scholarly journals Global analysis of the effects of the V2 receptor antagonist satavaptan on protein phosphorylation in collecting duct

2014 ◽  
Vol 306 (4) ◽  
pp. 410-421 ◽  
Author(s):  
Jason D. Hoffert ◽  
Trairak Pisitkun ◽  
Fahad Saeed ◽  
Justin L. Wilson ◽  
Mark A. Knepper
Keyword(s):  
Protein Kinase ◽  
Receptor Antagonist ◽  
Signaling Pathways ◽  
Time Course ◽  
Collecting Duct ◽  
Global Analysis ◽  
Cell Junctions ◽  
Mitogen Activated Protein Kinase ◽  
V2 Receptor ◽  
V2 Receptor Antagonist

Satavaptan (SR121463) is a vasopressin V2 receptor antagonist that has been shown to improve hyponatremia in patients with cirrhosis, congestive heart failure, and syndrome of inappropriate antidiuresis. While known to inhibit adenylyl cyclase-mediated accumulation of intracellular cyclic AMP and potentially recruit β-arrestin in kidney cell lines, very little is known regarding the signaling pathways that are affected by this drug. To this end, we carried out a global quantitative phosphoproteomic analysis of native rat inner medullary collecting duct cells pretreated with satavaptan or vehicle control followed by the V2 receptor agonist desmopressin (dDAVP) for 0.5, 2, 5, or 15 min. A total of 2,449 unique phosphopeptides from 1,160 proteins were identified. Phosphopeptides significantly changed by satavaptan included many of the same kinases [protein kinase A, phosphoinositide 3-kinase, mitogen-activated protein kinase kinase kinase 7 (TAK1), and calcium/calmodulin-dependent kinase kinase 2] and channels (aquaporin-2 and urea transporter UT-A1) regulated by vasopressin. Time course clustering and kinase motif analysis suggest that satavaptan blocks dDAVP-mediated activation of basophilic kinases, while also blocking dDAVP-mediated inhibition of proline-directed kinases. Satavaptan affects a variety of dDAVP-mediated processes including regulation of cell-cell junctions, actin cytoskeleton dynamics, and signaling through Rho GTPases. These results demonstrate that, overall, satavaptan acts as a selective V2 receptor antagonist and affects many of the same signaling pathways regulated by vasopressin. This study represents the first “systems-wide” analysis of a “vaptan”-class drug and provides a wealth of new data regarding the effects of satavaptan on vasopressin-mediated phosphorylation events.

scholarly journals TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii

2004 ◽  
Vol 72 (10) ◽  
pp. 5662-5667 ◽  
Author(s):  
Nicola J. Mason ◽  
Jim Fiore ◽  
Takashi Kobayashi ◽  
Katherine S. Masek ◽  
Yongwon Choi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Toxoplasma Gondii ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Interleukin 12 ◽  
Wild Type ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-κB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-κB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-κB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6−/− mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6−/− mice failed to produce IL-12(p40) in response to STAg, and TRAF6−/− macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6−/− macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.

scholarly journals Epstein-Barr Virus (EBV) Tegument Protein BGLF2 Promotes EBV Reactivation through Activation of the p38 Mitogen-Activated Protein Kinase

2015 ◽  
Vol 90 (2) ◽  
pp. 1129-1138 ◽  
Author(s):  
XueQiao Liu ◽  
Jeffrey I. Cohen
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Epstein Barr Virus ◽  
Virus Production ◽  
Mitogen Activated Protein Kinase ◽  
Tegument Protein ◽  
Barr Virus ◽  
Ebv Reactivation ◽  
Mitogen Activated Protein ◽  
Epstein Barr

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus associated with both B cell and epithelial cell malignancies. EBV infection of B cells triggers activation of several signaling pathways that are critical for cell survival, virus latency, and growth transformation. To identify EBV proteins important for regulating cell signaling, we used a proteomic approach to screen viral proteins for AP-1 and NF-κB promoter activity in AP-1– and NF-κB–luciferase reporter assays. We found that EBV BGLF2 activated AP-1 but not NF-κB reporter activity. Expression of EBV BGLF2 in cells activated p38 and c-Jun N-terminal kinase (JNK), both of which are important for mitogen-activated protein kinase (MAPK) signaling. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of BGLF2 to activate JNK and p38. Expression of BGLF2 enhanced BZLF1 expression in latently EBV-infected lymphoblastoid cell lines, and knockdown of BGLF2 reduced EBV reactivation induced by IgG cross-linking. Expression of BGLF2 induced BZLF1 expression and virus production in EBV-infected gastric carcinoma cells. BGLF2 enhanced BZLF1 expression and EBV production by activating p38; chemical inhibition of p38 and MAPK/ERK kinases 1 and 2 (MEK1/2) reduced expression of BZLF1 and virus production induced by BGLF2. In summary, the EBV tegument protein BGLF2, which is delivered to the cell at the onset of virus infection, activates the AP-1 pathway and enhances EBV reactivation and virus production.IMPORTANCEEpstein-Barr virus (EBV) is associated with both B cell and epithelial cell malignancies, and the virus activates multiple signaling pathways important for its persistence in latently infected cells. We identified a viral tegument protein, BGLF2, which activates members of the mitogen-activated protein kinase signaling pathway. Expression of BGLF2 increased expression of EBV BZLF1, which activates a switch from latent to lytic virus infection, and increased production of EBV. Inhibition of BGFL2 expression or inhibition of p38/MAPK, which is activated by BGLF2, reduced virus reactivation from latency. These results indicate that a viral tegument protein which is delivered to cells upon infection activates signaling pathways to enhance virus production and facilitate virus reactivation from latency.

scholarly journals The Insulin Receptor: A Potential Target of Amarogentin Isolated from Gentiana rigescens Franch That Induces Neurogenesis in PC12 Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 581
Author(s):  
Lihong Cheng ◽  
Hiroyuki Osada ◽  
Tianyan Xing ◽  
Minoru Yoshida ◽  
Lan Xiang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Insulin Receptor ◽  
Signaling Pathways ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Mitogen Activated Protein Kinase ◽  
Potential Target ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Gentiana Rigescens

Amarogentin (AMA) is a secoiridoid glycoside isolated from the traditional Chinese medicine, Gentiana rigescens Franch. AMA exhibits nerve growth factor (NGF)-mimicking and NGF-enhancing activities in PC12 cells and in primary cortical neuron cells. In this study, a possible mechanism was found showing the remarkable induction of phosphorylation of the insulin receptor (INSR) and protein kinase B (AKT). The potential target of AMA was predicted by using a small-interfering RNA (siRNA) and the cellular thermal shift assay (CETSA). The AMA-induced neurite outgrowth was reduced by the siRNA against the INSR and the results of the CETSA suggested that the INSR showed a significant thermal stability-shifted effect upon AMA treatment. Other neurotrophic signaling pathways in PC12 cells were investigated using specific inhibitors, Western blotting and PC12(rasN17) and PC12(mtGAP) mutants. The inhibitors of the glucocorticoid receptor (GR), phospholipase C (PLC) and protein kinase C (PKC), Ras, Raf and mitogen-activated protein kinase (MEK) significantly reduced the neurite outgrowth induced by AMA in PC12 cells. Furthermore, the phosphorylation reactions of GR, PLC, PKC and an extracellular signal-regulated kinase (ERK) were significantly increased after inducing AMA and markedly decreased after treatment with the corresponding inhibitors. Collectively, these results suggested that AMA-induced neuritogenic activity in PC12 cells potentially depended on targeting the INSR and activating the downstream Ras/Raf/ERK and PI3K/AKT signaling pathways. In addition, the GR/PLC/PKC signaling pathway was found to be involved in the neurogenesis effect of AMA.

scholarly journals A new selective, and sensitive method for the determination of lixivaptan, a vasopressin 2 (V2)-receptor antagonist, in mouse plasma and its application in a pharmacokinetic study

2018 ◽  
Vol 16 (1) ◽  
pp. 614-620
Author(s):  
Haitham Alrabiah ◽  
Mohammed Abunassif ◽  
Sabry Attia ◽  
Gamal Abdel-Hafiz Mostafa
Keyword(s):  
Receptor Antagonist ◽  
Pharmacokinetic Study ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Hplc Method ◽  
Maximum Plasma ◽  
Mouse Plasma ◽  
V2 Receptor ◽  
V2 Receptor Antagonist

AbstractA new, selective and sensitive HPLC method for the determination of lixivaptan, an oral selective vasopressin 2 (V2)-receptor antagonist, was investigated and validated. A Waters symmetry C18 column was used as a stationary phase in isocratic elution mode using a mobile phase composed of KH2PO4 (100 mM)-acetonitrile (40: 60, v/v) at a flow rate of 1.5 mL min-1. Diclofenac was used as the internal standard (IS). Lixivaptan and the IS were extracted from plasma by protein precipitation and were detected at 260 nm. Lixivaptan and diclofenac were eluted at 3.6 and 6.2 min, respectively. The developed method showed good linearity over the calibration range of 50 -1000 ng mL-1 with a lower limit of detection of 16.5 ng mL-1. The extraction percentage of lixivaptan in the mouse plasma was in the range of 88.88 - 114.43%, which indicates acceptable extraction. The aforementioned method was validated according to guidelines of the International Council on Harmonization (ICH). The intra- and inter-day coefficients of variation did not exceed 5.5%. This method was presented to be simple, sensitive, and accurate and was successfully adapted in a pharmacokinetic study of the profile of lixivaptan in mouse plasma. A mean maximum plasma concentration of lixivaptan of 113.82 ng mL-1 was achieved in 0.5 h after oral administration of a 10 mg kg-1 dose in mouse as determined using the developed method.

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