The covalent modification and regulation of TLR8 in HEK-293 cells stimulated with imidazoquinoline agonists

2007 ◽  
Vol 409 (1) ◽  
pp. 275-287 ◽  
Author(s):  
Raj Rajagopal ◽  
Andrew S. Waller ◽  
James D. Mendoza ◽  
Paul D. Wightman
Keyword(s):  
Immune Response ◽  
Peptide Sequencing ◽  
Covalent Modification ◽  
Regulatory Subunit ◽  
Binding Motif ◽  
Reporter System ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
293 Cells

The mammalian TLRs (Toll-like receptors) mediate the rapid initial immune response to pathogens through recognition of pathogen-associated molecular patterns. The pathogen pattern to which TLR8 responds is ssRNA (single-stranded RNA) commonly associated with ssRNA viruses. TLR8 also responds to small, purine-like molecules including the imidazoquinoline IRMs (immune-response modifiers). The IRMs include molecules that selectively activate TLR7, selectively activate TLR8 or non-selectively activate both TLR7 and TLR8. Using HEK-293 cells (human embryonic kidney cells) stably expressing an NF-κB (nuclear factor κB)/luciferase promoter-reporter system as a model system, we have examined the regulation of TLR8 using the non-selective TLR7/8 agonist, 3M-003. Using conservative tyrosine to phenylalanine site-directed mutation, we show that of the 13 tyrosine residues resident in the cytosolic domain of TLR8, only three appear to be critical to TLR8 signalling. Two of these, Tyr898 and Tyr904, reside in the Box 1 motif and the third, Tyr1048, lies in a YXXM putative p85-binding motif. TLR8 is tyrosine-phosphorylated following 3M-003 treatment and TLR8 signalling is inhibited by tyrosine kinase inhibitors. Treatment with 3M-003 results in the association of the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) with TLR8 and this association is inhibited by tyrosine to phenylalanine mutation of either the YXXM or Box 1 motifs. As a further consequence of activation by 3M-003, TLR8 is modified to yield both higher and lower molecular mass species. These species include a monoubiquitinated form as deduced from ubiquitin peptide sequencing by HPLC/MS/MS (tandem MS).

scholarly journals Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm

2007 ◽  
Vol 406 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Nobuhito Ono ◽  
Ingrid Van der Heijden ◽  
George L. Scheffer ◽  
Koen Van de Wetering ◽  
Elizabeth Van Deemter ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Kidney Cells ◽  
Cell Fractionation ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
Drug Conjugates ◽  
293 Cells ◽  
Boar Sperm ◽  
Alternatively Spliced

The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.

scholarly journals Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cϵ

2007 ◽  
Vol 405 (3) ◽  
pp. 591-596 ◽  
Author(s):  
Jun-ichi Saito ◽  
Shinnosuke Toriumi ◽  
Kenjiro Awano ◽  
Hidenori Ichijo ◽  
Keiichi Sasaki ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Feedback Regulation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Regulation Mechanism ◽  
H2o2 Treatment ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
293 Cells

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFα (tumour necrosis factor α). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cϵ (protein phosphatase 2Cϵ), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cϵ inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cϵ mutant enhanced AP-1 activity. Exogenous PP2Cϵ associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cϵ and ASK1 was also observed in mouse brain extracts. PP2Cϵ directly dephosphorylated ASK1 at Thr845in vitro. In contrast with PP5, PP2Cϵ transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cϵ maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cϵ and PP5 play different roles in H2O2-induced regulation of ASK1 activity.

scholarly journals Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3

2007 ◽  
Vol 403 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Yoko Nakano ◽  
Botond Banfi ◽  
Algirdas J. Jesaitis ◽  
Mary C. Dinauer ◽  
Lee-Ann H. Allen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Targeting ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Regulatory Subunits ◽  
Hek 293 Cells ◽  
293 Cells ◽  
A Subunit ◽  
And Function

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ∼50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22phox. Moreover, the glycosylation and maturation of Nox3 required p22phox expression, suggesting that p22phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.

scholarly journals Holocarboxylase synthetase catalyzes biotinylation of heat shock protein 72, thereby inducing RANTES expression in HEK-293 cells

2013 ◽  
Vol 305 (12) ◽  
pp. C1240-C1245 ◽  
Author(s):  
Jing Xue ◽  
Jie Zhou ◽  
Janos Zempleni
Keyword(s):  
Mass Spectrometry ◽  
Immune Response ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Limited Proteolysis ◽  
Holocarboxylase Synthetase ◽  
Heat Shock Protein 72 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

In a recent mass spectrometry screen, we identified 108 new proteins that were modified endogenously by covalent binding of biotin; members of the heat shock superfamily of proteins, including heat shock protein 72 (HSP72), were overrepresented among the biotinylated proteins. Mammals respond to infections by secreting extracellular HSP72 (eHSP72), which elicits an immune response. Here, using mass spectrometry and site-directed mutagenesis, we identified five biotinylation sites in HSP72. We used coimmunoprecipitation, mass spectrometry, and limited proteolysis assays to demonstrate that HSP72 interacts physically with the protein biotin ligase holocarboxylase synthetase (HLCS), leading to biotinylation of residues K112, K128 K348, K361, K415, and, probably, additional lysines. Finally, we demonstrated that HLCS-dependent biotinylation of eHSP72 increases expression of the chemokine regulated on activation normal T-expressed and presumably secreted (RANTES) by human embryonic kidney (HEK-293) cells. In conclusion, we report a novel endogenous modification of HSP72 and demonstrated that binding of biotin to eHSP72 prepares cells for a strong immune response.

scholarly journals K201 (JTV519) suppresses spontaneous Ca2+ release and [3H]ryanodine binding to RyR2 irrespective of FKBP12.6 association

2007 ◽  
Vol 404 (3) ◽  
pp. 431-438 ◽  
Author(s):  
Donald J. Hunt ◽  
Peter P. Jones ◽  
Ruiwu Wang ◽  
Wenqian Chen ◽  
Jeff Bolstad ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
Fk506 Binding Protein ◽  
293 Cells

K201 (JTV519), a benzothiazepine derivative, has been shown to possess anti-arrhythmic and cardioprotective properties, but the mechanism of its action is both complex and controversial. It is believed to stabilize the closed state of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest, Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292–296]. In the present study, we investigated the effect of K201 on spontaneous Ca2+ release induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac myocytes in a concentration-dependent manner. Treating ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt (wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden death, N4104K, in the absence of FKBP12.6. These observations demonstrate that FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+ release. Our results also suggest that suppression of spontaneous Ca2+ release and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic properties of K201.

Incorporation of a gephyrin-binding motif targets NMDA receptors to gephyrin-rich domains in HEK 293 cells

1999 ◽  
Vol 11 (2) ◽  
pp. 740-744 ◽  
Author(s):  
Stefan Kins ◽  
Jochen Kuhse ◽  
Bodo Laube ◽  
Heinrich Betz ◽  
Joachim Kirsch
Keyword(s):  
Nmda Receptors ◽  
Binding Motif ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1006 ◽  
Author(s):  
Abdalla Abdrabou ◽  
Daniel Brandwein ◽  
Changyu Liu ◽  
Zhixiang Wang
Keyword(s):  
Subcellular Localization ◽  
Signaling Pathways ◽  
Binding Motif ◽  
Rho Proteins ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Rac1 Activity ◽  
Mutual Regulation ◽  
Cytoskeleton Remodeling

Both 14-3-3 proteins (14-3-3s) and Rho proteins regulate cytoskeleton remodeling and cell migration, which suggests a possible interaction between the signaling pathways regulated by these two groups of proteins. Indeed, more and more emerging evidence indicates the mutual regulation of these two signaling pathways. However, all of the data regarding the interaction between Rac1 signaling pathways and 14-3-3 signaling pathways are through either the upstream regulators or downstream substrates. It is not clear if Rac1 could interact with 14-3-3s directly. It is interesting to notice that the Rac1 sequence 68RPLSYP73 is likely a 14-3-3 protein binding motif following the phosphorylation of S71 by Akt. Thus, we hypothesize that Rac1 directly interacts with 14-3-3s. We tested this hypothesis in this research. By using mutagenesis, co-immunoprecipitation (co-IP), Rac1 activity assay, immunoblotting, and indirect immunofluorescence, we demonstrate that 14-3-3s interact with Rac1. This interaction is mediated by Rac1 S71 in both phosphorylation-dependent and -independent manners, but the phosphorylation-dependent interaction is much stronger. Epidermal growth factor (EGF) strongly stimulates the phosphorylation of Rac1 S71 and the interaction between 14-3-3s and Rac1. Mutating S71 to A completely abolishes both phosphorylation-dependent and -independent interactions between 14-3-3s and Rac1. The interaction between 14-3-3s and Rac1 mostly serve to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -σ, and -θ showed interactions with Rac1 in both Cos-7 and HEK 293 cells. 14-3-3γ also binds to Rac1 in HEK 293 cells, but not in Cos-7 cells. We conclude that 14-3-3s interact with Rac1. This interaction is mediated by Rac1 S71 in both phosphorylation-dependent and -independent manners. The interaction between 14-3-3 and Rac1 mostly serves to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -γ, -σ, and -θ interact with Rac1.

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

2003 ◽  
Vol 285 (2) ◽  
pp. C467-C479 ◽  
Author(s):  
Mu-Lan He ◽  
Hana Zemkova ◽  
Taka-aki Koshimizu ◽  
Melanija Tomić ◽  
Stanko S. Stojilkovic
Keyword(s):  
Purinergic Receptors ◽  
Host Cells ◽  
P2x Receptor ◽  
Wild Type ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Voltage Gated ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intracellular Ca

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.

Agouti regulation of intracellular calcium: role of melanocortin receptors

1997 ◽  
Vol 272 (3) ◽  
pp. E379-E384 ◽  
Author(s):  
J. H. Kim ◽  
L. L. Kiefer ◽  
R. P. Woychik ◽  
W. O. Wilkison ◽  
A. Truesdale ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Cell Types ◽  
Maximal Response ◽  
Melanocortin Receptors ◽  
Response Curves ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
293 Cells

Several dominant mutations at the murine agouti locus cause a syndrome of marked obesity and insulin resistance. We have recently reported that intracellular free Ca2+ concentration ([Ca2+]i) is elevated in viable yellow mice. Because [Ca2+]i has a key role in the pathogenesis of insulin resistance, obesity, and hypertension, the role of the purified agouti gene product in regulating [Ca2+]i was evaluated in a number of cell types. Purified murine agouti induced slow, sustained increases in [Ca2+]i in A7r5 vascular smooth muscle cells and 3T3-L1 adipocytes in a dose-dependent fashion. In L6 skeletal myocytes, agouti stimulated an increase in [Ca2+]i with an apparent concentration eliciting 50% of the maximal response (EC50) of 62 nM. This response was substantially inhibited by Ca2+ entry blockade with nitrendipine. To determine whether melanocortin receptors play a role in agouti regulation of [Ca2+]i, we examined the effect of melanocortin peptides and agouti in cells stably transfected with human melanocortin receptors. Human embryonic kidney cells (HEK-293 cells) transfected with either the human melanocortin 1 receptor (MC1R) or melanocortin 3 receptor responded to human agouti with slow, sustained increases in [Ca2+]i, whereas nontransfected HEK-293 cells with no melanocortin receptors did not respond to agouti. Dose-response curves in the MC1R line showed that agouti had an EC50 of 18 nM, which is comparable to that for agouti antagonism of (125)I-Nle,D-Phe-alpha-melanocyte-stimulating hormone binding in the same cell line. This direct effect of agouti on stimulating increases in [Ca2+]i suggests a potential mechanism for agouti-induced insulin resistance.

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