Therapeutic target validation of protein kinase C(PKC)-ζ for asthma using a mouse model

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.

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Disruption of adenylyl cyclase type V does not rescue the phenotype of cardiac-specific overexpression of Gαq protein-induced cardiomyopathy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01208.2009 ◽

2010 ◽

Vol 299 (5) ◽

pp. H1459-H1467 ◽

Cited By ~ 9

Author(s):

Valeriy Timofeyev ◽

Cliff A. Porter ◽

Dipika Tuteja ◽

Hong Qiu ◽

Ning Li ◽

...

Keyword(s):

Heart Failure ◽

Protein Kinase C ◽

Protein Kinase ◽

Mouse Model ◽

Transgenic Mice ◽

Cardiac Function ◽

Adenylyl Cyclase ◽

Electrical Remodeling ◽

Kinase C ◽

Progressive Heart Failure

Adenylyl cyclase (AC) is the principal effector molecule in the β-adrenergic receptor pathway. ACV and ACVI are the two predominant isoforms in mammalian cardiac myocytes. The disparate roles among AC isoforms in cardiac hypertrophy and progression to heart failure have been under intense investigation. Specifically, the salutary effects resulting from the disruption of ACV have been established in multiple models of cardiomyopathy. It has been proposed that a continual activation of ACV through elevated levels of protein kinase C could play an integral role in mediating a hypertrophic response leading to progressive heart failure. Elevated protein kinase C is a common finding in heart failure and was demonstrated in murine cardiomyopathy from cardiac-specific overexpression of Gαq protein. Here we assessed whether the disruption of ACV expression can improve cardiac function, limit electrophysiological remodeling, or improve survival in the Gαq mouse model of heart failure. We directly tested the effects of gene-targeted disruption of ACV in transgenic mice with cardiac-specific overexpression of Gαq protein using multiple techniques to assess the survival, cardiac function, as well as structural and electrical remodeling. Surprisingly, in contrast to other models of cardiomyopathy, ACV disruption did not improve survival or cardiac function, limit cardiac chamber dilation, halt hypertrophy, or prevent electrical remodeling in Gαq transgenic mice. In conclusion, unlike other established models of cardiomyopathy, disrupting ACV expression in the Gαq mouse model is insufficient to overcome several parallel pathophysiological processes leading to progressive heart failure.

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Essential role of protein kinase C ζ in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP

The Journal of Cell Biology ◽

10.1083/jcb.200607019 ◽

2007 ◽

Vol 176 (7) ◽

pp. 1049-1060 ◽

Cited By ~ 20

Author(s):

Kageaki Kuribayashi ◽

Kiminori Nakamura ◽

Maki Tanaka ◽

Tsutomu Sato ◽

Junji Kato ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Morphological Changes ◽

Specific Inhibitor ◽

Squamous Carcinoma ◽

Inflammatory Cells ◽

Dominant Negative ◽

Chemotactic Peptide ◽

Kinase C ◽

Pkc Ζ

Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) ζ was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCζ in SASH1 cells, myristoylated PKCζ peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.

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

Biochemical Journal ◽

10.1042/bj3450297 ◽

2000 ◽

Vol 345 (2) ◽

pp. 297-306 ◽

Cited By ~ 55

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.

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FP557DUAL INHIBITION OF CLASSICAL PROTEIN KINASE C-ALPHA AND PROTEIN KINASE C-BETA ISOFORMS REDUCES EPITHELIAL-TO-MESENCHYMAL TRANSITION OF MESOTHELIAL CELLS AND PREVENTS PERITONEAL DAMAGE IN A MOUSE MODEL OF CHRONIC PERITONEAL EXPOSE TO HIGH GLUCOSE DIALYSATE

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfv180.09 ◽

2015 ◽

Vol 30 (suppl_3) ◽

pp. iii260-iii260

Author(s):

Le Wang ◽

Michael Balzer ◽

Marcus Hiss ◽

Hermann Haller ◽

Nelli Shushakova

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Mouse Model ◽

High Glucose ◽

Epithelial To Mesenchymal Transition ◽

Mesothelial Cells ◽

Mesenchymal Transition ◽

Kinase C ◽

Protein Kinase C Alpha ◽

Protein Kinase C Beta

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

Biochemical Journal ◽

10.1042/bj3470781 ◽

2000 ◽

Vol 347 (3) ◽

pp. 781-785 ◽

Cited By ~ 20

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.

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