scholarly journals Insulin-induced phospholipase D1 and phospholipase D2 activity in human embryonic kidney-293 cells mediated by the phospholipase Cγ and protein kinase Cα signalling cascade

2000 ◽  
Vol 351 (3) ◽  
pp. 613-619 ◽  
Author(s):  
Rita SLAABY ◽  
Guangwei DU ◽  
Yelena M. ALTSHULLER ◽  
Michael A. FROHMAN ◽  
Klaus SEEDORF
Keyword(s):  
Protein Kinase ◽  
Cell Types ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Phospholipase D1

Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKCα results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)γ has the same effect, while co-expression of PLD2 with PLCγ allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLCγ and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKCα is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKCα and PLCγ, are expressed at sufficient levels.

scholarly journals Calcitonin stimulates expression of the rat 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter in HEK-293 cells expressing calcitonin receptor: identification of signaling pathways

2004 ◽  
Vol 32 (1) ◽  
pp. 87-98 ◽  
Author(s):  
XH Gao ◽  
PP Dwivedi ◽  
JL Omdahl ◽  
HA Morris ◽  
BK May
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Basal Expression ◽  
293 Cells ◽  
Gc Box

Regulation of the gene for renal 25-hydroxyvitamin D-24-hydroxylase (CYP24) is important for controlling the level of circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We report here for the first time that the peptide hormone calcitonin significantly stimulates expression of a rat CYP24 promoter-luciferase construct in both transiently and stably transfected kidney HEK-293 cells. A GC box at -114/-101 and a CCAAT box at -62/-51 have been identified that underlie both basal expression of the CYP24 promoter and the calcitonin inductive response. Data from overexpression studies suggested that Sp1 and NF-Y are the proteins that function through the GC and CCAAT boxes respectively. ERK1/2 signaling pathways were not involved in the calcitonin-mediated response, since stimulation of the promoter was unaffected by the pharmacological ERK1/2 inhibitor PD98059 and by a dominant negative mutant of ERK1/2 (ERK1K71R). In contrast, calcitonin induction but not basal expression was dependent on protein kinase A and protein kinase C (PKC) activities with the inhibitors H89 and calphostin C lowering induction by 50-60%. The atypical PKC, PKCzeta contributes to calcitonin induction, but not to basal expression of the CYP24 promoter, since overexpression of a dominant negative clone PKCzetaK281 M lowered induction by 50%. Cotransfection of a dominant negative form of Ras resulted in calcitonin-mediated induction being reduced also by about 50%. A Ras-PKCzeta signaling pathway for calcitonin action is proposed, which acts through the GC box. The findings have been extrapolated to the in vivo situation where we suggest that induction of renal CYP24 by calcitonin could be important under hypercalcemic conditions thus contributing to the lowering of circulating 1,25(OH)2D3 levels.

scholarly journals Characterization of the reversible phosphorylation and activation of ERK8

2006 ◽  
Vol 394 (1) ◽  
pp. 365-373 ◽  
Author(s):  
Iva V. Klevernic ◽  
Margaret J. Stafford ◽  
Nicholas Morrice ◽  
Mark Peggie ◽  
Simon Morton ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Growth Factor ◽  
Protein Kinase ◽  
Insect Cells ◽  
Osmotic Shock ◽  
Tyrosine Phosphatase ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15–20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.

scholarly journals Nephroprotective activity of Annona Squamosa leaves against paracetamol-induced nephrotoxicity in rats: in vitro and in vivo experiments

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
S. Neelima ◽  
P. Dwarakanadha Reddy ◽  
Chandra Sekhar Kothapalli Bannoth
Keyword(s):  
Kidney Tissue ◽  
Ethanolic Extract ◽  
Tissue Homogenate ◽  
Annona Squamosa ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
In Vivo Experiments ◽  
293 Cells

Abstract Background Paracetamol (PCM), being extensively adapted analgesic and anti-inflammatory drug all over the world, beyond therapeutic dosages, the oxidative stress-involved nephrotoxicity has been evidenced. However, herbal plants are the windfall for the humankind providing solution for most of the wellness breakdowns. Annona squamosa (AS) is one of such plants with enormous therapeutic and nutraceutical potencies. The main aspiration of the current investigation is to evaluate the nephroprotective ability of ethanolic extract of Annona squamosa (EEAS) leaves against paracetamol-induced nephrotoxicity using in vitro human embryonic kidney (HEK)-293 cells and in vivo experiments in Wistar rats through biochemical parameters, oxidative parameters, and histopathological findings. Results When HEK-293 cells were incubated with PCM, an increased cell death associated with alterations in the morphology of normal cells was observed. At variable concentrations, HEK-293 cells co-treated with PCM and EEAS extracts gave a significant improvement in cell growth on comparing with PCM treatment showing cytoprotective feature of EEAS with an IC50 28.75 μg/mL. In vivo nephroprotective property was assessed from the amount of blood urea nitrogen (BUN) along with creatinine and uric acid which were reduced (P < 0.001) within serum and compact levels of glutathione, catalase, and superoxide dismutase which were termed as GSH, CAT, and SOD, respectively, were increased (P < 0.001) in kidney tissue homogenate in the treated groups than the PCM alone group. Results were additionally supported by histopathological observations. Conclusion The results exhibited that EEAS has impending benefits against PCM-induced nephrotoxicity through in vitro and in vivo experiments.

scholarly journals In vitro 3D Spheroid Culture Developed on the Parafilm Surface Using HEK-293 Cells

2020 ◽  
Vol 3 (1) ◽  
pp. 220-227
Author(s):  
Erdal Eroğlu
Keyword(s):  
Three Dimensional ◽  
Culture Method ◽  
In Vivo Studies ◽  
Preclinical Research ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Hanging Drop Culture

Preclinical research to predict the effects of drugs and chemicals on humans is commonly carried out either by cell culture studies in vitro condition or on animals in vivo condition. While drug studies tested on cells cultured as a monolayer in plastic flasks are incompatible with realistic results, falsifying findings can also be achieved from in vivo studies performed on different species. In recent years, research on drug tests using spheroid cultures formed by growing cells in three-dimensional (3D) in vitro has attracted great interest. 3D spheroid structures are formed by growing the cells in a drop suspended on superhydrophobic surfaces. In this study, HEK-293 cells were investigated on parafilm surfaces displaying superhydrophobic properties by growing in 2 &amp;micro;l volume using hanging drop culture method in terms of spheroid formation. Light microscopy images from spheroid structures were taken on different incubation days and the area of spheroids was measured using the ImageJ program. Our study holds important findings for a chip platform that can be developed for use in vitro drug tests.

334 ADENOVIRAL VECTOR-MEDIATED EXPRESSION OF RECOMBINANT HUMAN GLUCOCEREBROSIDASE IN THE MAMMARY GLAND OF RATS

2013 ◽  
Vol 25 (1) ◽  
pp. 314
Author(s):  
K. C. S. Tavares ◽  
C. Feltrin ◽  
I. S. Carneiro ◽  
A. S. Morais ◽  
C. D. Medeiros ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Mammary Glands ◽  
Santa Cruz ◽  
Cmv Promoter ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Control Milk

Glucocerebrosidase is a lysosomal enzyme that plays a key role in sphingolipid cleavage, an intermediate in glycolipid metabolism. A recessive mutation in the glucocerebrosidase gene leads to the accumulation of glucosylceramide in macrophages (sphingolipidosis), a lysosomal storage disease known in humans as the Gaucher disease. The enzyme replacement treatment with recombinant human glucocerebrosidase (hGCase) dramatically reduces and reverses symptoms, with the need of lifelong treatment for patients to attain a normal life. Currently, hGCase is very costly, being produced through in vitro expression in Chinese hamster ovary cells or in vivo, in plants. The aim of this study was to develop a model for the production of hGCase in the mammary gland of rats transiently transduced with recombinant adenovirus. A replication-defective adenovirus carrying hGCase was generated using the AdEasy™ adenoviral vector system (Stratagene, La Jolla, CA, USA). The hGCase cDNA (NM_001005741) was in vitro-synthesized and ligated in the XhoI site of the pAdTrack-CMV vector (pAdT-hGCase). The resulting plasmid was recombined with the pAdEasy™ vector in BJ5183 electro-competent cells. The purified pAdE-pAdT-hGCase vector was linearized and transfected into HEK-293 cells for the production of a primary viral stock. Further amplifications and the titration assay were done in HEK-293 cells, monitoring the transduction by the qualitative evaluation of green fluorescent protein (GFP) expression. Following transfection, the HEK-293 cells increasingly expressed the GFP reporter, regulated by a CMV promoter, in tandem with the hGCase cDNA, under another CMV promoter. On Day 18 of gestation, a female rat (Rattus norvegicus) was anesthetized and the 2 left caudal mammary glands were infused with 109 GTU mL–1 of the pAdE-pAdT-hGCase in PBS solution supplemented with 36 mM EGTA. The 2 right caudal mammary glands were infused only with PBS-EGTA (control milk). Milk samples collected from Days 2 through 9 post-partum were mixed with separation buffer (10 mM Tris-HCl, pH 8.0; 10 mM CaCl2) and centrifuged, with the supernatant assayed for hGCase by Western blot using a monoclonal anti-human glucocerebrosidase antibody (sc-166407, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Relative quantification of the hGCase expression was done using the FluorChem FC2 system (Alpha Innotech, San Leandro, CA, USA), with hGCase band intensity being normalized against GAPDH expression. The in vivo expression assay confirmed the production of hGCase in the secreted portion of the rat milk, with a specific band between 50 to 60 kDa observed on the Western blot, and no detection of the protein in the control milk. The hGCase peak production occurred in Days 5 and 6 of lactation, with levels being 35 times greater than on Day 9. An ELISA quantification assay and an enzymatic activity assay for the recombinant hGCase are currently in development. In conclusion, the use of the rat for hGCase transient expression in the milk was proven a valid model for testing the potential use of a mammary gland expression system for the production of a functional human glucocerebrosidase protein.

scholarly journals The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo

1999 ◽  
Vol 344 (3) ◽  
pp. 929-936 ◽  
Author(s):  
Alexander GRAY ◽  
Jeroen V AN DER KAAY ◽  
C. Peter DOWNES
Keyword(s):  
Protein Kinase ◽  
Fusion Proteins ◽  
Protein Kinase B ◽  
Pleckstrin Homology ◽  
3T3 Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Swiss 3T3

We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P3 levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H2O2). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H2O2. The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz–Jeghers cancer syndrome

2002 ◽  
Vol 362 (2) ◽  
pp. 481-490 ◽  
Author(s):  
Gopal P. SAPKOTA ◽  
Jérôme BOUDEAU ◽  
Maria DEAK ◽  
Agnieszka KIELOCH ◽  
Nick MORRICE ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Wild Type ◽  
Cancer Syndrome ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Increased Risk ◽  
In Cells

Peutz—Jeghers syndrome is an inherited cancer syndrome, which results in a greatly increased risk of developing tumours in those affected. The causative gene encodes a nuclear-localized protein kinase, termed LKB1, which is predicted to function as a tumour suppressor. The mechanism by which LKB1 is regulated in cells is not known, and nor have any of its physiological substrates been identified. Recent studies have demonstrated that LKB1 is phosphorylated in cells. As a first step towards identifying the roles that phosphorylation of LKB1 play, we have mapped the residues that are phosphorylated in human embryonic kidney (HEK)-293 cells, as well as the major in vitro autophosphorylation sites. We demonstrate that LKB1 expressed in HEK-293 cells, in addition to being phosphorylated at Ser431, a previously characterized phosphorylation site, is also phosphorylated at Ser31, Ser325 and Thr366. Incubation of wild-type LKB1, but not a catalytically inactive mutant, with manganese-ATP in vitro resulted in the phosphorylation of LKB1 at Thr336 as well as at Thr366. We were unable to detect autophosphorylation at Thr189, a site previously claimed to be an LKB1 autophosphorylation site. A catalytically inactive mutant of LKB1 was phosphorylated at Ser31 and Ser325 in HEK-293 cells to the same extent as the wild-type enzyme, indicating that LKB1 does not phosphorylate itself at these residues. We show that phosphorylation of LKB1 does not directly affect its nuclear localization or its catalytic activity in vitro, but that its phosphorylation at Thr336, and perhaps to a lesser extent at Thr366, inhibits LKB1 from suppressing cell growth.

scholarly journals AMP-activated protein kinase regulates the vacuolar H+-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney

2013 ◽  
Vol 305 (7) ◽  
pp. F943-F956 ◽  
Author(s):  
Rodrigo Alzamora ◽  
Mohammad M. Al-Bataineh ◽  
Wen Liu ◽  
Fan Gong ◽  
Hui Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Collecting Duct ◽  
Phosphorylation Site ◽  
Intercalated Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
A Subunit ◽  
Amp Activated Protein Kinase

The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibited V-ATPase-dependent H+ secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3−-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.

scholarly journals Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449

2009 ◽  
Vol 284 (24) ◽  
pp. 16562-16574 ◽  
Author(s):  
Rosalyn P. Johnson ◽  
Ahmed F. El-Yazbi ◽  
Morgan F. Hughes ◽  
David C. Schriemer ◽  
Emma J. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Delayed Rectifier ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Maldi Tof ◽  
293 Cells ◽  
Kinase A

Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.

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