Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKC␦ actions in cardiomyocytes. Rather, tyrosine phosphorylation regulates PKC␦ kinase activity. PKC␦ is recovered from the soluble fraction of H 2 O 2 -treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKC␦ phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKC␦ substrate specificity. The stimulus-specific modes for PKC␦ signaling identified in this study allow for distinct PKC␦-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes. Protein kinase C (PKC)1 comprises a multigene family of at least 10 structurally distinct phospholipid-dependent serinethreonine kinases that regulate cardiac contraction, play a role in ischemic preconditioning, and contribute to the pathogenesis of cardiac hypertrophy and heart failure (1, 2). PKC isoforms are single polypeptide chains with structurally homologous C-terminal catalytic domains and more variable N-terminal regulatory domains. This diverse group of enzymes is subdivided into three distinct subfamilies based upon structural differences in their N-terminal regulatory domain that confer distinct patterns of cofactor activation. Conventional PKC isoforms (cPKCs; ␣, I, II, ␥) contain an autoinhibitory pseudosubstrate domain followed by membrane-targeting C1 and C2 domains that are regulated by diacylglycerol (DAG) and calcium, respectively. Novel PKCs (nPKCs; ␦, ⑀, , and ) lack a calcium-binding C2 domain and are maximally activated by DAG and phorbol ester, in the absence of calcium. Atypical PKCs (aPKCs; and /) are regulated by lipids, but are not activated by second messengers such as calcium and DAG. Current models of PKC isoform activation in the heart have focused largely on the conformational changes induced by cofactor interactions with N-terminal membrane-targeting modules that anchor the enzyme to membranes, expel the autoinhibitory pseudosubstrate domain from the substrate-binding pocket, and thereby relieve autoinhibition. According to this model, individual PKC isoforms elicit distinct (and occasionally functionally opposing) cellular responses as a result of cofactorinduced compartmentation to distinct membrane subdomains, in close proximity to their unique sets of target protein substrates (1).Recent studies identify an additional mechanism for PKC regulation via sequential phosphorylations on a conserved threonine in the activation loop and two conserved serine/threonines in turn and hydrophobic motifs in the C terminus (3). For cPKCs, these phosphorylation even...
Extracellular ligands stimulate cardiac growth and differentiation by activating a network of protein kinases that phosphorylate transcription factors and alter gene expression. Many of these mechanisms are resurrected in the damaged or failing heart in an attempt to compensate for contractile dysfunction. Our previous studies focused on the cellular actions of thrombin, a serine protease that is generated at sites of cardiac injury and proteolytically activates protease-activated receptor-1 (PAR-1), 3 a G protein-coupled receptor that activates a spectrum of effectors that contribute to cardiac fibroblast proliferation and cardiomyocyte hypertrophy (1). Certain aspects of PAR-1 signaling are cell-specific; PAR-1 activates ERK primarily via an epidermal growth factor receptor (EGFR) transactivation pathway in cardiac fibroblasts and a distinct pathway that does not require EGFR kinase activity in cardiomyocytes. Of note, the PAR-1 signaling pathway in cardiomyocytes triggers a form of cellular remodeling that resembles the changes observed in dilated cardiomyopathies (with pronounced cell elongation and relatively little increased cell width). This hypertrophic phenotype is morphologically distinct from the concentric hypertrophy induced by ␣ 1 -AR agonists such as norepinephrine (NE) or P. multocida toxin (PMT, a direct G␣ q agonist); NE and PMT induce very pronounced increases in overall cell size in association with enhanced sarcomeric organization and atrial natriuretic factor expression (2). cAMP response element-binding protein (CREB) is a bZip transcription factor that forms homo-or heterodimers with itself or with other CREB/ATF family members and binds to specific DNA elements (termed cAMP response elements or CREs) within the regulatory regions of CREB target genes. CREB has been implicated in the maintenance of normal ventricular structure and function; cardiac-specific overexpression of dominant-negative CREB leads to dilated cardiomyopathy and interstitial fibrosis (3). CREB also has been implicated in the electrophysiological remodeling that accompanies pacinginduced cardiac memory in dogs (4). CREB is regulated via phosphorylation at Ser 133 , which activates CREB-dependent gene transcription by recruiting a coactivator (CREB-binding protein, or CBP) to the promoters of CREB target genes.* This work was supported, in whole or in part, by National Institutes of Health Grants HL77860, HL-67101, and HL-28958. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Protease-activated receptor (PAR)-4 is a low affinity thrombin receptor with slow activation and desensitization kinetics relative to PAR-1. This study provides novel evidence that cardiomyocytes express functional PAR-4 whose signaling phenotype is distinct from PAR-1 in cardiomyocytes. AYPGKF, a modified PAR-4 agonist with increased potency at PAR-4, activates p38-mitogenactivated protein kinase but is a weak activator of phospholipase C, extracellular signal-regulated kinase, and cardiomyocyte hypertrophy; AYPGKF and thrombin, but not the PAR-1 agonist SFLLRN, activate Src. The observation that AYPGKF and thrombin activate Src in cardiomyocytes cultured from PAR-1 ؊/؊ mice establishes that Src activation is via PAR-4 (and not PAR-1) in cardiomyocytes. Further studies implicate Src and epidermal growth factor receptor (EGFR) kinase activity in the PAR-4-dependent p38-mitogen-activated protein kinase signaling pathway. Thrombin phosphorylates EGFRs and ErbB2 via a PP1-sensitive pathway in PAR-1 ؊/؊ cells that stably overexpress PAR-4; the Src-mediated pathway for EGFR/ErbB2 transactivation underlies the protracted phases of thrombin-dependent extracellular signal-regulated kinase activation in PAR-1 ؊/؊ cells that overexpress PAR-4 and in cardiomyocytes. These studies identify a unique signaling phenotype for PAR-4 (relative to other cardiomyocyte G protein-coupled receptors) that is predicted to contribute to cardiac remodeling and influence the functional outcome at sites of cardiac inflammation.
Cathepsin G is a neutrophil-derived serine protease that contributes to tissue damage at sites of inflammation. The actions of cathepsin G are reported to be mediated by protease-activated receptor (PAR)-4 (a thrombin receptor) in human platelets. This study provides the first evidence that cathepsin G promotes inositol 1,4,5-trisphosphate accumulation, activates ERK, p38 MAPK, and AKT, and decreases contractile function in cardiomyocytes. Because some cathepsin G responses mimic cardiomyocyte activation by thrombin, a role for PARs was considered. Cathepsin G markedly activates phospholipase C and p38 MAPK in cardiomyocytes from PAR-1 ؊/؊ mice, but it fails to activate phospholipase C, ERK, p38 MAPK, or AKT in PAR-1-or PAR-4-expressing PAR-1 ؊/؊ fibroblasts (which display robust responses to thrombin). These results argue that PAR-1 does not mediate the actions of cathepsin G in cardiomyocytes, and neither PAR-1 nor PAR-4 mediates the actions of cathepsin G in fibroblasts. Of note, prolonged incubation of cardiomyocytes with cathepsin G results in the activation of caspase-3, cleavage of FAK and AKT, sarcomeric disassembly, cell rounding, cell detachment from underlying matrix, and morphologic features of apoptosis. Inhibition of Src family kinases or caspases (with PP1 or benzyloxycarbonyl-VAD-fluoromethyl ketone, respectively) delays FAK and AKT cleavage and cardiomyocyte detachment from substrate. Collectively, these studies describe novel cardiac actions of cathepsin G that do not require PARs and are predicted to assume functional importance at sites of interstitial inflammation in the heart.
Caveolin-3 the muscle-specific caveolin isoform, acts like the more ubiquitously expressed caveolin-1 to sculpt caveolae, specialized membrane microdomains that serve as platforms to organize signal transduction pathways. Caveolin-2 is a structurally related isoform that alone does not drive caveolae biogenesis; rather, caveolin-2 cooperates with caveolin-1 to form caveolae in nonmuscle cells. Although caveolin-2 might be expected to interact in an fashion analogous to that of caveolin-3, it generally has not been detected in cardiomyocytes. This study shows that caveolin-2 and caveolin-3 are detected at low levels in ventricular myocardium and increase dramatically with age or when neonatal cardiomyocytes are placed in culture. In contrast, flotillins (caveolin functional homologs) are expressed at relatively constant levels in these preparations. In neonatal cardiac cultures, caveolin-2 and -3 expression is not influenced by thyroid hormone (a postnatal regulator of other cardiac gene products). The further evidence that caveolin-2 coimmunoprecipitates with caveolin-3 and floats with caveolin-3 by isopycnic centrifugation in cardiomyocyte cultures suggests that caveolin-2 may play a role in caveolae biogenesis and influence cardiac muscle physiology.
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