Genome-Wide Gene Expression Analysis Shows AKAP13-Mediated PKD1 Signaling Regulates the Transcriptional Response to Cardiac Hypertrophy.

In the heart, scaffolding proteins such as A-Kinase Anchoring Proteins (AKAPs) play a crucial role in normal cellular function by serving as a signaling hub for multiple protein kinases including protein kinase D1 (PKD1). Under cardiac hypertrophic conditions AKAP13 anchored PKD1 activates the transcription factor MEF2 leading to subsequent fetal gene activation and hypertrophic response. We used an expression microarray to identify the global transcriptional response in the hearts of wild-type mice expressing the native form of AKAP13 compared to a gene-trap mouse model expressing a truncated form of AKAP13 that is unable to bind PKD1 (AKAP13-ΔPKD1). Microarray analysis showed that AKAP13-ΔPKD1 mice broadly failed to exhibit the transcriptional profile normally associated with compensatory cardiac hypertrophy following trans-aortic constriction (TAC). The identified differentially expressed genes in WT and AKAP13-ΔPKD1 hearts are vital for the compensatory hypertrophic response to pressure-overload and include myofilament, apoptotic, and cell growth/differentiation genes in addition to genes not previously identified as affected by AKAP13-anchored PKD1. Our results show that AKAP13-PKD1 signaling is critical for transcriptional regulation of key contractile, cell death, and metabolic pathways during the development of compensatory hypertrophy in vivo.

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity.

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic ß-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response.

UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy.

Hypertrophy increases the risk of heart failure and arrhythmia. Prevention or reversal of the maladaptive hypertrophic phenotype has thus been proposed to treat heart failure. Chronic ß-adrenergic receptor (ß-AR) stimulation induces cardiomyocyte hypertrophy by elevating 3',5'-cyclic adenosine monophosphate (cAMP) levels and activating downstream effectors such protein kinase A (PKA). Conversely, hydrolysis of cAMP by phosphodiesterases (PDEs) spatiotemporally restricts cAMP signaling. Here, we demonstrate that PDE4, but not PDE3, is critical in regulating cardiomyocyte hypertrophy, and may represent a potential target for preventing maladaptive hypertrophy. We identify a sequence within the upstream conserved region 1 of PDE4D, termed UCR1C, as a novel activator of PDE4 long isoforms. UCR1C activates PDE4 in complex with A-kinase anchoring protein (AKAP)-Lbc resulting in decreased PKA signaling facilitated by AKAP-Lbc. Expression of UCR1C in cardiomyocytes inhibits hypertrophy in response to chronic ß-AR stimulation. This effect is partially due to inhibition of nuclear PKA activity, which decreases phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In conclusion, PDE4 activation by UCR1C attenuates cardiomyocyte hypertrophy by specifically inhibiting nuclear PKA activity.

Moesin and myosin phosphatase confine neutrophil orientation in a chemotactic gradient.

Neutrophils respond to invading bacteria by adopting a polarized morphology, migrating in the correct direction, and engulfing the bacteria. How neutrophils establish and precisely orient this polarity toward pathogens remains unclear. Here we report that in resting neutrophils, the ERM (ezrin, radixin, and moesin) protein moesin in its active form (phosphorylated and membrane bound) prevented cell polarization by inhibiting the small GTPases Rac, Rho, and Cdc42. Attractant-induced activation of myosin phosphatase deactivated moesin at the prospective leading edge to break symmetry and establish polarity. Subsequent translocation of moesin to the trailing edge confined the formation of a prominent pseudopod directed toward pathogens and prevented secondary pseudopod formation in other directions. Therefore, both moesin-mediated inhibition and its localized deactivation by myosin phosphatase are essential for neutrophil polarization and effective neutrophil tracking of pathogens.

The C-terminus of the long AKAP13 isoform (AKAP-Lbc) is critical for development of compensatory cardiac hypertrophy.

The objective of this study was to determine the role of A-Kinase Anchoring Protein (AKAP)-Lbc in the development of heart failure, by investigating AKAP-Lbc-protein kinase D1 (PKD1) signaling in vivo in cardiac hypertrophy. Using a gene-trap mouse expressing a truncated version of AKAP-Lbc (due to disruption of the endogenous AKAP-Lbc gene), that abolishes PKD1 interaction with AKAP-Lbc (AKAP-Lbc-ΔPKD), we studied two mouse models of pathological hypertrophy: i) angiotensin (AT-II) and phenylephrine (PE) infusion and ii) transverse aortic constriction (TAC)-induced pressure overload. Our results indicate that AKAP-Lbc-ΔPKD mice exhibit an accelerated progression to cardiac dysfunction in response to AT-II/PE treatment and TAC. AKAP-Lbc-ΔPKD mice display attenuated compensatory cardiac hypertrophy, increased collagen deposition and apoptosis, compared to wild-type (WT) control littermates. Mechanistically, reduced levels of PKD1 activation are observed in AKAP-Lbc-ΔPKD mice compared to WT mice, resulting in diminished phosphorylation of histone deacetylase 5 (HDAC5) and decreased hypertrophic gene expression. This is consistent with a reduced compensatory hypertrophy phenotype leading to progression of heart failure in AKAP-Lbc-ΔPKD mice. Overall, our data demonstrates a critical in vivo role for AKAP-Lbc-PKD1 signaling in the development of compensatory hypertrophy to enhance cardiac performance in response to TAC-induced pressure overload and neurohumoral stimulation by AT-II/PE treatment.

Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

AKAP13 Rho-GEF and PKD-binding domain deficient mice develop normally but have an abnormal response to ß-adrenergic-induced cardiac hypertrophy.

BACKGROUND: A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during ß-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction. CONCLUSIONS: These results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or ß-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of ß-adrenergic-induced cardiac hypertrophy.

Src homology 2 domain-containing phosphatase 2 (Shp2) is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex and is inhibited by protein kinase A (PKA) under pathological hypertrophic conditions in the heart.

BACKGROUND: AKAP-Lbc is a scaffold protein that coordinates cardiac hypertrophic signaling. RESULTS: AKAP-Lbc interacts with Shp2, facilitating its regulation by PKA. CONCLUSION: AKAP-Lbc integrates PKA and Shp2 signaling in the heart. Under pathological hypertrophic conditions Shp2 is phosphorylated by PKA, and phosphatase activity is inhibited. SIGNIFICANCE: Inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote pathological cardiac hypertrophy. Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Our results identify a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that the tyrosine phosphatase, Shp2, is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits its protein-tyrosine phosphatase activity. Given the important cardiac roles of both AKAP-Lbc and Shp2, we investigated the AKAP-Lbc-Shp2 interaction in the heart. AKAP-Lbc-tethered PKA is implicated in cardiac hypertrophic signaling; however, mechanism of PKA action is unknown. Mutations resulting in loss of Shp2 catalytic activity are also associated with cardiac hypertrophy and congenital heart defects. Our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic ß-adrenergic stimulation and PKA activation. Thus, while induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote compensatory cardiac hypertrophy.

Molecular evolution of A-kinase anchoring protein (AKAP)-7: implications in comparative PKA compartmentalization.

BACKGROUND: A-Kinase Anchoring Proteins (AKAPs) are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA), directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18) are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport. RESULTS: Shown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction) corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels) that promote the production of AKAP7γ instead of AKAP7δ. CONCLUSIONS: This AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described.

Intersectin (ITSN) family of scaffolds function as molecular hubs in protein interaction networks.

Members of the intersectin (ITSN) family of scaffold proteins consist of multiple modular domains, each with distinct ligand preferences. Although ITSNs were initially implicated in the regulation of endocytosis, subsequent studies have revealed a more complex role for these scaffold proteins in regulation of additional biochemical pathways. In this study, we performed a high throughput yeast two-hybrid screen to identify additional pathways regulated by these scaffolds. Although several known ITSN binding partners were identified, we isolated more than 100 new targets for the two mammalian ITSN proteins, ITSN1 and ITSN2. We present the characterization of several of these new targets which implicate ITSNs in the regulation of the Rab and Arf GTPase pathways as well as regulation of the disrupted in schizophrenia 1 (DISC1) interactome. In addition, we demonstrate that ITSN proteins form homomeric and heteromeric complexes with each other revealing an added level of complexity in the function of these evolutionarily conserved scaffolds.

A-kinase anchoring proteins that regulate cardiac remodeling.

In response to injury or stress, the adult heart undergoes maladaptive changes, collectively defined as pathological cardiac remodeling. Here, we focus on the role of A-kinase anchoring proteins (AKAPs) in 3 main areas associated with cardiac remodeling and the progression of heart failure: excitation-contraction coupling, sarcomeric regulation, and induction of pathological hypertrophy. AKAPs are a diverse family of scaffold proteins that form multiprotein complexes, integrating cAMP signaling with protein kinases, phosphatases, and other effector proteins. Many AKAPs have been characterized in the heart, where they play a critical role in modulating cardiac function.

A-kinase anchoring proteins: from protein complexes to physiology and disease.

Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review, we focus on recent advances in the elucidation of AKAP function.

AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.

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.

Dynamic regulation of cAMP synthesis through anchored PKA-adenylyl cyclase V/VI complexes.

Spatiotemporal organization of cAMP signaling begins with the tight control of second messenger synthesis. In response to agonist stimulation of G protein-coupled receptors, membrane-associated adenylyl cyclases (ACs) generate cAMP that diffuses throughout the cell. The availability of cAMP activates various intracellular effectors, including protein kinase A (PKA). Specificity in PKA action is achieved by the localization of the enzyme near its substrates through association with A-kinase anchoring proteins (AKAPs). Here, we provide evidence for interactions between AKAP79/150 and ACV and ACVI. PKA anchoring facilitates the preferential phosphorylation of AC to inhibit cAMP synthesis. Real-time cellular imaging experiments show that PKA anchoring with the cAMP synthesis machinery ensures rapid termination of cAMP signaling upon activation of the kinase. This protein configuration permits the formation of a negative feedback loop that temporally regulates cAMP production.

AKAP-Lbc nucleates a protein kinase D activation scaffold.

The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

Protein phosphatase 4 interacts with the Survival of Motor Neurons complex and enhances the temporal localisation of snRNPs.

Protein phosphatase 4 (PPP4) is a ubiquitous essential protein serine/threonine phosphatase found in higher eukaryotes. Coordinate variation of the levels of the catalytic subunit (PPP4c) and the regulatory subunit (R2) suggests that PPP4c and R2 form a heterodimeric core to which other regulatory subunits bind. Two proteins that specifically co-purify with Flag-epitope-tagged R2 expressed in HEK-293 cells were identified as Gemin3 and Gemin4. These two proteins have been identified previously as components of the Survival of Motor Neurons (SMN) protein complex, which is functionally defective in the hereditary disorder spinal muscular atrophy. Immuno-sedimentation of the epitope-tagged SMN protein complex from HeLa cells expressing CFP-SMN showed that the SMN protein interacts, as previously reported, with Gemin2 (SIP1), Gemin3 and Gemin4 and in addition associates with PPP4c. The SMN complex has been implicated in the assembly and maturation of small nuclear ribonucleoproteins (snRNPs). Expression of GFP-R2-PPP4c in HeLa cells enhances the temporal localisation of newly formed snRNPs, which is consistent with an association of R2-PPP4c with the SMN protein complex.