Lysophosphatidic acid rapidly induces protein kinase D activation through a pertussis toxin-sensitive pathway

2000 ◽  
Vol 278 (1) ◽  
pp. C33-C39 ◽  
Author(s):  
Lina Paolucci ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt
Keyword(s):  
Protein Kinase ◽  
Pertussis Toxin ◽  
Lysophosphatidic Acid ◽  
Kinase Inhibitor ◽  
Structural Features ◽  
Protein Kinase D ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Swiss 3T3

Protein kinase D (PKD) is a serine-threonine protein kinase with distinct structural features and enzymological properties. Herein we demonstrate that lysophosphatidic acid (LPA) induces rapid PKD activation in mouse Swiss 3T3 and Rat-1 cells. LPA induced PKD activation in a concentration-dependent fashion with maximal stimulation (7.6-fold) achieved at 5 μM. Treatment of Swiss 3T3 cells with the protein kinase C (PKC) inhibitors GF-I, Ro-31–8220, and Gö-7874 completely abrogated PKD activation induced by LPA at concentrations that did not inhibit PKD activity when added directly to the in vitro kinase assays. PKD activation induced by LPA was attenuated markedly and selectively by prior exposure of either Swiss 3T3 or Rat-1 cells to pertussis toxin (PTx) in a concentration-dependent manner. In contrast, treatment with the protein tyrosine kinase inhibitor genistein, the MEK inhibitor PD-098059, or the phosphoinositide 3-kinase inhibitor wortmannin did not affect PKD activation in response to LPA. These results provide the first example of PTx-sensitive and PKC-dependent PKD activation and identify a novel Gi-dependent event in the action of LPA.

scholarly journals P2X7 receptors activate protein kinase D and p42/p44 mitogen-activated protein kinase (MAPK) downstream of protein kinase C

2002 ◽  
Vol 366 (3) ◽  
pp. 745-755 ◽  
Author(s):  
Michelle D. BRADFORD ◽  
Stephen P. SOLTOFF
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinases ◽  
P2x7 Receptor ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase D ◽  
Dependent Manner ◽  
P2x7 Receptors ◽  
High Concentrations

Protein kinase D (PKD), also called protein kinase Cμ (PKCμ), is a serine/threonine kinase that has unique enzymic and structural properties distinct from members of the PKC family of proteins. In freshly isolated rat parotid acinar salivary cells, extracellular ATP rapidly increased the activity and phosphorylation of PKD. The stimulation by ATP required high concentrations, was mimicked by the P2X7 receptor ligand BzATP [2′- and 3′-O-(4-benzoylbenzoyl)ATP], and was blocked by Mg2+ and 4,4′-di-isothiocyano-2,2′-stilbene disulphonate (DIDS), suggesting that activation of PKD was mediated by P2X7 receptors, which are ligand-gated non-selective cation channels. Phorbol ester (PMA) and the activation of muscarinic and substance P receptors also increased PKD activity. PKC inhibitors blocked ligand-dependent PKD activation and phosphorylation, determined by in vitro phosphorylation studies and by phospho-specific antibodies to two activation loop sites (Ser744 and Ser748) and an autophosphorylation site (Ser916). ATP and BzATP also increased the tyrosine phosphorylation and activity of PKCΔ, and these stimuli also increased extracellular signal-regulated protein kinase (ERK) 1/2 activity in a PKC-dependent manner. PKD activation was not promoted by pervanadate (an inhibitor of tyrosine phosphatases) and was not blocked by PP1 (an inhibitor of Src family kinases) or genistein (a tyrosine kinase inhibitor), suggesting that tyrosine kinases and phosphatases did not play a major role in PKD activation. P2X7 receptor-mediated signalling events were not dependent on Ca2+ entry. These studies indicate that PKC is involved in cellular signalling initiated by P2X7 receptors as well as by G-protein-coupled receptors, and demonstrate that PKD and ERK1/2 are activated in similar PKC-dependent signalling pathways initiated by these diverse receptor types.

scholarly journals Reversal of Cancer Multidrug Resistance (MDR) Mediated by ATP-Binding Cassette Transporter G2 (ABCG2) by AZ-628, a RAF Kinase Inhibitor

2020 ◽  
Vol 8 ◽  
Author(s):  
Jing-Quan Wang ◽  
Qiu-Xu Teng ◽  
Zi-Ning Lei ◽  
Ning Ji ◽  
Qingbin Cui ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Kinase Inhibitor ◽  
Efflux Pump ◽  
Simulation Analysis ◽  
Chemotherapeutic Agents ◽  
Dynamics Simulation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Raf Kinase

Overexpression of ABCG2 remains a major impediment to successful cancer treatment, because ABCG2 functions as an efflux pump of chemotherapeutic agents and causes clinical multidrug resistance (MDR). Therefore, it is important to uncover effective modulators to circumvent ABCG2-mediated MDR in cancers. In this study, we reported that AZ-628, a RAF kinase inhibitor, effectively antagonizes ABCG2-mediated MDR in vitro. Our results showed that AZ-628 completely reversed ABCG2-mediated MDR at a non-toxic concentration (3 μM) without affecting ABCB1-, ABCC1-, or ABCC10 mediated MDR. Further studies revealed that the reversal mechanism was by attenuating ABCG2-mediated efflux and increasing intracellular accumulation of ABCG2 substrate drugs. Moreover, AZ-628 stimulated ABCG2-associated ATPase activity in a concentration-dependent manner. Docking and molecular dynamics simulation analysis showed that AZ-628 binds to the same site as ABCG2 substrate drugs with higher score. Taken together, our studies indicate that AZ-628 could be used in combination chemotherapy against ABCG2-mediated MDR in cancers.

Platelet-derived lysophosphatidic acid decreases endothelial permeability in vitro

1998 ◽  
Vol 274 (1) ◽  
pp. H115-H122 ◽  
Author(s):  
J. S. Alexander ◽  
W. F. Patton ◽  
B. W. Christman ◽  
L. L. Cuiper ◽  
F. R. Haselton
Keyword(s):  
Lysophosphatidic Acid ◽  
Endothelial Permeability ◽  
Enzyme Inactivation ◽  
Continuous Exposure ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Solute Permeability ◽  
Methanol Extracts ◽  
Endothelial Monolayers

We previously reported that platelets release a soluble factor that decreases the solute permeability of cultured bovine aortic endothelial monolayers. This factor was characterized as heat stable, trypsin sensitive, and not serotonin, adenosine, ADP, or ATP [F. R. Haselton and J. S. Alexander. Am. J. Physiol. 263 ( Lung Cell Mol. Physiol. 7): L670–L678, 1992]. We now report its identity as lysophosphatidic acid (LPA). Endothelial permeability decreases rapidly, reversibly, and repeatedly when exposed to platelet supernatants. Continuous exposure produces a sustained decrease in permeability. Methanol extracts of platelet supernatants also decrease endothelial permeability. Treatment of methanol extracts of platelet supernatants with phospholipase B or alkaline phosphatase, which modify the structure of LPA, abolishes the permeability-decreasing activity. However, activity is unaffected by treatment with phospholipase A2. This pattern of enzyme inactivation is consistent with the structure of LPA. Furthermore, synthetic 1-oleoyl-LPA rapidly and significantly decreases endothelial permeability in a concentration-dependent manner. Platelet activation does not appear to be required to produce activity in supernatants from platelet isolations, since P-selectin expression is not increased and thromboxane B2 is <14 pg/6,000 platelets. Our data show that platelets release a methanol-extractable compound with an enzyme degradation profile consistent with LPA, which decreases the permeability of endothelial monolayers in vitro. In vivo, LPA derived from platelets may be an important mediator of the transport barrier formed by the vascular endothelium.

scholarly journals G-protein βγ subunits antagonize protein kinase C-dependent phosphorylation and inhibition of phospholipase C-β1

1997 ◽  
Vol 326 (3) ◽  
pp. 701-707 ◽  
Author(s):  
Irene LITOSCH
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
G Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Negative Feedback Regulation ◽  
Kinase C ◽  
Stimulation Of

Protein kinase C (PKC) isoforms phosphorylated phospholipase C-β1 (PLC-β1) in vitro as follows: PKCα ≫ PKCϵ; not PKCζ. PLC-β3 was not phosphorylated by PKCα. G-protein βγ subunits inhibited the PKCα phosphorylation of PLC-β1 in a concentration-dependent manner. Half-maximal inhibition occurred with 500 nM βγ. G-protein βγ subunits also antagonized the PKCα-mediated inhibition of PLC-β1 enzymic activity. PKCα, in turn, inhibited the stimulation of PLC-β1 activity by βγ. There was little effect of PKCα on the stimulation of PLC-β1 by αq/11–guanosine 5′[γ-thio]triphosphate (GTP[S]). These findings demonstrate that G protein βγ subunits antagonize PKCα regulation of PLC-β1. Thus βγ subunits might have a role in modulating the negative feedback regulation of this signalling system by PKC.

The role of protein kinase C in LHRH-induced LH and FSH release and LHRH self-priming in rat anterior pituitary glands in vitro

1988 ◽  
Vol 116 (2) ◽  
pp. 231-239 ◽  
Author(s):  
M. S. Johnson ◽  
R. Mitchell ◽  
G. Fink
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Polymyxin B ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Pituitary Glands

ABSTRACT We have investigated the role of protein kinase C (PKC) in LHRH-induced LH and FSH secretion and LHRH priming. Hemipituitary glands from pro-oestrous rats were incubated with agents known to affect PKC and with or without LHRH, during which time the secretion of gonadotrophins was measured. Phorbol esters and phospholipase C, activators of PKC, released LH and FSH in a concentration-dependent manner and potentiated the LHRH-induced secretion of gonadotrophins in parallel with their ability to release these hormones alone. Inhibitors of PKC had either no effect on LH release (1-(5-isoquinolinesulphonyl)-2-methylpiperazine hydrochloride) or they augmented LHRH-induced gonadotrophin release (polymyxin B and 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate). Neither the activators nor the inhibitors of PKC, when present with LHRH, caused any change in LHRH priming, even though the activators alone produced a release of gonadotrophins that showed a temporal pattern similar to that produced by LHRH priming. The profiles of effects on LH and FSH secretion were always qualitatively similar. These results show that PKC may be involved in general regulation of gonadotrophin release but that it is not important in acute responses to LHRH nor in LHRH self-priming. J. Endocr. (1988) 116, 231–239

scholarly journals Phorbol ester stimulates choline uptake in Swiss 3T3 fibroblasts following introduction of the gene encoding protein kinase Cα

1995 ◽  
Vol 305 (2) ◽  
pp. 621-626 ◽  
Author(s):  
B E Slack ◽  
J Breu ◽  
E Livneh ◽  
H Eldar ◽  
R J Wurtman
Keyword(s):  
Protein Kinase ◽  
Expression System ◽  
Choline Uptake ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner ◽  
3T3 Fibroblasts ◽  
Swiss 3T3 ◽  
Pkc Alpha ◽  
Swiss 3T3 Fibroblasts

Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation into phosphatidylcholine (PtdCho) in a time- and concentration-dependent manner in wild-type NIH 3T3 fibroblasts. The accumulation of labelled choline induced by PMA was paralled by an increase in choline mass. The results implicate protein kinase C (PKC) in the regulation of choline uptake. In order to address the PKC-subtype specificity of this response, a study was undertaken in Swiss 3T3 fibroblast cells, which normally express very low levels of PKC alpha. A retroviral expression system was used to introduce the genes for PKC alpha and neomycin resistance (used for selection) into the cells. Two resulting lines expressed PKC alpha at levels that were 20-fold higher than those found in the control (neomycin-resistant) line, or in the wild-type cells. In control Swiss 3T3 fibroblasts, 1 microM PMA elevated choline levels by only 30%, whereas, in Swiss 3T3 cell lines that stably over-expressed PKC alpha, PMA caused a 5-fold enhancement in [14C]choline accumulation. This concentration of PMA significantly increased [14C]PtdCho levels in both control and PKC alpha-over-expressing lines, although the effect in the latter was significantly greater. The effects of PMA were inhibited by the PKC antagonist sphingosine. These results implicate PKC alpha in the regulation of choline accumulation and phospholipid synthesis in fibroblasts. Although additional PKC subtypes appear to participate in the control of PtdCho synthesis in these cells, PMA-stimulated choline uptake in Swiss 3T3 fibroblasts is almost entirely dependent on the presence of PKC alpha.

scholarly journals Structural Features of Three Hetero-Galacturonans from Passiflora foetida Fruits and Their in Vitro Immunomodulatory Effects

Polymers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 615 ◽  
Author(s):  
Ya Song ◽  
Peng Wen ◽  
Huili Hao ◽  
Minqian Zhu ◽  
Yuanming Sun ◽  
...  
Keyword(s):  
Structural Features ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Minimum Effective Concentration ◽  
Enhancing Effect ◽  
Traditional Chinese Herbal Medicine ◽  
Raw264.7 Macrophages ◽  
Pharmaceutical Industries ◽  
Passiflora Foetida

Passiflora foetida is a horticultural plant and vital traditional Chinese herbal medicine. In our previous study, the characterization and immuno-enhancing effect of fruits polysaccharide 1 (PFP1), a water-eluted hetero-mannan from wild Passiflora foetida fruits, were investigated. Herein, another three salt-eluted novel polysaccharides, namely PFP2, PFP3, and PFP4, were obtained and structurally characterized. The results showed that PFP2, PFP3, and PFP4 were three structurally similar hetero-galacturonans with different molecular weights of 6.11 × 104, 4.37 × 104, and 3.48 × 105 g/mol, respectively. All three of these hetero-galacturonans are mainly composed of galacturonic acid, galactose, arabinose (75.69%, 80.39%, and 74.30%, respectively), and other monosaccharides including mannose, fucose, glucose, ribose, xylose, and glucuronic acid (24.31%, 19.61, and 25.70%, respectively), although differences in their backbone structure exist. Additionally, immunomodulatory assay indicated that the three hetero-galacturonans possess the ability to promote the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in RAW264.7 macrophages in a concentration-dependent manner (p < 0.05). Especially, PFP3 displayed a stronger enhancing effect than PFP2 and PFP4 at the minimum effective concentration. Therefore, the results suggested that the obtained three salt-eluted hetero-galacturonans, especially PFP3, could be utilized as immunomodulatory effectivity ingredients in nutritional/pharmaceutical industries.

scholarly journals Melatonin Sensitizes Human Myometrial Cells to Oxytocin in a Protein Kinase Cα/Extracellularly Regulated Kinase-Dependent Manner

2010 ◽  
Vol 24 (5) ◽  
pp. 1106-1106
Author(s):  
James T. Sharkey ◽  
Casey Cable ◽  
James Olcese
Keyword(s):  
Protein Kinase ◽  
Phospholipase C ◽  
Signaling Pathway ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Human Myometrium ◽  
Dependent Manner ◽  
Uterine Contractility ◽  
Myometrial Cells

abstract Context: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night. Objective: The purpose of this study was to determine the signaling pathway underlying the effects of MEL on contractility and the contractile machinery in immortalized human myometrial cells. Design: To ascertain the signaling pathway of MEL leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodo-MEL (I-MEL) with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on inositol trisphosphate (IP3)/diacylglycerol (DAG)/protein kinase C (PKC) signaling were also investigated. Additionally, we assayed for caldesmon phosphorylation and ERK1/2 activation. Results: I-MEL was found to activate PKCα via the phospholipase C/IP3/DAG signaling pathway, which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity, and its effects on contractility were insensitive to Rho kinase inhibition. I-MEL did increase phosphorylation of ERK1/2 and caldesmon, which was inhibited by the MAPK kinase inhibitor PD98059 or the PKC inhibitor C1. Conclusions: MEL sensitizes myometrial cells to subsequent procontractile signals in vitro through activation of the phospholipase C/IP3/DAG signaling pathway, resulting in specific activation of PKCα and ERK1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and cross-bridging. In vivo, this sensitization would provide a mechanism for the increased nocturnal uterine contractility and labor that has been observed in late-term human pregnancy.

scholarly journals Looking for a generic inhibitor of amyloid-like fibril formation among flavone derivatives

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1271 ◽  
Author(s):  
Tomas Šneideris ◽  
Lina Baranauskienė ◽  
Jonathan G. Cannon ◽  
Rasa Rutkienė ◽  
Rolandas Meškys ◽  
...  
Keyword(s):  
Beta Amyloid ◽  
Structural Features ◽  
Alpha Synuclein ◽  
Fibril Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Amyloid Fibril Formation ◽  
Flavone Derivatives ◽  
Alpha Synuclein Aggregation

A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrilsin vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid.

scholarly journals Exocytosis in electropermeabilized neutrophils. Responsiveness to calcium and guanosine 5′-[γ-thio]triphosphate

1992 ◽  
Vol 287 (3) ◽  
pp. 695-700 ◽  
Author(s):  
G J J C Boonen ◽  
J van Steveninck ◽  
T M A R Dubbelman ◽  
P J A van den Broek ◽  
J G R Elferink
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Pertussis Toxin ◽  
Binding Protein ◽  
Enzyme Release ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Inorganic Phosphates

Electropermeabilized neutrophils were used to study the exocytotic response in rabbit neutrophils. Enzyme release from electropermeabilized neutrophils could be induced by elevating the Ca2+ concentration. Ca(2+)-induced secretion was significantly enhanced by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) in a concentration-dependent manner. The effect of GTP[S] could be blocked by guanosine 5′-[beta-thio]diphosphate (GDP[S]) and was not affected by pertussis toxin. GTP[S] did not induce enzyme release in the absence of Ca2+. Induction of an exocytotic response did not require addition of ATP. However, neutrophils permeabilized in the absence of ATP became refractory to stimulation due to a reduction in their affinity for Ca2+. Responsiveness to the effectors Ca2+ or Ca2+ + GTP[S] could be prolonged or restored by ATP. ATP was not the only agent that prolonged responsiveness; other nucleotides and inorganic phosphates were also effective. The protein kinase C inhibitors staurosporine and 1-O-hexadecyl-2-methyl-sn-glycerol did not inhibit exocytosis and had only a small effect on the prolongation and restoration of responsiveness by ATP. A hypothesis is presented suggesting that the loss of responsiveness is caused by dephosphorylation and that the restoration or prolongation of responsiveness is not mediated by protein kinase C. It is possible that an as yet unidentified Ca(2+)-binding protein is dephosphorylated, resulting in a decrease in Ca2+ affinity.

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