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

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 β-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.     

A Small Novel A-Kinase Anchoring Protein (AKAP) That Localizes Specifically Protein Kinase A-Regulatory Subunit I (PKA-RI) to the Plasma Membrane

Protein kinase A-anchoring proteins (AKAPs) provide spatio-temporal specificity for the omnipotent cAMP-dependent protein kinase (PKA) via high affinity interactions with PKA regulatory subunits (PKA-RI, RII). Many PKA-RII-AKAP complexes are heavily tethered to cellular substructures, whereas PKA-RI-AKAP complexes have remained largely undiscovered. Here, using a cAMP affinity-based chemical proteomics strategy in human heart and platelets, we uncovered a novel, ubiquitously expressed AKAP, termed small membrane (sm)AKAP due to its specific localization at the plasma membrane via potential myristoylation/palmitoylation anchors. In vitro binding studies revealed specificity of smAKAP for PKA-RI (K_d = 7 nm) over PKA-RII (K_d = 53 nm) subunits, co-expression of smAKAP with the four PKA R subunits revealed an even more exclusive specificity of smAKAP for PKA-RIα/β in the cellular context. Applying the singlet oxygen-generating electron microscopy probe miniSOG indicated that smAKAP is tethered to the plasma membrane and is particularly dense at cell-cell junctions and within filopodia. Our preliminary functional characterization of smAKAP provides evidence that, like PKA-RII, PKA-RI can be tightly tethered by a novel repertoire of AKAPs, providing a new perspective on spatio-temporal control of cAMP signaling.     

Cardiac troponin T, a sarcomeric AKAP, tethers protein kinase A at the myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to β-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.     

Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (R_Select) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of R_Select proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.     

Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.     

Phospholipase C-related but catalytically inactive protein (PRIP) modulates synaptosomal-associated protein 25 (SNAP-25) phosphorylation and exocytosis

Exocytosis is one of the most fundamental cellular events. The basic mechanism of the final step, membrane fusion, is mediated by the formation of the SNARE complex, which is modulated by the phosphorylation of proteins controlled by the concerted actions of protein kinases and phosphatases. We have previously shown that a protein phosphatase-1 (PP1) anchoring protein, phospholipase C-related but catalytically inactive protein (PRIP), has an inhibitory role in regulated exocytosis. The current study investigated the involvement of PRIP in the phospho-dependent modulation of exocytosis. Dephosphorylation of synaptosome-associated protein of 25 kDa (SNAP-25) was mainly catalyzed by PP1, and the process was modulated by wild-type PRIP but not by the mutant (F97A) lacking PP1 binding ability in in vitro studies. We then examined the role of PRIP in phospho-dependent regulation of exocytosis in cell-based studies using pheochromocytoma cell line PC12 cells, which secrete noradrenalin. Exogenous expression of PRIP accelerated the dephosphorylation process of phosphorylated SNAP-25 after forskolin or phorbol ester treatment of the cells. The phospho-states of SNAP-25 were correlated with noradrenalin secretion, which was enhanced by forskolin or phorbol ester treatment and modulated by PRIP expression in PC12 cells. Both SNAP-25 and PP1 were co-precipitated in anti-PRIP immunocomplex isolated from PC12 cells expressing PRIP. Collectively, together with our previous observation regarding the roles of PRIP in PP1 regulation, these results suggest that PRIP is involved in the regulation of the phospho-states of SNAP-25 by modulating the activity of PP1, thus regulating exocytosis.     

Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.     

Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.     

AMP-activated protein kinase phosphorylates cardiac troponin I at Ser-150 to increase myofilament calcium sensitivity and blunt PKA-dependent function

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme central to the regulation of metabolic homeostasis. In the heart AMPK is activated during cardiac stress-induced ATP depletion and functions to stimulate metabolic pathways that restore the AMP/ATP balance. Recently it was demonstrated that AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vitro. We sought to determine if the metabolic regulatory kinase AMPK phosphorylates cTnI at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrates that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo-phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA-dependent increases in length dependent activation. Thus, in addition to the defined role of AMPK as a cardiac metabolic energy gauge, these data demonstrate AMPK Ser-150 phosphorylation of cTnI directly links the regulation of cardiac metabolic demand to myofilament contractile energetics. Furthermore, the blunting effect of cTnI Ser-150 phosphorylation cross-talk can uncouple the effects of myofilament PKA-dependent phosphorylation from β-adrenergic signaling as a novel thin filament contractile regulatory signaling mechanism.     

Ezrin-anchored protein kinase A coordinates phosphorylation-dependent disassembly of a NHERF1 ternary complex to regulate hormone-sensitive phosphate transport

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Loss-of-function mutations stabilize an inactive NHERF1 conformation that we show is refractory to PKA phosphorylation and impairs assembly of the ternary complex. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action. These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex.     

Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.     

Anchoring of Protein Kinase A by ERM (Ezrin-Radixin-Moesin) Proteins Is Required for Proper Netrin Signaling through DCC (Deleted in Colorectal Cancer)

Netrin-1, acting through its principal receptor DCC (deleted in colorectal cancer), serves as an axon guidance cue during neural development and also contributes to vascular morphogenesis, epithelial migration, and the pathogenesis of some tumors. Several lines of evidence suggest that netrin-DCC signaling can regulate and be regulated by the cAMP-dependent protein kinase, PKA, although the molecular details of this relationship are poorly understood. Specificity in PKA signaling is often achieved through differential subcellular localization of the enzyme by interaction with protein kinase A anchoring proteins (AKAPs). Here, we show that AKAP function is required for DCC-mediated activation of PKA and phosphorylation of cytoskeletal regulatory proteins of the Mena/VASP (vasodilator-stimulated phosphoprotein) family. Moreover, we show that DCC and PKA physically interact and that this association is mediated by the ezrin-radixin-moesin (ERM) family of plasma membrane-actin cytoskeleton cross-linking proteins. Silencing of ERM protein expression inhibits DCC-PKA interaction, DCC-mediated PKA activation, and phosphorylation of Mena/VASP proteins as well as growth cone morphology and neurite outgrowth. Finally, although expression of wild-type radixin partially rescued growth cone morphology and tropism toward netrin in ERM-knockdown cells, expression of an AKAP-deficient mutant of radixin did not fully rescue growth cone morphology and switched netrin tropism from attraction to repulsion. These data support a model in which ERM-mediated anchoring of PKA activity to DCC is required for proper netrin/DCC-mediated signaling.     

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E₂), couple to the G proteins G_s and G_i/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E₂ and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of G_i/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of G_i/o and retains receptors in the plasma membrane.     

Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ～200-pS open state while simultaneously reducing transitions between this open state and a ～65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ～200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.     

Anchored protein kinase A recruitment of active Rac GTPase

Protein kinase A-anchoring proteins (AKAPs) influence fundamental cellular processes by directing the cAMP-dependent protein kinase (PKA) toward its intended substrates. In this report we describe the identification and characterization of a ternary complex of AKAP220, the PKA holoenzyme, and the IQ domain GTPase-activating protein 2 isoform (IQGAP2) that is enriched at cortical regions of the cell. Formation of an IQGAP2-AKAP220 core complex initiates a subsequent phase of protein recruitment that includes the small GTPase Rac. Biochemical and molecular biology approaches reveal that PKA phosphorylation of Thr-716 on IQGAP2 enhances association with the active form of the Rac GTPase. Cell-based experiments indicate that overexpression of an IQGAP2 phosphomimetic mutant (IQGAP2 T716D) enhances the formation of actin-rich membrane ruffles at the periphery of HEK 293 cells. In contrast, expression of a nonphosphorylatable IQGAP2 T716A mutant or gene silencing of AKAP220 suppresses formation of membrane ruffles. These findings imply that IQGAP2 and AKAP220 act synergistically to sustain PKA-mediated recruitment of effectors such as Rac GTPases that impact the actin cytoskeleton.     

Ca2+/calmodulin-dependent protein kinase II inhibitors disrupt AKAP79-dependent PKC signaling to GluA1 AMPA receptors

GluA1 (formerly GluR1) AMPA receptor subunit phosphorylation at Ser-831 is an early biochemical marker for long-term potentiation and learning. This site is a substrate for Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and protein kinase C (PKC). By directing PKC to GluA1, A-kinase anchoring protein 79 (AKAP79) facilitates Ser-831 phosphorylation and makes PKC a more potent regulator of GluA1 than CaMKII. PKC and CaM bind to residues 31-52 of AKAP79 in a competitive manner. Here, we demonstrate that common CaMKII inhibitors alter PKC and CaM interactions with AKAP79(31-52). Most notably, the classical CaMKII inhibitors KN-93 and KN-62 potently enhanced the association of CaM to AKAP79(31-52) in the absence (apoCaM) but not the presence of Ca(2+). In contrast, apoCaM association to AKAP79(31-52) was unaffected by the control compound KN-92 or a mechanistically distinct CaMKII inhibitor (CaMKIINtide). In vitro studies demonstrated that KN-62 and KN-93, but not the other compounds, led to apoCaM-dependent displacement of PKC from AKAP79(31-52). In the absence of CaMKII activation, complementary cellular studies revealed that KN-62 and KN-93, but not KN-92 or CaMKIINtide, inhibited PKC-mediated phosphorylation of GluA1 in hippocampal neurons as well as AKAP79-dependent PKC-mediated augmentation of recombinant GluA1 currents. Buffering cellular CaM attenuated the ability of KN-62 and KN-93 to inhibit AKAP79-anchored PKC regulation of GluA1. Therefore, by favoring apoCaM binding to AKAP79, KN-62 and KN-93 derail the ability of AKAP79 to efficiently recruit PKC for regulation of GluA1. Thus, AKAP79 endows PKC with a pharmacological profile that overlaps with CaMKII.     

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 β-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.     

Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase

The NAD(+)-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that is DBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD(+). We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex.     

Plasmin overcomes resistance to prostaglandin E2 in fibrotic lung fibroblasts by reorganizing protein kinase A signaling

Collagen deposition by fibroblasts contributes to scarring in fibrotic diseases. Activation of protein kinase A (PKA) by cAMP represents a pivotal brake on fibroblast activation, and the lipid mediator prostaglandin E(2) (PGE(2)) exerts its well known anti-fibrotic actions through cAMP signaling. However, fibrotic fibroblasts from the lungs of patients with idiopathic pulmonary fibrosis, or of mice with bleomycin-induced fibrosis, are resistant to the normal collagen-inhibiting action of PGE(2). In this study, we demonstrate that plasminogen activation to plasmin restores PGE(2) sensitivity in fibrotic lung fibroblasts from human and mouse. This involves amplified PKA signaling resulting from the promotion of new interactions between AKAP9 and PKA regulatory subunit II in the perinuclear region as well as from the inhibition of protein phosphatase 2A. This is the first report to show that an extracellular mediator can dramatically reorganize and amplify the intracellular PKA-A-kinase anchoring protein signaling network and suggests a new strategy to control collagen deposition by fibrotic fibroblasts.