Protein kinase C-ζ modulates thromboxane A2-mediated apoptosis in adult ventricular myocytes via Akt

2002 ◽  
Vol 282 (1) ◽  
pp. H320-H327 ◽  
Author(s):  
Yukitaka Shizukuda ◽  
Peter M. Buttrick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Adult Rat ◽  
Kinase C ◽  
Adult Cardiac Myocytes ◽  
Pkc Ζ

We hypothesized that thromboxane A2 (TxA2) receptor stimulation directly induces apoptosis in adult cardiac myocytes. To investigate this, we exposed cultured adult rat ventricular myocytes (ARVM) to a TxA2 mimetic [1S-[1α,2α(Z),3β(1E,3S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) for 24 h. Stimulation with I-BOP induced apoptosis in a dose-dependent manner and was completely prevented by a TxA2 receptor antagonist, SQ-29548. We further investigated the role of protein kinase C (PKC) in this process. TxA2 stimulation resulted in membrane translocation of PKC-ζ but not PKC-α, -βII, -δ, and -ε at 3 min and 1 h. The activation of PKC-ζ by I-BOP was confirmed using an immune complex kinase assay. Treatment of ARVM with a cell-permeable PKC-ζ pseudosubstrate peptide (ζ-PS) significantly attenuated apoptosis by I-BOP. In addition, I-BOP treatment decreased baseline Akt activity and its decrease was reversed by treatment with ζ-PS. The inhibition of phosphatidylinositol 3-kinase upstream of Akt by wortmannin or LY-294002 abolished the antiapoptotic effect of ζ-PS. Therefore, our results suggest that the activation of PKC-ζ modulates TxA2 receptor-mediated apoptosis at least, in part, through Akt activity in adult cardiac myocytes.

Protein kinase C-δ modulates apoptosis induced by hyperglycemia in adult ventricular myocytes

2002 ◽  
Vol 282 (5) ◽  
pp. H1625-H1634 ◽  
Author(s):  
Yukitaka Shizukuda ◽  
Mary E. Reyland ◽  
Peter M. Buttrick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Kinase Assay ◽  
Adult Rat ◽  
Kinase C ◽  
Inhibitor Peptide ◽  
Labeling Method ◽  
Immune Complex Kinase Assay

We evaluated the direct effect of hyperglycemia on apoptosis of adult rat ventricular myocytes (ARVM) in vitro. Hyperglycemia (16.5 mM) for 24 h increased apoptosis by greater than threefold (48.2 ± 4.4%, by the TdT-mediated dUTP nick-end labeling method) compared with baseline (14.7 ± 2.5%). Hyperosmolarity with mannitol (11.0 mM) in the presence of 5.5 mM glucose also increased apoptosis by approximately twofold of baseline. Both glucose and mannitol treatment resulted in the membrane translocation of protein kinase C (PKC)-δ, and the activation of PKC-δ was confirmed by immune complex kinase assay. PKC-δ-specific translocation inhibitor peptide (δV1-1) attenuated only apoptosis induced by hyperglycemia but not by mannitol. A PKC-ɛ-specific translocation inhibitor peptide (ɛV1-1) affected neither type of apoptosis. Moderate overexpression of PKC-δ by adenovirus gene transfer prevented the antiapoptotic effect of δV1-1. Furthermore, δV1-1 attenuated the production of reactive oxygen species (ROS) by glucose. Taken together, our results indicate that increased ROS production regulated by PKC-δ is in part responsible for the induction of apoptosis by hyperglycemia and that apoptosis by hyperglycemia is mechanistically different from that by hyperosmolarity.

14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation

2000 ◽  
Vol 345 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Paulus C. J. VAN DER HOEVEN ◽  
José C. M. VAN DER WAL ◽  
Paula RUURS ◽  
Marc C. M. VAN DIJK ◽  
Wim J. VAN BLITTERSWIJK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Dependent Manner ◽  
Cos Cells ◽  
Kinase C ◽  
Pkc Ζ ◽  
Co Precipitation

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.

Calmodulin kinase II and protein kinase C mediate the effect of increased intracellular calcium to augment late sodium current in rabbit ventricular myocytes

2012 ◽  
Vol 302 (8) ◽  
pp. C1141-C1151 ◽  
Author(s):  
Jihua Ma ◽  
Antao Luo ◽  
Lin Wu ◽  
Wei Wan ◽  
Peihua Zhang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Dependent Manner ◽  
Open Probability ◽  
Late Sodium Current ◽  
Kinase C ◽  
Open Time ◽  
Rabbit Ventricular Myocytes

An increase in intracellular Ca2+ concentration ([Ca2+]i) augments late sodium current ( INa.L) in cardiomyocytes. This study tests the hypothesis that both Ca2+-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) mediate the effect of increased [Ca2+]i to increase INa.L. Whole cell and open cell-attached patch clamp techniques were used to record INa.L in rabbit ventricular myocytes dialyzed with solutions containing various concentrations of [Ca2+]i. Dialysis of cells with [Ca2+]i from 0.1 to 0.3, 0.6, and 1.0 μM increased INa.L in a concentration-dependent manner from 0.221 ± 0.038 to 0.554 ± 0.045 pA/pF ( n = 10, P < 0.01) and was associated with an increase in mean Na+ channel open probability and prolongation of channel mean open-time ( n = 7, P < 0.01). In the presence of 0.6 μM [Ca2+]i, KN-93 (10 μM) and bisindolylmaleimide (BIM, 2 μM) decreased INa.L by 45.2 and 54.8%, respectively. The effects of KN-93 and autocamtide-2-related inhibitory peptide II (2 μM) were not different. A combination of KN-93 and BIM completely reversed the increase in INa.L as well as the Ca2+-induced changes in Na+ channel mean open probability and mean open-time induced by 0.6 μM [Ca2+]i. Phorbol myristoyl acetate increased INa.L in myocytes dialyzed with 0.1 μM [Ca2+]i; the effect was abolished by Gö-6976. In summary, both CaMKII and PKC are involved in [Ca2+]i-mediated augmentation of INa.L in ventricular myocytes. Inhibition of CaMKII and/or PKC pathways may be a therapeutic target to reduce myocardial dysfunction and cardiac arrhythmias caused by calcium overload.

Protein kinase C activation by acidic proteins including 14-3-3

2000 ◽  
Vol 347 (3) ◽  
pp. 781-785 ◽  
Author(s):  
Paulus C. J. VAN DER HOEVEN ◽  
José C. M. VAN DER WAL ◽  
Paula RUURS ◽  
Wim J. VAN BLITTERSWIJK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Dose Dependent Fashion ◽  
Acidic Proteins ◽  
Pkc Ζ

14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-ζ and facilitate coupling of PKC-ζ to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-ζ activity. Using recombinant 14-3-3 proteins and purified PKC-ζ in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-ζ activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-ζ by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-ζ with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-ζ. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-ζ activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-ζ, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.

scholarly journals Nesfatin-1 Suppresses Cardiac L-type Ca2+ Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway

2015 ◽  
Vol 36 (2) ◽  
pp. 555-568 ◽  
Author(s):  
Jiaoqian Ying ◽  
Yuan Zhang ◽  
Shan Gong ◽  
Zhigang Chang ◽  
Xiaofeng Zhou ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Dependent Manner ◽  
The Novel ◽  
Inotropic Effects ◽  
Kinase C ◽  
Melanocortin 4 Receptor ◽  
Dose Dependent ◽  
Ca2 Channels

Background/Aims: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. Methods: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca2+ currents (ICa,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased ICa,L in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-β-S or anti-Gβ antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on ICa,L. Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca2+-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKCθ), and PKCθ inhibition abolished the decrease in ICa,L induced by NF-1. Conclusion: These data suggest that NF-1 suppresses L-type Ca2+ channels via the MC4-R that couples sequentially to the βγ subunits of Gi/o-protein and the novel PKCθ isoform in adult ventricular myocytes.

Do adult rat ventricular myocytes express protein kinase C-alpha?

1997 ◽  
Vol 272 (5) ◽  
pp. H2485-H2491 ◽  
Author(s):  
V. Rybin ◽  
S. F. Steinberg
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Chromatographic Purification ◽  
Adult Rat ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

Although calcium-insensitive protein kinase C (PKC) isoforms (PKC-epsilon and PKC-delta) are consistently detected in adult ventricular myocytes, the evidence that adult ventricular myocytes also express calcium-sensitive PKC-alpha is inconsistent. The current study used four different anti-PKC-alpha-antibodies to resolve some of the uncertainties regarding the immunodetection of PKC-alpha in adult ventricular myocytes. Three of the antibodies used in this study barely (GIBCO-BRL) or rather faintly (Transduction Laboratories and Seikagaku America) recognize PKC-alpha in crude preparations from adult ventricular myocytes. Although each of these antibodies recognizes a prominent 80-kDa band, which is similar in size to PKC-alpha, this represents nonspecific immunoreactivity and should not be confused with PKC-alpha. This conclusion is based on peptide-blocking experiments (GIBCO-BRL), the absence of the requisite sensitivity to calcium- and phorbol 12-myristate 13-acetate-induced translocation (Seikagaku America and Transduction Laboratories), and/or the failure to copurify with PKC-alpha on DEAE-Sephacel chromatography. Nevertheless, an antibody from Upstate Biotechnology clearly recognizes PKC-alpha and not other unrelated nonspecific immunoreactive species in crude preparations from adult ventricular myocytes. Each of the antisera used in this study could detect PKC-alpha immunoreactivity following chromatographic purification of the samples to enrich for PKC-alpha and remove nonspecific immunoreactive proteins. These results suggest that PKC-alpha is expressed by adult ventricular myocytes and argue that differences in the sensitivity and/or specificity of available antisera contribute to at least some of the confusion regarding PKC-alpha expression in adult ventricular myocytes.

scholarly journals The Positive Inotropic and Calcium-Mobilizing Effects of Growth Hormone-Releasing Peptides on Rat Heart

Endocrinology ◽  
2003 ◽  
Vol 144 (11) ◽  
pp. 5050-5057 ◽  
Author(s):  
Xiang-Bin Xu ◽  
Ji-Min Cao ◽  
Jing-Jiang Pang ◽  
Rong-Kun Xu ◽  
Chao Ni ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Phase 1 ◽  
Dependent Manner ◽  
Phase 2 ◽  
Perfused Heart ◽  
Voltage Gated ◽  
Kinase C ◽  
Phase Phase

Abstract GH-releasing peptides (GHRP) are synthetic peptides exerting GH-dependent or GH-independent effects via GH secretagogue receptor on many organs, including the heart. The underlying mechanisms of the cardiotropic properties of GHRP are poorly understood. This study investigates these effects of four GHRP in isolated perfused heart preparations and isolated neonatal and adult ventricular myocytes. The calcium response of cardiocytes to GHRP was visualized using confocal microscopy. All tested GHRP facilitated both ventricular contraction and relaxation in a dose-dependent manner, moderately decreasing coronary flow, but not modifying heart rate. GHRP induced a biphasic increase in intracellular free Ca2+ of the cardiocytes, consisting of a transient phase (phase 1), followed by a plateau phase (phase 2). Phase 1 was abolished by pretreatment with thapsigargin, a Ca2+-adenosine triphosphatase inhibitor of the sarcoplasmic reticulum. The phase 2 response was eliminated by removing extracellular free Ca2+, by verapamil, a voltage-gated Ca2+ channel blocker, or by 24-h pretreatment with phorbol 12-myristate 13-acetate, down-regulating protein kinase C. In isolated (denervated) heart, GHRP have a direct cardiotropic, without chronotropic, effect. GHRP elevate myocardial intracellular free Ca2+ through activating Ca2+ influx via voltage-gated Ca2+ channels and triggering Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores. Protein kinase C mediates the GHRP-induced Ca2+ influx, but not Ca2+ release. These finding support a number of roles for GHRP in the cardiovascular system.

scholarly journals Protein Kinase C-ζ Phosphorylates Insulin Receptor Substrate-1, -3, and -4 But Not -2: Isoform Specific Determinants of Specificity in Insulin Signaling

Endocrinology ◽  
2008 ◽  
Vol 149 (5) ◽  
pp. 2451-2458 ◽  
Author(s):  
Sihoon Lee ◽  
Edward G. Lynn ◽  
Jeong-a Kim ◽  
Michael J. Quon
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Insulin Receptor ◽  
Negative Feedback ◽  
Insulin Signaling ◽  
Kinase Assay ◽  
Insulin Receptor Substrate ◽  
Kinase C ◽  
Pi3k Activity ◽  
Pkc Ζ

Protein kinase C-ζ, a downstream effector of phosphatidylinositol 3-kinase (PI3K), phosphorylates insulin receptor substrate (IRS)-1 on serine residues impairing activation of PI3K in response to insulin. Because IRS-1 is upstream from PI3K, this represents a negative feedback mechanism that may contribute to signal specificity in insulin action. To determine whether similar feedback pathways exist for other IRS isoforms, we evaluated IRS-2, -3, and -4 as substrates for PKC-ζ. In an in vitro kinase assay, purified recombinant PKC-ζ phosphorylated IRS-1, -3 and -4 but not IRS-2. Similar results were obtained with an immune-complex kinase assay demonstrating that wild-type, but not kinase-deficient mutant PKC-ζ, phosphorylated IRS-1, -3, and -4 but not IRS-2. We evaluated functional consequences of serine phosphorylation of IRS isoforms by PKC-ζ in NIH-3T3IR cells cotransfected with epitope-tagged IRS proteins and either PKC-ζ or empty vector control. Insulin-stimulated IRS tyrosine phosphorylation was impaired by overepxression of PKC-ζ for IRS-1, -3, and -4 but not IRS-2. Significant insulin-stimulated increases in PI3K activity was coimmunoprecipitated with all IRS isoforms. In cells overexpressing PKC-ζ there was marked inhibition of insulin-stimulated PI3K activity associated with IRS-1, -3 and -4 but not IRS-2. That is, PI3K activity associated with IRS-2 in response to insulin was similar in control cells and cells overexpressing PKC-ζ. We conclude that IRS-3 and -4 are novel substrates for PKC-ζ that may participate in a negative feedback pathway for insulin signaling similar to IRS-1. The inability of PKC-ζ to phosphorylate IRS-2 may help determine specific functional roles for IRS-2.

Sign in / Sign up

Export Citation Format

Share Document