N-Tertaining a New Signaling Paradigm for the Cardiomyocyte ß 1 -Adrenergic Receptor.

ABSTRACT: ß 1 -adrenergic receptors (ß 1 ARs) are the principle mediators of catecholamine actions in cardiomyocytes. ß 1 ARs rapidly adjust cardiac output and provide short-term hemodynamic support for the failing heart by activating a Gs-adenylyl cyclase pathway that increases 3'-5'-cyclic adenosine monophosphate and leads to the activation of protein kinase A and the phosphorylation of substrates involved in excitation-contraction coupling. However, chronic persistent ß 1 AR activation in the setting of heart failure leads to a spectrum of maladaptive changes that contribute to the evolution of heart failure. The molecular basis for ß 1 AR-driven maladaptive responses remains uncertain because chronic persistent ß 1 AR activation has been linked to the activation of both proapoptotic and antiapoptotic signaling pathways. Of note, studies to date have been predicated on the assumption that ß 1 ARs signal exclusively as full-length receptor proteins. Our recent studies show that ß 1 ARs are detected as both full-length and N-terminally truncated species in cardiomyocytes, that N-terminal cleavage is regulated by O-glycan modifications at specific sites on the ß 1 AR N-terminus, and that N-terminally truncated ß 1 ARs remain signaling competent, but their signaling properties differ from those of the full-length ß 1 AR. The N-terminally truncated form of the ß 1 AR constitutively activates the protein kinase B signaling pathway and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. These studies identify a novel signaling paradigm for the ß 1 AR, implicating the N-terminus as a heretofore-unrecognized structural determinant of ß 1 AR responsiveness that could be pharmacologically targeted for therapeutic advantage.

Decoding the Cardiac Actions of Protein Kinase D Isoforms.

Protein kinase D (PKD) consists of a family of three structurally related enzymes that play key roles in a wide range of biological functions that contribute to the evolution of cardiac hypertrophy and heart failure. PKD1 (the founding member of this enzyme family) has been implicated in the phosphorylation of substrates that regulate cardiac hypertrophy, contraction, and susceptibility to ischemia/reperfusion injury, and de novo PRKD1 (protein kinase D1 gene) mutations have been identified in patients with syndromic congenital heart disease. However, cardiomyocytes coexpress all three PKDs. Although stimulus-specific activation patterns for PKD1, PKD2, and PKD3 have been identified in cardiomyocytes, progress toward identifying PKD isoform-specific functions in the heart have been hampered by significant gaps in our understanding of the molecular mechanisms that regulate PKD activity. This review incorporates recent conceptual breakthroughs in our understanding of various alternative mechanisms for PKD activation, with an emphasis on recent evidence that PKDs activate certain effector responses as dimers, to consider the role of PKD isoforms in signaling pathways that drive cardiac hypertrophy and ischemia/reperfusion injury. The focus is on whether the recently identified activation mechanisms that enhance the signaling repertoire of PKD family enzymes provide novel therapeutic strategies to target PKD enzymes and prevent or slow the evolution of cardiac injury and pathological cardiac remodeling. SIGNIFICANCE STATEMENT: PKD isoforms regulate a large number of fundamental biological processes, but the understanding of the biological actions of individual PKDs (based upon studies using adenoviral overexpression or gene-silencing methods) remains incomplete. This review focuses on dimerization, a recently identified mechanism for PKD activation, and the notion that this mechanism provides a strategy to develop novel PKD-targeted pharmaceuticals that restrict proliferation, invasion, or angiogenesis in cancer and prevent or slow the evolution of cardiac injury and pathological cardiac remodeling.

ß1-adrenergic receptor N-terminal cleavage by ADAM17; the mechanism for redox-dependent downregulation of cardiomyocyte ß1-adrenergic receptors.

ß1-adrenergic receptors (ß1ARs) are the principle mediators of catecholamine action in cardiomyocytes. We previously showed that the ß1AR extracellular N-terminus is a target for post-translational modifications that impact on signaling responses. Specifically, we showed that the ß1AR N-terminus carries O-glycan modifications at Ser37/Ser41, that O-glycosylation prevents ß1AR N-terminal cleavage, and that N-terminal truncation influences ß1AR signaling to downstream effectors. However, the site(s) and mechanism for ß1AR N-terminal cleavage in cells was not identified. This study shows that ß1ARs are expressed in cardiomyocytes and other cells types as both full-length and N-terminally truncated species and that the truncated ß1AR species is formed as a result of an O-glycan regulated N-terminal cleavage by ADAM17 at R31↓L32. We identify Ser41 as the major O-glycosylation site on the ß1AR N-terminus and show that an O-glycan modification at Ser41 prevents ADAM17-dependent cleavage of the ß1-AR N-terminus at S41↓L42, a second N-terminal cleavage site adjacent to this O-glycan modification (and it attenuates ß1-AR N-terminal cleavage at R31↓L32). We previously reported that oxidative stress leads to a decrease in ß1AR expression and catecholamine responsiveness in cardiomyocytes. This study shows that redox-inactivation of cardiomyocyte ß1ARs is via a mechanism involving N-terminal truncation at R31↓L32 by ADAM17. In keeping with the previous observation that N-terminally truncated ß1ARs constitutively activate an AKT pathway that affords protection against doxorubicin-dependent apoptosis, overexpression of a cleavage resistant ß1AR mutant exacerbates doxorubicin-dependent apoptosis. These studies identify the ß1AR N-terminus as a structural determinant of ß1AR responses that can be targeted for therapeutic advantage.

Telangiectasia-ectodermal dysplasia-brachydactyly-cardiac anomaly syndrome is caused by de novo mutations in protein kinase D1.

BACKGROUND: We describe two unrelated patients who display similar clinical features including telangiectasia, ectodermal dysplasia, brachydactyly and congenital heart disease. METHODS: We performed trio whole exome sequencing and functional analysis using in vitro kinase assays with recombinant proteins. RESULTS: We identified two different de novo mutations in protein kinase D1 (PRKD1, NM_002742.2): c.1774G>C, p.(Gly592Arg) and c.1808G>A, p.(Arg603His), one in each patient. PRKD1 (PKD1, HGNC:9407) encodes a kinase that is a member of the protein kinase D (PKD) family of serine/threonine protein kinases involved in diverse cellular processes such as cell differentiation and proliferation and cell migration as well as vesicle transport and angiogenesis. Functional analysis using in vitro kinase assays with recombinant proteins showed that the mutation c.1808G>A, p.(Arg603His) represents a gain-of-function mutation encoding an enzyme with a constitutive, lipid-independent catalytic activity. The mutation c.1774G>C, p.(Gly592Arg) in contrast shows a defect in substrate phosphorylation representing a loss-of-function mutation. CONCLUSION: The present cases represent a syndrome, which associates symptoms from several different organ systems: skin, teeth, bones and heart, caused by heterozygous de novo mutations in PRKD1 and expands the clinical spectrum of PRKD1 mutations, which have hitherto been linked to syndromic congenital heart disease and limb abnormalities.

Carvedilol Prevents Redox Inactivation of Cardiomyocyte Β1-Adrenergic Receptors.

The mechanism that leads to a decrease in ß1-adrenergic receptor (ß1AR) expression in the failing heart remains uncertain. This study shows that cardiomyocyte ß1AR expression and isoproterenol responsiveness decrease in response to oxidative stress. Studies of mechanisms show that the redox-dependent decrease in ß1AR expression is uniquely prevented by carvedilol and not other ßAR ligands. Carvedilol also promotes the accumulation of N-terminally truncated ß1ARs that confer protection against doxorubicin-induced apoptosis in association with activation of protein kinase B. The redox-induced molecular controls for cardiomyocyte ß1ARs and pharmacologic properties of carvedilol identified in this study have important clinical and therapeutic implications.

Post-translational modifications at the ATP-positioning G-loop that regulate protein kinase activity.

Protein kinases are a superfamily of enzymes that control a wide range of cellular functions. These enzymes share a highly conserved catalytic core that folds into a similar bilobar three-dimensional structure. One highly conserved region in the protein kinase core is the glycine-rich loop (or G-loop), a highly flexible loop that is characterized by a consensus GxGxxG sequence. The G-loop points toward the catalytic cleft and functions to bind and position ATP for phosphotransfer. Of note, in many protein kinases, the second and third glycine residues in the G-loop triad flank residues that can be targets for phosphorylation (Ser, Thr, or Tyr) or other post-translational modifications (ubiquitination, acetylation, O-GlcNAcylation, oxidation). There is considerable evidence that cyclin-dependent kinases are held inactive through inhibitory phosphorylation of the conserved Thr/Tyr residues in this position of the G-loop and that dephosphorylation by cellular phosphatases is required for CDK activation and progression through the cell cycle. This review summarizes literature that identifies residues in or adjacent to the G-loop in other protein kinases that are targets for functionally important post-translational modifications.

ß1-adrenergic receptor O-glycosylation regulates N-terminal cleavage and signaling responses in cardiomyocytes.

ß1-adrenergic receptors (ß1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Structural studies of the ß1AR define ligand-binding sites in the transmembrane helices and effector docking sites at the intracellular surface of the ß1AR, but the extracellular N-terminus, which is a target for post-translational modifications, typically is ignored. This study identifies ß1AR N-terminal O-glycosylation at Ser37/Ser41 as a mechanism that prevents ß1AR N-terminal cleavage. We used an adenoviral overexpression strategy to show that both full-length/glycosylated ß1ARs and N-terminally truncated glycosylation-defective ß1ARs couple to cAMP and ERK-MAPK signaling pathways in cardiomyocytes. However, a glycosylation defect that results in N-terminal truncation stabilizes ß1ARs in a conformation that is biased toward the cAMP pathway. The identification of O-glycosylation and N-terminal cleavage as novel structural determinants of ß1AR responsiveness in cardiomyocytes could be exploited for therapeutic advantage.

Protein kinase C mechanisms that contribute to cardiac remodelling.

Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure.

The protein kinase D1 COOH terminus: marker or regulator of enzyme activity?

Protein kinase D1 (PKD1) is a Ser/Thr kinase implicated in a wide variety of cellular responses. PKD1 activation is generally attributed to a PKC-dependent pathway that leads to phosphorylation of the activation loop at Ser(744)/Ser(748). This modification increases catalytic activity, including that toward an autophosphorylation site (Ser(916)) in a postsynaptic density-95/disks large/zonula occludens-1 (PDZ)-binding motif at the extreme COOH terminus. However, there is growing evidence that PKD1 activation can also result from a PKC-independent autocatalytic reaction at Ser(744)/Ser(748) and that certain stimuli increase in PKD1 phosphorylation at Ser(744)/S(748) without an increase in autophosphorylation at Ser(916). This study exposes a mechanism that results in a discrepancy between PKD1 COOH-terminal autocatalytic activity and activity toward other substrates. We show that PKD1 constructs harboring COOH-terminal epitope tags display high levels of in vitro activation loop autocatalytic activity and activity toward syntide-2 (a peptide substrate), but no Ser(916) autocatalytic activity. Cell-based studies show that the COOH-terminal tag, adjacent to PKD1's PDZ1-binding motif, does not grossly influence PKD1 partitioning between soluble and particulate fractions in resting cells or PKD1 translocation to the particulate fraction following treatment with PMA. However, a COOH-terminal tag that confers a high level of activation loop autocatalytic activity decreases the PKC requirement for agonist-dependent PKD1 activation in cells. The recognition that COOH-terminal tags alter PKD1's pharmacological profile is important from a technical standpoint. The altered dynamics and activation mechanisms for COOH-terminal-tagged PKD1 enzymes also could model the signaling properties of localized pools of enzyme anchored through the COOH terminus to PDZ domain-containing scaffolding proteins.

S49G and R389G polymorphisms of the ß1-adrenergic receptor influence signaling via the cAMP-PKA and ERK pathways.

Two functionally important ß1-adrenergic receptor (ß1AR) polymorphisms have been identified. The R389G polymorphism influences coupling to the Gs-cAMP pathway. R(389)-ß1ARs display enhanced activation of cAMP/PKA; they provide short-term inotropic support but also cause a predisposition to cardiomyopathic decompensation. A second S49G polymorphism is implicated in the evolution of heart failure, but the mechanism remains uncertain. This study shows that position 49 and 389 polymorphisms function in a coordinate manner to influence agonist-dependent cAMP/PKA and ERK responses. cAMP/PKA and ERK responses are more robust in HEK293 cells that heterologously overexpress G(49)-ß1ARs, compared with S(49)-ß1ARs. However, this phenotype is most obvious on a G(389)-ß1AR background; the more robust agonist-dependent cAMP/PKA and ERK responses in R(389)-ß1AR cells effectively obscure the effect of the S49G polymorphism. We also show that isoproterenol (Iso) and carvedilol activate ERK via a similar EGFR-independent mechanism in cells expressing various ß1AR haplotypes. However, Iso activates ERK via an Src-independent pathway, but carvedilol-dependent ERK activation requires Src. Since the S49G polymorphism has been linked to changes in ß1AR trafficking, we examined whether ß1AR polymorphisms influence partitioning to lipid raft membranes. Biochemical fractionation studies show that all four ß1AR variants are recovered in buoyant flotillin-enriched membranes; the distinct signaling phenotypes of the different ß1AR variants could not be attributed to any gross differences in basal compartmentalization to lipid raft membranes. The allele-specific differences in ß1AR signaling phenotypes identified in this study could underlie interindividual differences in responsiveness to ß-blocker therapy and clinical outcome in heart failure.

Regulation of protein kinase D1 activity.

Protein kinase D1 (PKD1) is a stress-activated serine/threonine kinase that plays a vital role in various physiologically important biological processes, including cell growth, apoptosis, adhesion, motility, and angiogenesis. Dysregulated PKD1 expression also contributes to the pathogenesis of certain cancers and cardiovascular disorders. Studies to date have focused primarily on the canonical membrane-delimited pathway for PKD1 activation by G protein-coupled receptors or peptide growth factors. Here, agonist-dependent increases in diacylglycerol accumulation lead to the activation of protein kinase C (PKC) and PKC-dependent phosphorylation of PKD1 at two highly conserved serine residues in the activation loop; this modification increases PKD1 catalytic activity, as assessed by PKD1 autophosphorylation at a consensus phosphorylation motif at the extreme C terminus. However, recent studies expose additional controls and consequences for PKD1 activation loop and C-terminal phosphorylation as well as additional autophosphorylation reactions and trans-phosphorylations (by PKC and other cellular enzymes) that contribute to the spatiotemporal control of PKD1 signaling in cells. This review focuses on the multisite phosphorylations that are known or predicted to influence PKD1 catalytic activity and may also influence docking interactions with cellular scaffolds and trafficking to signaling microdomains in various subcellular compartments. These modifications represent novel targets for the development of PKD1-directed pharmaceuticals for the treatment of cancers and cardiovascular disorders.

Cardiomyocyte lipids impair ß-adrenergic receptor function via PKC activation.

Normal hearts have increased contractility in response to catecholamines. Because several lipids activate PKCs, we hypothesized that excess cellular lipids would inhibit cardiomyocyte responsiveness to adrenergic stimuli. Cardiomyocytes treated with saturated free fatty acids, ceramide, and diacylglycerol had reduced cellular cAMP response to isoproterenol. This was associated with increased PKC activation and reduction of ß-adrenergic receptor (ß-AR) density. Pharmacological and genetic PKC inhibition prevented both palmitate-induced ß-AR insensitivity and the accompanying reduction in cell surface ß-ARs. Mice with excess lipid uptake due to either cardiac-specific overexpression of anchored lipoprotein lipase, PPARγ, or acyl-CoA synthetase-1 or high-fat diet showed reduced inotropic responsiveness to dobutamine. This was associated with activation of protein kinase C (PKC)α or PKCδ. Thus, several lipids that are increased in the setting of lipotoxicity can produce abnormalities in ß-AR responsiveness. This can be attributed to PKC activation and reduced ß-AR levels.

Reactive oxygen species decrease cAMP response element binding protein expression in cardiomyocytes via a protein kinase D1-dependent mechanism that does not require Ser133 phosphorylation.

Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

Raf-1: a novel cardiac troponin T kinase.

Phosphorylation of cardiac troponin is a key mechanism involved in regulation of contractile function. In vitro kinase assays revealed that lysates prepared from resting cardiomyocytes contain cardiac troponin I (cTnI) and cTnT kinase activity. cTnI phosphorylation is inhibited by pharmacologic inhibitors of PKA, PKC, Rho kinase and PKC effectors such as RSK and PKD; these kinase inhibitors do not inhibit phosphorylation of cTnT. Rather, cTnT phosphorylation is decreased by the Raf inhibitor GW5074. In vitro kinase assays show that recombinant Raf phosphorylates cTnT, and that Raf-dependent cTnT phosphorylation is abrogated by a T206E substitution; Raf does not phosphorylate cTnI. These studies identify Raf-dependent cTnT-Thr(206) phosphorylation as a novel mechanism that would link growth factor-dependent signaling pathways to dynamic changes in cardiac contractile function.

p66Shc links alpha1-adrenergic receptors to a reactive oxygen species-dependent AKT-FOXO3A phosphorylation pathway in cardiomyocytes.

p66Shc is an adapter protein that is induced by hypertrophic stimuli and has been implicated as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. This study implicates p66Shc in an alpha(1)-adrenergtic receptor (alpha(1)-AR) pathway that requires the cooperative effects of protein kinase (PK)Cepsilon and PKCdelta and leads to AKT-FOXO3a phosphorylation in cardiomyocytes. alpha(1)-ARs promote p66Shc-YY(239/240) phosphorylation via a ROS-dependent mechanism that is localized to caveolae and requires epidermal growth factor receptor (EGFR) and PKCepsilon activity. alpha(1)-ARs also increase p66Shc-S(36) phosphorylation via an EGFR transactivation pathway involving PKCdelta. p66Shc links alpha(1)-ARs to an AKT signaling pathway that selectively phosphorylates/inactivates FOXO transcription factors and downregulates the ROS-scavenging protein manganese superoxide dismutase (MnSOD); the alpha(1)-AR-p66Shc-dependent pathway involving AKT does not regulate GSK3. Additional studies show that RNA interference-mediated downregulation of endogenous p66Shc leads to the derepression of FOXO3a-regulated genes such as MnSOD, p27Kip1, and BIM-1. p66Shc downregulation also increases proliferating cell nuclear antigen expression and induces cardiomyocyte hypertrophy, suggesting that p66Shc exerts an antihypertrophic action in neonatal cardiomyocytes. The novel alpha(1)-AR- and ROS-dependent pathway involving p66Shc identified in this study is likely to contribute to cardiomyocyte remodeling and the evolution of heart failure.

Protein kinase D1 autophosphorylation via distinct mechanisms at Ser744/Ser748 and Ser916.

Protein kinase D1 (PKD1) is a physiologically important signaling enzyme that is activated via protein kinase C-dependent trans-phosphorylation of the activation loop at Ser744 and Ser748 followed by PKD1 autophosphorylation at Ser916. Although PKD-Ser916 autophosphorylation is widely used to track cellular PKD activity, this study exposes conditions leading to increased PKD-Ser(P)916 immunoreactivity without an associated increase in PKD activity in cardiomyocytes that heterologously overexpress catalytically inactive PKD1 and in cardiomyocytes treated with Gö6976 (a PKD inhibitor that competes with ATP). In each case, PKD1 is detected as a Ser916-phosphorylated enzyme that lacks kinase activity. In vitro kinase assays reconcile these seemingly discrepant findings by demonstrating that PKD1-Ser916 autophosphorylation can proceed via either an intermolecular reaction or an intramolecular autophosphorylation that requires only very low ATP concentrations that do not support target substrate phosphorylation. Additional studies show that Ser744 and Ser748 are targets for a protein kinase C-independent autocatalytic phosphorylation and that the PKD1-S744A/S748A mutant is a Ser916-phosphorylated enzyme that is not active toward heterologous substrates. In contrast, PKD1-S916A is an active kinase that autophosphorylates at Ser744. However, the S916A substitution leads to a Ser748 phosphorylation defect and a prolonged cellular PKD1 signaling response. Collectively, these results implicate PKD1-Ser744 phosphorylation in the phorbol 12-myristate 13-acetate-dependent mechanism that increases PKD1 activity toward physiologically relevant substrates. We show that PKD1-Ser916 autophosphorylation does not necessarily correlate with PKD1 activity. Rather, autophosphorylation at Ser916 is required for subsequent autophosphorylation at Ser748. Finally, this study exposes a novel role for Ser916 and/or Ser748 autophosphorylation to terminate the cellular PKD1 signaling response.

Protein kinase D links Gq-coupled receptors to cAMP response element-binding protein (CREB)-Ser133 phosphorylation in the heart.

Many growth regulatory stimuli promote cAMP response element-binding protein (CREB) Ser(133) phosphorylation, but the physiologically relevant CREB-Ser(133) kinase(s) in the heart remains uncertain. This study identifies a novel role for protein kinase D (PKD) as an in vivo cardiac CREB-Ser(133) kinase. We show that thrombin activates a PKCdelta-PKD pathway leading to CREB-Ser(133) phosphorylation in cardiomyocytes and cardiac fibroblasts. alpha(1)-Adrenergic receptors also activate a PKCdelta-PKD-CREB-Ser(133) phosphorylation pathway in cardiomyocytes. Of note, while the epidermal growth factor (EGF) promotes CREB-Ser(133) phosphorylation via an ERK-RSK pathway in cardiac fibroblasts, the thrombin-dependent EGFR transactivation pathway leading to ERK-RSK activation does not lead to CREB-Ser(133) phosphorylation in this cell type. Adenoviral-mediated overexpression of PKCdelta (but not PKCepsilon or PKCalpha) activates PKD; PKCdelta and PKD1-S744E/S748E overexpression both promote CREB-Ser(133) phosphorylation. Pasteuralla multocida toxin (PMT), a direct Galpha(q) agonist that induces robust cardiomyocyte hypertrophy, also activates the PKD-CREB-Ser(133) phosphorylation pathway, leading to the accumulation of active PKD and Ser(133)-phosphorylated CREB in the nucleus, activation of a CRE-responsive promoter, and increased Bcl-2 (CREB target gene) expression in cardiomyocyte cultures. Cardiac-specific Galpha(q) overexpression also leads to an increase in PKD-Ser(744)/Ser(748) and CREB-Ser(133) phosphorylation as well as increased Bcl-2 protein expression in the hearts of transgenic mice. Collectively, these studies identify a novel Galpha(q)-PKCdelta-PKD-CREB-Ser(133) phosphorylation pathway that is predicted to contribute to cardiac remodeling and could be targeted for therapeutic advantage in the setting of heart failure phenotypes.

Phorbol 12-myristate 13-acetate-dependent protein kinase C delta-Tyr311 phosphorylation in cardiomyocyte caveolae.

Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCdelta to caveolae and increase PKCdelta-Tyr(311) phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.

Novel mode for neutrophil protease cathepsin G-mediated signaling: membrane shedding of epidermal growth factor is required for cardiomyocyte anoikis.

Neutrophils are thought to orchestrate myocardial remodeling during the early progression to cardiac failure through the release of reactive oxygen species, antimicrobial peptides, and proteases. Although neutrophil activation may be beneficial at early stages of disease, excessive neutrophil infiltration can induce cardiomyocyte death and tissue damage. The neutrophil-derived serine protease cathepsin G (Cat.G) has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis. However, the involved signaling mechanisms for Cat.G are not well understood. This study identifies epidermal growth factor receptor (EGFR) transactivation as a mechanism whereby Cat.G induces signaling in cardiomyocytes. Cat.G induced a rapid and transient increase in EGFR tyrosine phosphorylation, and inhibition of EGFR kinase activity, either with AG1478 or by expression of kinase inactive EGFR mutants (EGFR-CD533), markedly attenuated EGFR downstream signaling and myocyte anoikis induced by Cat.G. Consistent with this effect of EGFR, high level expression of wild-type EGFR was sufficient to promote myocyte apoptosis. We also found that matrix metalloproteinase-dependent membrane shedding of heparin-binding EGF was involved in Cat.G signaling and that membrane type 1 matrix metalloproteinase activation may constitute a potential target that entails matrix metalloproteinase activation induced by Cat.G. The paradoxical proapoptotic effect of EGFR appeared to be dependent on protein tyrosine phosphatase SHP2 (Src homology domain 2-containing tyrosine phosphatase 2) activation and focal adhesion kinase downregulation. These results show that Cat.G-induced cardiomyocyte apoptosis involves an increase in EGFR-dependent activation of SHP2 that promotes focal adhesion kinase dephosphorylation and subsequent cardiomyocyte anoikis.

Protein kinase Cepsilon (PKCepsilon) and Src control PKCdelta activation loop phosphorylation in cardiomyocytes.

Protein kinase Cdelta (PKCdelta) is unusual among AGC kinases in that it does not require activation loop (Thr(505)) phosphorylation for catalytic competence. Nevertheless, Thr(505) phosphorylation has been implicated as a mechanism that influences PKCdelta activity. This study examines the controls of PKCdelta-Thr(505) phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCdelta autophosphorylation in the "priming" maturational PKCdelta-Thr(505) phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCdelta-Thr(505) phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the alpha(1)-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCepsilon overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCdelta-Thr(505) autophosphorylation and PKCdelta-Thr(505) phosphorylation in trans by PKCepsilon. Our studies show that adenovirus-mediated PKCepsilon overexpression leads to an increase in PKCdelta-Thr(505) phosphorylation. However, this cannot be attributed to an effect of PKCepsilon to function as a direct PKCdelta-Thr(505) kinase, since the PKCepsilon-dependent increase in PKCdelta-Thr(505) phosphorylation is accompanied by (and dependent upon) increased PKCdelta phosphorylation at Tyr(311) and Tyr(332). Further studies implicate Src in this mechanism, showing that 1) PKCepsilon overexpression increases PKCdelta-Thr(505) phosphorylation in cardiomyocytes and Src(+) cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCdelta-Tyr(311)/Tyr(332) phosphorylation), and 2) in vitro PKCdelta-Thr(505) autophosphorylation is augmented in assays performed with Src (which promotes PKCdelta-Tyr(311)/Tyr(332) phosphorylation). Collectively, these results identify a novel PKCdelta-Thr(505) autophosphorylation mechanism that is triggered by PKCepsilon overexpression and involves Src-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation.