AT1Rs) in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly reduces the ability of angiotensin II to promote RhoA activation, differentiation of cardiac fibroblasts to myofibroblasts, collagen deposition as well as myofibroblast migration. Interestingly, AT1Rs promote AKAP-Lbc activation via a pathway that requires the α subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as a key Rho-guanine nucleotide exchange factor modulating profibrotic responses in cardiac fibroblasts., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In response to stress, the heart undergoes a remodeling process associated with cardiac hypertrophy that eventually leads to heart failure. A-kinase anchoring proteins (AKAPs) have been shown to coordinate numerous prohypertrophic signaling pathways in cultured cardiomyocytes. However, it remains to be established whether AKAP-based signaling complexes control cardiac hypertrophy and remodeling in vivo. In the current study, we show that AKAP-Lbc assembles a signaling complex composed of the kinases PKN, MLTK, MKK3, and p38α that mediates the activation of p38 in cardiomyocytes in response to stress signals. To address the role of this complex in cardiac remodeling, we generated transgenic mice displaying cardiomyocyte-specific overexpression of a molecular inhibitor of the interaction between AKAP-Lbc and the p38-activating module. Our results indicate that disruption of the AKAP-Lbc/p38 signaling complex inhibits compensatory cardiomyocyte hypertrophy in response to aortic banding-induced pressure overload and promotes early cardiac dysfunction associated with increased myocardial apoptosis, stress gene activation, and ventricular dilation. Attenuation of hypertrophy results from a reduced protein synthesis capacity, as indicated by decreased phosphorylation of 4E-binding protein 1 and ribosomal protein S6. These results indicate that AKAP-Lbc enhances p38-mediated hypertrophic signaling in the heart in response to abrupt increases in the afterload.

In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In response to stress, the heart undergoes a pathological remodeling process associated with hypertrophy and the reexpression of a fetal gene program that ultimately causes cardiac dysfunction and heart failure. In this study, we show that A-kinase-anchoring protein (AKAP)-Lbc and the inhibitor of NF-κB kinase subunit β (IKKβ) form a transduction complex in cardiomyocytes that controls the production of proinflammatory cytokines mediating cardiomyocyte hypertrophy. In particular, we can show that activation of IKKβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukin-6 (IL-6), which in turn enhances fetal gene expression and cardiomyocyte growth. These findings provide a new mechanistic hypothesis explaining how hypertrophic signals are coordinated and conveyed to interleukin-mediated transcriptional reprogramming events in cardiomyocytes.

Spatial regulation of tyrosine phosphorylation is important for many aspects of cell biology. However, phosphotyrosine accounts for less than 1% of all phosphorylated substrates, and it is typically a very transient event in vivo. These factors complicate the identification of key tyrosine kinase substrates, especially in the context of their extraordinary spatial organization. Here, we describe an approach to identify tyrosine kinase substrates based on their subcellular distribution from within cells. This method uses an unnatural amino acid-modified Src homology 2 (SH2) domain that is expressed within cells and can covalently trap phosphotyrosine proteins on exposure to light. This SH2 domain-based photoprobe was targeted to cellular structures, such as the actin cytoskeleton, mitochondria, and cellular membranes, to capture tyrosine kinase substrates unique to each cellular region. We demonstrate that RhoA, one of the proteins associated with actin, can be phosphorylated on two tyrosine residues within the switch regions, suggesting that phosphorylation of these residues might modulate RhoA signaling to the actin cytoskeleton. We conclude that expression of SH2 domains within cellular compartments that are capable of covalent phototrapping can reveal the spatial organization of tyrosine kinase substrates that are likely to be important for the regulation of subcellular structures.

The adrenergic receptors are among the best characterized G protein-coupled receptors (GPCRs) and knowledge on this receptor family has provided several important paradigms about GPCR function and regulation. One of the most recent paradigms initially supported by studies on adrenergic receptors is that both βarrestins and G proteincoupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. In this review we will briefly summarize the main features of βarrestin binding to the adrenergic receptor subtypes and we will review more in detail the main proteins found to selectively interact with distinct AR subtype. At the end, we will review the main findings on oligomerization of the AR subtypes.

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. Scaffolds that bind protein kinase A are called "A-kinase anchoring proteins" (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease.

The mitogen-activated protein kinases (MAPKs) pathways are highly organized signaling systems that transduce extracellular signals into a variety of intracellular responses. In this context, it is currently poorly understood how kinases constituting these signaling cascades are assembled and activated in response to receptor stimulation to generate specific cellular responses. Here, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critically involved in the activation of the p38α MAPK downstream of α(1b)-adrenergic receptors (α(1b)-ARs). Our results indicate that AKAP-Lbc can assemble a novel transduction complex containing the RhoA effector PKNα, MLTK, MKK3, and p38α, which integrates signals from α(1b)-ARs to promote RhoA-dependent activation of p38α. In particular, silencing of AKAP-Lbc expression or disrupting the formation of the AKAP-Lbc·p38α signaling complex specifically reduces α(1)-AR-mediated p38α activation without affecting receptor-mediated activation of other MAPK pathways. These findings provide a novel mechanistic hypothesis explaining how assembly of macromolecular complexes can specify MAPK signaling downstream of α(1)-ARs.

AKAP-Lbc is a member of the A-kinase anchoring protein (AKAP) family that has been recently associated with the development of pathologies, such as cardiac hypertrophy and cancer. We have previously demonstrated that, at the molecular level, AKAP-Lbc functions as a guanine nucleotide exchange factor (GEF) that promotes the specific activation of RhoA. In the present study, we identified the ubiquitin-like protein LC3 as a novel regulatory protein interacting with AKAP-Lbc. Mutagenesis studies revealed that LC3, through its NH(2)-terminal alpha-helical domain, interacts with two binding sites located within the NH(2)-terminal regulatory region of AKAP-Lbc. Interestingly, LC3 overexpression strongly reduced the ability of AKAP-Lbc to interact with RhoA, profoundly impairing the Rho-GEF activity of the anchoring protein and, as a consequence, its ability to promote cytoskeletal rearrangements associated with the formation of actin stress fibers. Moreover, AKAP-Lbc mutants that fail to interact with LC3 show a higher basal Rho-GEF activity as compared with the wild type protein and become refractory to the inhibitory effect of LC3. This suggests that LC3 binding maintains AKAP-Lbc in an inactive state that displays a reduced ability to promote downstream signaling. Collectively, these findings provide evidence for a previously uncharacterized role of LC3 in the regulation of Rho signaling and in the reorganization of the actin cytoskeleton.

Elevated catecholamines in the heart evoke transcriptional activation of the Myocyte Enhancer Factor (MEF) pathway to induce a cellular response known as pathological myocardial hypertrophy. We have discovered that the A-Kinase Anchoring Protein (AKAP)-Lbc is upregulated in hypertrophic cardiomyocytes. It coordinates activation and movement of signaling proteins that initiate MEF2-mediated transcriptional reprogramming events. Live-cell imaging, fluorescent kinase activity reporters, and RNA interference techniques show that AKAP-Lbc couples activation of protein kinase D (PKD) with the phosphorylation-dependent nuclear export of the class II histone deacetylase HDAC5. These studies uncover a role for AKAP-Lbc in which increased expression of the anchoring protein selectively amplifies a signaling pathway that drives cardiac myocytes toward a pathophysiological outcome.

In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth have been the subject of intensive investigation. It has been known for more than a decade that the small molecular weight GTPase RhoA is involved in the signaling pathways leading to cardiomyocyte hypertrophy. Although some of the hypertrophic pathways activated by RhoA have now been identified, the identity of the exchange factors that modulate its activity in cardiomyocytes is currently unknown. In this study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critical for activating RhoA and transducing hypertrophic signals downstream of alpha1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbc-specific short hairpin RNAs strongly reduces both alpha1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, alpha1-ARs promote AKAP-Lbc activation via a pathway that requires the alpha subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor (GEF) involved in the signaling pathways leading to cardiomyocytes hypertrophy.

A Kinase-anchoring proteins (AKAPs) are a family of functionally related proteins involved in the targeting of the PKA holoenzyme towards specific physiological substrates. We have recently identified a novel anchoring protein expressed in cardiomyocytes, called AKAP-Lbc, that functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) that activates the GTPase RhoA. Here, we discuss the most recent findings elucidating the molecular mechanisms and the transduction pathways involved in the regulation of the AKAP-Lbc signaling complex inside cells. We could show that AKAP-Lbc is regulated in a bi-directional manner by signals that activate or deactivate its Rho-GEF activity. Activation of AKAP-Lbc occurs in response to agonists that stimulate G proteins coupled receptors linked to the heterotrimeric G protein G12, whereas inactivation occurs through mechanisms that require phosphorylation of AKAP-Lbc by anchored PKA and subsequent recruitment of the regulatory protein 14-3-3. Interestingly, we could demonstrate that AKAP-Lbc can form homo-oligomers inside cells and that 14-3-3 can inhibit the Rho-GEF activity of AKAP-Lbc only when the anchoring protein adopts an oligomeric conformation. These findings reveal the molecular architecture of the AKAP-Lbc transduction complex and provide a mechanistic explanation of how upstream signaling pathways can be integrated within the AKAP-Lbc transduction complex to precisely modulate the activation of Rho.

Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the alpha1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The alpha1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected alpha1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the alpha1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired alpha1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (DeltaR8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G protein-coupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.

AKAP-Lbc is a novel member of the A-kinase anchoring protein (AKAPs) family, which functions as a cAMP-dependent protein kinase (PKA)-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We recently demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha-subunit of the heterotrimeric G protein G(12), whereas phosphorylation of AKAP-Lbc by the anchored PKA induces the recruitment of 14-3-3, which inhibits its GEF function. In the present report, using co-immunoprecipitation approaches, we demonstrated that AKAP-Lbc can form homo-oligomers inside cells. Mutagenesis studies revealed that oligomerization is mediated by two adjacent leucine zipper motifs located in the C-terminal region of the anchoring protein. Most interestingly, disruption of oligomerization resulted in a drastic increase in the ability of AKAP-Lbc to stimulate the formation of Rho-GTP in cells under basal conditions, suggesting that oligomerization maintains AKAP-Lbc in a basal-inactive state. Based on these results and on our previous findings showing that AKAP-Lbc is inactivated through the association with 14-3-3, we investigated the hypothesis that AKAP-Lbc oligomerization might be required for the regulatory action of 14-3-3. Most interestingly, we found that mutants of AKAP-Lbc impaired in their ability to undergo oligomerization were completely resistant to the inhibitory effect of PKA and 14-3-3. This suggests that 14-3-3 can negatively regulate the Rho-GEF activity of AKAP-Lbc only when the anchoring protein is in an oligomeric state. Altogether, these findings provide a novel mechanistic explanation of how oligomerization can regulate the activity of exchange factors of the Dbl family.

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

The aim of a large number of studies on G protein-coupled receptors was centered on understanding the structural basis of their main functional properties. Here, we will briefly review the results obtained on the alpha1-adrenergic receptor subtypes belonging to the rhodopsin-like family of receptors. These findings contribute, on the one hand, to further understand the molecular basis of adrenergic transmission and, on the other, to provide some generalities on the structure-functional relationship of G protein-coupled receptors.

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

Guanine nucleotide exchange factors of the Dbl family relay signals from membrane receptors to Rho family GTPases. We now demonstrate that a longer transcript of the Lbc gene encodes a chimeric molecule, which we have called AKAP-Lbc, that functions as an A-kinase-anchoring protein (AKAP) and a Rho-selective guanine nucleotide exchange factor. Expression of AKAP-Lbc in fibroblasts favors the formation of stress fibers in a Rho-dependent manner. Application of lysophosphatidic acid or selective expression of Galpha(12) enhances cellular AKAP-Lbc activation. Furthermore, biochemical studies indicate that AKAP-Lbc functions as an adaptor protein to selectively couple Galpha(12) to Rho. Thus, AKAP-Lbc anchors PKA and nucleates the assembly of a Rho-mediated signaling pathway.

Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division [1,2] and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA) [3,4]. Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs) [5,6]. AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Here, we show that pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. We demonstrate that both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo.

We compared the phosphorylation and internalization properties of constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of the third intracellular loop and at D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of agonist-independent phosphorylation were 150% and 50% higher than those of the wild-type alpha-1b AR, respectively. On the other hand, for the constitutively active D142A and D142T mutants, the basal levels of phosphorylation were similar to those of the wild-type alpha-1b AR and did not appear to be further stimulated by epinephrine. Overexpression of the guanyl nucleotide binding regulatory protein-coupled receptor kinase GRK2 further increases the basal phosphorylation of the A293E mutant, but not that of D142A mutant. Both the wild-type alpha-1b AR and the A293E mutant could undergo beta-arrestin-mediated internalization. The epinephrine-induced internalization of the constitutively active A293E mutant was significantly higher than that of the wild-type alpha-1b AR. In contrast, the D142A mutant was impaired in its ability to interact with beta-arrestin and to undergo agonist-induced internalization. Interestingly, a double mutant A293E/D142A retained very high constitutive activity and regulatory properties of both the A293E and D142A receptors. These findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.