Zoya Gertsberg

p66Shc Links α1-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 &agr;1-adrenergtic receptor (&agr;1-AR) pathway that requires the cooperative effects of protein kinase (PK)Cϵ and PKC&dgr; and leads to AKT-FOXO3a phosphorylation in cardiomyocytes. &agr;1-ARs promote p66Shc-YY239/240 phosphorylation via a ROS-dependent mechanism that is localized to caveolae and requires epidermal growth factor receptor (EGFR) and PKCϵ activity. &agr;1-ARs also increase p66Shc-S36 phosphorylation via an EGFR transactivation pathway involving PKC&dgr;. p66Shc links &agr;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 &agr;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 &agr;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.

Neuronal Serotonin Regulates Growth of the Intestinal Mucosa in Mice

BACKGROUND & AIMS The enteric abundance of serotonin (5-HT), its ability to promote proliferation of neural precursors, and reports that 5-HT antagonists affect crypt epithelial proliferation led us to investigate whether 5-HT affects growth and maintenance of the intestinal mucosa in mice. METHODS cMice that lack the serotonin re-uptake transporter (SERTKO mice) and wild-type mice were given injections of selective serotonin re-uptake inhibitors (gain-of-function models). We also analyzed mice that lack tryptophan hydroxylase-1 (TPH1KO mice, which lack mucosal but not neuronal 5-HT) and mice deficient in tryptophan hydroxylase-2 (TPH2KO mice, which lack neuronal but not mucosal 5-HT) (loss-of-function models). Wild-type and SERTKO mice were given ketanserin (an antagonist of the 5-HT receptor, 5-HT(2A)) or scopolamine (an antagonist of the muscarinic receptor). 5-HT(2A) receptors and choline acetyltransferase were localized by immunocytochemical analysis. RESULTS Growth of the mucosa and proliferation of mucosal cells were significantly greater in SERTKO mice and in mice given selective serotonin re-uptake inhibitors than in wild-type mice, but were diminished in TPH2KO (but not in TPH1KO) mice. Ketanserin and scopolamine each prevented the ability of SERT knockout or inhibition to increase mucosal growth and proliferation. Cholinergic submucosal neurons reacted with antibodies against 5-HT(2A). CONCLUSIONS 5-HT promotes growth and turnover of the intestinal mucosal epithelium. Surprisingly, these processes appear to be mediated by neuronal, rather than mucosal, 5-HT. The 5-HT(2A) receptor activates cholinergic neurons, which provide a muscarinic innervation to epithelial effectors.

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

Protein kinase Cδ (PKCδ) is unusual among AGC kinases in that it does not require activation loop (Thr505) phosphorylation for catalytic competence. Nevertheless, Thr505 phosphorylation has been implicated as a mechanism that influences PKCδ activity. This study examines the controls of PKCδ-Thr505 phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCδ autophosphorylation in the “priming” maturational PKCδ-Thr505 phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCδ-Thr505 phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the α1-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCϵ overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCδ-Thr505 autophosphorylation and PKCδ-Thr505 phosphorylation in trans by PKCϵ. Our studies show that adenovirus-mediated PKCϵ overexpression leads to an increase in PKCδ-Thr505 phosphorylation. However, this cannot be attributed to an effect of PKCϵ to function as a direct PKCδ-Thr505 kinase, since the PKCϵ-dependent increase in PKCδ-Thr505 phosphorylation is accompanied by (and dependent upon) increased PKCδ phosphorylation at Tyr311 and Tyr332. Further studies implicate Src in this mechanism, showing that 1) PKCϵ overexpression increases PKCδ-Thr505 phosphorylation in cardiomyocytes and Src+ cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCδ-Tyr311/Tyr332 phosphorylation), and 2) in vitro PKCδ-Thr505 autophosphorylation is augmented in assays performed with Src (which promotes PKCδ-Tyr311/Tyr332 phosphorylation). Collectively, these results identify a novel PKCδ-Thr505 autophosphorylation mechanism that is triggered by PKCϵ overexpression and involves Src-dependent PKCδ-Tyr311/Tyr332 phosphorylation.

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 Ser133 in cardiomyocytes. Although CREB-Ser133 phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser133 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-Ser133 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.

Distinct Signaling Functions for Shc Isoforms in the Heart

Thrombin activates protease-activated receptor-1 (PAR-1) and engages signaling pathways that influence the growth and survival of cardiomyocytes as well as extracellular matrix remodeling by cardiac fibroblasts. This study examines the role of Shc proteins in PAR-1-dependent signaling pathways that influence ventricular remodeling. We show that thrombin increases p46Shc/p52Shc phosphorylation at Tyr239/Tyr240 and Tyr317 (and p66Shc-Ser36 phosphorylation) via a pertussis toxin-insensitive epidermal growth factor receptor (EGFR) transactivation pathway in cardiac fibroblasts; p66Shc-Ser36 phosphorylation is via a MEK-dependent mechanism. In contrast, cardiac fibroblasts express β2-adrenergic receptors that activate ERK through a pertussis toxin-sensitive EGFR transactivation pathway that does not involve Shc isoforms or lead to p66Shc-Ser36 phosphorylation. In cardiomyocytes, thrombin triggers MEK-dependent p66Shc-Ser36 phosphorylation, but this is not via EGFR transactivation (or associated with Shc-Tyr239/Tyr240 and/or Tyr317 phosphorylation). Importantly, p66Shc protein expression is detected in neonatal, but not adult, cardiomyocytes; p66Shc expression is induced (via a mechanism that requires protein kinase C and MEK activity) by Pasteurella multocida toxin, a Gαq agonist that promotes cardiomyocyte hypertrophy. These results identify novel regulation of individual Shc isoforms in receptor-dependent pathways leading to cardiac hypertrophy and the transition to heart failure. The observations that p66Shc expression is induced by a Gαq agonist and that PAR-1 activation leads to p66Shc-Ser36 phosphorylation identifies p66Shc as a novel candidate hypertrophy-induced mediator of cardiomyocyte apoptosis and heart failure.

Protein Kinase D Isoforms Are Activated in an Agonist-specific Manner in Cardiomyocytes

Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α1-adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the α1-adrenergic receptor pathway that activates PKD1 and the thrombin- and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser133 and cTnI-Ser23/Ser24 phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser133 phosphorylation; there is no associated increase in cTnI-Ser23/Ser24 phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser133 phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.

Phorbol 12-Myristate 13-Acetate-dependent Protein Kinase Cδ-Tyr311 Phosphorylation in Cardiomyocyte Caveolae

Protein kinase Cδ (PKCδ) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCδ also is dynamically regulated through tyrosine phosphorylation in H2O2- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H2O2 activates Src and related Src-family kinases (SFKs), which function as dual PKCδ-Tyr311 and -Tyr332 kinases in vitro and contribute to H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCδ-Tyr311 phosphorylation, but this is not associated with SFK activation or PKCδ-Tyr332 phosphorylation. Rather, PMA increases PKCδ-Tyr311 phosphorylation by delivering PKCδ to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCδ-Tyr311 phosphorylation, without blocking H2O2-dependent PKCδ-Tyr311 phosphorylation. The enzyme that acts as a PKCδ-Tyr311 kinase without increasing PKCδ phosphorylation at Tyr332 in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCδ-Tyr311 kinase, PMA-dependent PKCδ-Tyr311 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 PKCδ to caveolae and increase PKCδ-Tyr311 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 PKCδ exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCδ localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.

Protein kinase C-δ regulates the subcellular localization of Shc in H2O2-treated cardiomyocytes

Protein kinase C-δ (PKCδ) exerts important cardiac actions as a lipid-regulated kinase. There is limited evidence that PKCδ also might exert an additional kinase-independent action as a regulator of the subcellular compartmentalization of binding partners such as Shc (Src homologous and collagen), a family of adapter proteins that play key roles in growth regulation and oxidative stress responses. This study shows that native PKCδ forms complexes with endogenous Shc proteins in H(2)O(2)-treated cardiomyocytes; H(2)O(2) treatment also leads to the accumulation of PKCδ and Shc in a detergent-insoluble cytoskeletal fraction and in mitochondria. H(2)O(2)-dependent recruitment of Shc isoforms to cytoskeletal and mitochondrial fractions is amplified by wild-type-PKCδ overexpression, consistent with the notion that PKCδ acts as a signal-regulated scaffold to anchor Shc in specific subcellular compartments. However, overexpression studies with kinase-dead (KD)-PKCδ-K376R (an ATP-binding mutant of PKCδ that lacks catalytic activity) are less informative, since KD-PKCδ-K376R aberrantly localizes as a constitutively tyrosine-phosphorylated enzyme to detergent-insoluble and mitochondrial fractions of resting cardiomyocytes; relatively little KD-PKCδ-K376R remains in the cytosolic fraction. The aberrant localization and tyrosine phosphorylation patterns for KD-PKCδ-K376R do not phenocopy the properties of native PKCδ, even in cells chronically treated with GF109203X to inhibit PKCδ activity. Hence, while KD-PKCδ-K376R overexpression increases Shc localization to the detergent-insoluble and mitochondrial fractions, the significance of these results is uncertain. Our studies suggest that experiments using KD-PKCδ-K376R overexpression as a strategy to competitively inhibit the kinase-dependent actions of native PKCδ or to expose the kinase-independent scaffolding functions of PKCδ should be interpreted with caution.