Effects of bisindolylmaleimide PKC inhibitors on p90 RSK activity in vitro and in adult ventricular myocytes

Self Cite

103

113

Abstract-The protein kinase D (PKD) family is a recent addition to the calcium/calmodulin-dependent protein kinase group of serine/threonine kinases, within the protein kinase complement of the mammalian genome. Relative to their alphabetically superior cousins in the AGC group of kinases, namely the various isoforms of protein kinase A, protein kinase B/Akt, and protein kinase C, PKD family members have to date received limited attention from cardiovascular investigators. Nevertheless, increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system, particularly in the regulation of myocardial contraction, hypertrophy and remodeling. This review provides a primer on PKD signaling, using information gained from studies in multiple cell types, and discusses recent data that suggest novel functions for PKD-mediated pathways in the heart and the circulation.

Section: Expression and Regulationmentioning

confidence: 86%

Section: Expression and Regulationmentioning

confidence: 99%

Protein Kinase D in the Cardiovascular System

Avkiran

Rowland

Cuello

et al. 2008

Self Cite

103

113

“…Figure 1D and 1E shows that NE induces a relatively modest increase in AKT phosphorylation (relative to the considerably more robust response triggered by heregulin or H 2 O 2 ) and that NE-dependent AKT activation is markedly augmented (approaching levels in heregulin-or H 2 O 2 -treated cultures) when cultures are pretreated with GF109203X, but not Go6976. Figure 2A shows that NE-dependent AKT activation is not influenced by U0126, effectively excluding a potential regulatory role for ribosomal S6 kinase (a GF109203X-sensitive enzyme and downstream target of the ␣ 1 -AR/nPKC signaling pathway 9 ). The effect of NEϩGF109203X to increase AKT phosphorylation is sustained for at least 30 minutes; GF109203X alone does not significantly alter basal AKT phosphorylation ( Figure 1E).…”

Section: Methodsmentioning

confidence: 99%

α ₁ -Adrenergic Receptors Activate AKT via a Pyk2/PDK-1 Pathway That Is Tonically Inhibited by Novel Protein Kinase C Isoforms in Cardiomyocytes

Guo

Sabri

Elouardighi

et al. 2006

Abstract-AKT is a potent antiapoptotic kinase, but its role in the cardioprotective actions of ␣ 1 -adrenergic receptors (ARs) remains uncertain, because ␣ 1 -ARs typically induce little-to-no AKT activation in most cardiomyocyte models. This study identifies a prominent ␣ 1 -AR-dependent AKT activation pathway that is under tonic inhibitory control by novel protein kinase Cs (nPKCs) in neonatal rat cardiomyocyte cultures. We also implicate Pyk2, Pyk2 complex formation with PDK-1 and paxillin, and increased PDK-1-Y373/376 phosphorylation as the mechanism that links ␣ 1 -AR activation to increased AKT phosphorylation. nPKCs (which are prominent ␣ 1 -AR effectors) interfere with this ␣ 1 -AR-dependent AKT activation by blocking Pyk2/PDK-1/paxillin complex formation and PDK-1-Y373/376 phosphorylation. Additional studies used an adenoviral-mediated overexpression strategy to show that Pyk2 exerts dual controls on antiapoptotic PDK-1/AKT and proapoptotic c-Jun N-terminal kinase (JNK) pathways. Although the high nPKC activity of most cardiomyocyte models favors Pyk2 signaling to JNK (and cardiac apoptosis), the cardioprotective actions of Pyk2 through the PDK-1/AKT pathway are exposed when PKC or JNK activation is prevented. Collectively, these studies identify JNK and AKT as functionally distinct downstream components of the ␣ 1 -AR/Pyk2 signaling pathway. We also implicate nPKCs as molecular switches that control the balance of signaling via proapoptotic JNK and antiapoptotic PDK-1/AKT pathways, exposing a novel mechanism for nPKC-dependent regulation of cardiac hypertrophy and failure. Key Words:KT is a potent antiapoptotic serine/threonine kinase that has been implicated in growth regulatory pathways in the heart. AKT is expressed as 3 isoforms (AKT1, AKT2, and AKT3) that share a similar N-terminal pleckstrin homology and C-terminal catalytic domain structure. AKT is stimulated by phosphatidylinositol 3-kinase, the enzyme that converts phosphatidylinositol 4,5-biphosphate to phosphatidylinositol 1,4,5-trisphosphate, a lipid that anchors AKT (through its pleckstrin homology domain) to the plasma membrane. Membrane-targeted AKT is then sequentially phosphorylated at highly conserved activation loop (T308) and C-terminal hydrophobic domain (S473) phosphorylation motifs. AKT-T308 phosphorylation has been attributed to 3-phosphoinositide-dependent protein kinase-1 (PDK-1), whereas the controls of AKT-S473 phosphorylation in any particular cell type remain controversial; many enzymes with S473 kinase activity (as well as a specific AKT-S473 phosphatase) have been identified, presumably evolving to allow for stimulus-and/or cell-specific regulatory controls. 1,2 Once activated, AKT phosphorylates a broad range of cellular substrates that are implicated in the control of cell proliferation, survival, and differentiation.AKT has attracted considerable interest as a therapeutic target for cardiac disorders because it provides meaningful cardioprotection in both in vitro models of cardiomyocyte apoptosis and in vivo experi...

“…7 Briefly, bacterial expression vectors (pGEX-KG or pGEX-3X) encoding aa 516-815, 516-630, 625-747, 748-815 or 625-815) of human NHE1 (the latter with or without the indicated Ser/Ala mutations), Nterminally linked to GST, were transformed into BL21 strain of Escherichia coli. Cultures were grown to sub-log phase and induced with 0.5 mmol/L isopropyl-β-D-thiogalactopyranoside.…”

Section: : Supplementary Materials and Methodsmentioning

confidence: 99%

“…2 Although basal activity of the sarcolemmal NHE is low under physiological conditions, 2 increased exchanger activity may mediate inotropic responses to neurohormonal stimuli, such as endothelin-1, 3 angiotensin II, 4 and ␣ 1 -adrenoceptor agonists, 5 principally through NHE1-mediated increases in intracellular sodium. 6,7 The regulation of NHE1 activity in such settings involves modification of the intracellular carboxyl-terminal regulatory domain of the exchanger, either by the binding of accessory proteins (such as calcineurin homologous protein, 8 carbonic anhydrase II, 9 and calmodulin [CaM] 10 ) and/or by phosphorylation by protein kinases (such as the extracellular signal-regulated kinases [ERKs], 11 p90 ribosomal S6 kinase [RSK], 12,13 Rho-associated kinase [p160-ROCK)], 14 and p38 mitogen-activated protein kinase [p38-MAPK] 15 ). Although the majority of studies have linked protein kinase-mediated phosphorylation of the NHE1 carboxyl-terminal regulatory domain to the upregulation of NHE1 activity, [11][12][13][14][15] there are reports that certain protein kinase pathways can inhibit NHE1.…”

Section: T He Namentioning

confidence: 99%

Protein Kinase B/Akt Phosphorylates and Inhibits the Cardiac Na ^+/ H ⁺ Exchanger NHE1

Snabaitis

Cuello

Avkiran

2008

Self Cite

110

Abstract-Sarcolemmal Naϩ /H ϩ exchanger (NHE) activity is mediated by NHE isoform 1 (NHE1), which is subject to regulation by protein kinases. Our objectives were to determine whether NHE1 is phosphorylated by protein kinase B (PKB), identify any pertinent phosphorylation site(s), and delineate the functional consequences of such phosphorylation. Active PKB␣ phosphorylated in vitro a glutathione S-transferase (GST)-NHE1 fusion protein comprising amino acids 516 to 815 of the NHE1 carboxyl-terminal regulatory domain. PKB␣-mediated phosphorylation of GST-NHE1 fusion proteins containing overlapping segments of this region localized the targeted residues to the carboxyl-terminal 190 amino acids (625 to 815) of NHE1. Mass spectrometry and phosphorylation analysis of mutated (Ser3 Ala) GST-NHE1 fusion proteins revealed that PKB␣-mediated phosphorylation of NHE1 occurred principally at Ser648. Far-Western assays demonstrated that PKB␣-mediated Ser648 phosphorylation abrogated calcium-activated calmodulin (CaM) binding to the regulatory domain of NHE1. In adult rat ventricular myocytes, adenovirus-mediated expression of myristoylated PKB␣ (myr-PKB␣) increased cellular PKB activity, as confirmed by increased glycogen synthase kinase 3␤ phosphorylation. Heterologously expressed myr-PKB␣ was present in the sarcolemma, colocalized with NHE1 at the intercalated disc regions, increased NHE1 phosphorylation, and reduced NHE1 activity following intracellular acidosis. Conversely, pharmacological inhibition of endogenous PKB increased NHE1 activity following intracellular acidosis. Our data suggest that NHE1 is a novel PKB substrate and that its PKB-mediated phosphorylation at Ser648 inhibits sarcolemmal NHE activity during intracellular acidosis, most likely by interfering with CaM binding and reducing affinity for intracellular H 15 ). Although the majority of studies have linked protein kinase-mediated phosphorylation of the NHE1 carboxyl-terminal regulatory domain to the upregulation of NHE1 activity, [11][12][13][14][15] there are reports that certain protein kinase pathways can inhibit NHE1. 16,17 Furthermore, although the functionally important phosphorylation sites in NHE1 have been established for some kinases (eg, RSK 12,18 ), they remain to be confirmed for many others.Interestingly, the carboxyl-terminal regulatory domain of NHE1 contains 3 putative phosphorylation sites that conform to the optimal protein kinase B (PKB) target motif (RxRxxS/ T 19 ), which suggests a potential regulatory interaction between PKB and NHE1. In the heart, 3 isoforms of PKB (PKB␣/Akt1, PKB␤/Akt2, and PKB␥/Akt3) are differentially expressed (␣ϭ␤Ͼ␥), and each constitutes a phosphoprotein of Ϸ57 kDa that consists of an amino-terminal pleckstrin homology (PH) domain, 20,21 a central kinase domain, 22,23 and a carboxyl-terminal hydrophobic motif. 24 The PH domain is crucial for the activation of PKB as it facilitates the phosphatidylinositol 3,4,5-triphosphate-dependent translocation of PKB to the inner surface of the cell membrane, where dua...