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

2007 ◽  
Vol 176 (7) ◽  
pp. 1049-1060 ◽  
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.

scholarly journals Protein kinase C bound to the Golgi apparatus supports the formation of constitutive transport vesicles

1996 ◽  
Vol 320 (2) ◽  
pp. 651-658 ◽  
Author(s):  
Peter WESTERMANN ◽  
Maria KNOBLICH ◽  
Olaf MAIER ◽  
Carsten LINDSCHAU ◽  
Hermann HALLER
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Hepg2 Cells ◽  
Heparan Sulphate ◽  
Specific Inhibitor ◽  
Marginal Effect ◽  
Calphostin C ◽  
Kinase C ◽  
Transport Vesicles ◽  
Pkc Ζ

Constitutive secretion of heparan sulphate proteoglycans (HSPGs) was stimulated in human hepatoma HepG2 cells by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC). To delineate more closely the site of PKC action, the packaging in vitro of 35SO4-labelled HSPGs into transport vesicles was investigated. Formation of transport vesicles at the trans-Golgi network was stimulated by PMA and inhibited by calphostin C or Ro 31-8220 by using a post-nuclear supernatant. Treatment of either isolated Golgi-enriched membranes or cytosolic proteins with calphostin C provided evidence that membrane-bound PKC forms strongly supported vesicle formation, whereas cytosolic PKC forms showed a marginal effect. The PKC isoforms PKC-α and PKC-ζ were attached to highly purified Golgi membranes, as shown by Western blotting. Both isoforms were localized by confocal immunofluorescence microscopy in the Golgi area of HepG2 cells. Immunoelectron microscopy of ultrathin cryosections of HepG2 cells showed that PKC-ζ predominantly attaches to the trans-Golgi region, whereas PKC-α binds to the cis- and trans-Golgi area.

Role of protein kinase C-η in cigarette smoke extract-induced apoptosis in MRC-5-cells

2014 ◽  
Vol 34 (9) ◽  
pp. 869-877 ◽  
Author(s):  
ES Son ◽  
SY Kyung ◽  
SP Lee ◽  
SH Jeong ◽  
JY Shin ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cigarette Smoke ◽  
Caspase 3 ◽  
Dominant Negative ◽  
Lung Fibroblasts ◽  
Apoptotic Pathway ◽  
Kinase C ◽  
Extrinsic Apoptotic Pathway ◽  
Infected Cells

Cigarette smoke (CS) is a major risk factor for emphysema, which causes cell death in structural cells of the lung by mechanisms that are still not completely understood. We demonstrated previously that CS extract (CSE) induces caspase activation in MRC-5 human lung fibroblasts, activated protein kinase C-η (PKC-η), and translocated PKC-η from the cytosol to the membrane. The objective of this study was to investigate the involvement of PKC-η activation in a CSE-induced extrinsic apoptotic pathway. We determined that CSE increases expression of caspase 3 and 8 cleavage in MRC-5 cells and overexpression of PKC-η significantly increased expression of caspase 3 and 8 cleavage compared with control LacZ-infected cells. In contrast, dominant negative (dn) PKC-η inhibited apoptosis in MRC-5 cells exposed to CSE and decreased expression of caspase 3 and 8 compared with control cells. Exposure to 10% CSE for >8 h significantly increased lactate dehydrogenase release in PKC-η-infected cells compared with LacZ-infected cells. Additionally, PKC-η-infected cells had an increased number of Hoechst 33342 stained nuclei compared with LacZ-infected cells, while dn PKC-η-infected cells exhibited fewer morphological changes than LacZ-infected cells under phase-contrast microscopy. In conclusion, PKC-η activation plays a pro-apoptotic role in CSE-induced extrinsic apoptotic pathway in MRC-5 cells. These results suggest that modulation of PKC-η may be a useful tool for regulating the extrinsic apoptosis of MRC-5 cells by CSE and may have therapeutic potential in the treatment of CS-induced lung injury.

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.

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.

scholarly journals Release of superoxide and change in morphology by neutrophils in response to phorbol esters: antagonism by inhibitors of calcium-binding proteins.

1985 ◽  
Vol 101 (3) ◽  
pp. 1052-1058 ◽  
Author(s):  
J M Robinson ◽  
J A Badwey ◽  
M L Karnovsky ◽  
M J Karnovsky
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Guinea Pig ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Phorbol Esters ◽  
Morphological Changes ◽  
Calcium Binding Proteins ◽  
Kinase C ◽  
Activate Protein Kinase

The ability of phorbol derivatives to function as stimulating agents for superoxide (O2-) release by guinea pig neutrophils has been evaluated and compared to the known ability of each compound to activate protein kinase C. Those that activate the kinase also stimulate O2- release, while those that are inactive with respect to the kinase have no effect on O2- release. The same correlation was observed with respect to the ability of phorbol esters to induce morphological changes in neutrophils, i.e., vesiculation and reduction in granule content. Certain phenothiazines and naphthalene sulfonamides that are known antagonists of calcium-binding proteins blocked both phorbol ester-induced O2- release and morphological changes in these cells.

scholarly journals Serine Phosphorylation Sites on IRS2 Activated by Angiotensin II and Protein Kinase C To Induce Selective Insulin Resistance in Endothelial Cells

2013 ◽  
Vol 33 (16) ◽  
pp. 3227-3241 ◽  
Author(s):  
Kyoungmin Park ◽  
Qian Li ◽  
Christian Rask-Madsen ◽  
Akira Mima ◽  
Koji Mizutani ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Serine Phosphorylation ◽  
Kinase C ◽  
Pkc Activation

Protein kinase C (PKC) activation, induced by hyperglycemia and angiotensin II (AngII), inhibited insulin-induced phosphorylation of Akt/endothelial nitric oxide (eNOS) by decreasing tyrosine phosphorylation of IRS2 (p-Tyr-IRS2) in endothelial cells. PKC activation by phorbol ester (phorbol myristate acetate [PMA]) reduced insulin-induced p-Tyr-IRS2 by 46% ± 13% and, similarly, phosphorylation of Akt/eNOS. Site-specific mutational analysis showed that PMA increased serine phosphorylation at three sites on IRS2 (positions 303, 343, and 675), which affected insulin-induced tyrosine phosphorylation of IRS2 at positions 653, 671, and 911 (p-Tyr-IRS2) and p-Akt/eNOS. Specific PKCβ2 activation decreased p-Tyr-IRS2 and increased the phosphorylation of two serines (Ser303 and Ser675) on IRS2 that were confirmed in cells overexpressing single point mutants of IRS2 (S303A or S675A) containing a PKCβ2-dominant negative or selective PKCβ inhibitor. AngII induced phosphorylation only on Ser303 of IRS2 and inhibited insulin-induced p-Tyr911 of IRS2 and p-Akt/eNOS, which were blocked by an antagonist of AngII receptor I, losartan, or overexpression of single mutant S303A of IRS2. Increases in p-Ser303 and p-Ser675 and decreases in p-Tyr911 of IRS2 were observed in vessels of insulin-resistant Zucker fatty rats versus lean rats. Thus, AngII or PKCβ activation can phosphorylate Ser303 and Ser675 in IRS2 to inhibit insulin-induced p-Tyr911 and its anti-atherogenic actions (p-Akt/eNOS) in endothelial cells.

The role of protein kinase C-δ in PTH stimulation of IGF-binding protein-5 mRNA in UMR-106–01 cells

2002 ◽  
Vol 282 (3) ◽  
pp. E534-E541 ◽  
Author(s):  
Mary S. Erclik ◽  
Jane Mitchell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Protein ◽  
Specific Inhibitor ◽  
Dominant Negative Mutant ◽  
Nuclear Fraction ◽  
Mrna Levels ◽  
Kinase C ◽  
Umr 106

We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106–01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 μM bovine PTH-(3–34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1–34) and PTH-(3–34) (100 nM) both stimulated PKC-δ translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-δ-specific inhibitor, and a dominant negative mutant of PKC-δ were both able to significantly inhibit PTH-(1–34) and PTH-(3–34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-δ in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-α translocation from the cytosol to the membrane and inhibited ∼50% of the PTH-(1–34), forskolin, and 8-bromoadenosine 3′,5′-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-α negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-δ is the primary mediator of the effects of PTH via the PKC pathway.

Ca2+-independent protein kinase C isoforms may modulate parietal cell HCl secretion

1997 ◽  
Vol 272 (2) ◽  
pp. G246-G256 ◽  
Author(s):  
C. S. Chew ◽  
C. J. Zhou ◽  
J. A. Parente
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Parietal Cell ◽  
Morphological Changes ◽  
Dependent Manner ◽  
Independent Manner ◽  
Gastric Glands ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Hcl Secretion

Although activation of adenosine 3',5'-cyclic monophosphate by histamine and of Ca2+-dependent signaling pathways by cholinergic agonists is a generally recognized mechanism for increasing parietal cell HCl secretion, the role of protein kinase C (PKC) in this process is controversial. In this study, acid-secretory responses of gastric glands from rabbits [measured as accumulation of aminopyrine (AP)] were found to be relatively resistant to the PKC inhibitors calphostin C, chelerythrine chloride, staurosporine, and the bisindolylmaleimide-like inhibitors Ro 31-8220, Go 6976, and bisindolylmaleimide I hydrochloride. Western analyses of the PKC isozyme profile in highly enriched parietal cells (98% purity) indicated that this cell type expresses abundant levels of the novel isoforms PKC-epsilon and PKC-mu and abundant levels of the atypical isoforms PKC-iota, PKC-lambda, and PKC-zeta. In contrast, there appeared to be low to undetectable expression of the classical isoforms PKC-alpha and PKC-beta1/beta2, respectively. Relatively high concentrations of Ro 31-8220 potentiated both carbachol- and histamine-stimulated AP accumulation (IC50 857 +/- 100 and 910 +/- 98 nM, respectively). There was a similar dose dependence for Ro 31-8220 inhibition of in situ phosphorylation of a parietal cell phosphoprotein, pp66 (IC50 750 +/- 120 nM). Similar concentrations of Ro 31-8220 also inhibited phosphorylation of the cytoskeletal, actin membrane cross-linking phosphoprotein ezrin, but not other phosphoproteins. Ezrin phosphorylation was increased by carbachol and 12-O-tetradecanoylphorbol 13-acetate (TPA). Because carbachol and TPA stimulate pp66 phosphorylation in a Ca2+-independent manner, our results suggest that one or more novel PKC isoforms may be involved in negative regulation of HCl secretion. In related experiments, PKC-epsilon, but not PKC-mu, was immunolocalized by confocal microscopy to a parietal cell compartment that bore a striking resemblance to that containing filamentous actin. Moreover, pp66 was enriched in a Triton X-100-insoluble parietal cell fraction, suggesting a potential cytoskeletal localization for this unknown protein. Given their location and sensitivity to Ro 31-8220, it is possible that pp66 and ezrin interact in a PKC-dependent manner to regulate the well-known morphological changes that occur in concert with agonist-dependent activation of parietal cell HCl secretion.

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