Involvement of Mitogen-Activated Protein Kinase in Agonist-Induced Phosphorylation of the μ-Opioid Receptor in HEK 293 Cells

The extracellular Ca2+-sensing receptor (CaR) responds to polycations, including Ca2+ and neomycin. This receptor is a physiological regulator of systemic Ca2+ metabolism and may also mediate the toxic effects of hypercalcemia. A number of divalent cations, including Pb2+, Co2+, Cd2+, and Fe2+, are toxic to the kidney, brain, and other tissues where the CaR is expressed. To determine which divalent cations can activate the CaR, we expressed the human CaR in HEK-293 cells and measured activation of phospholipase A2(PLA2) and the mitogen-activated protein kinase p42ERK in response to potential agonists for the receptor. HEK-293 cells expressing the nonfunctional mutant CaR R796W served as controls. Extracellular Ca2+, Ba2+, Cd2+, Co2+, Fe2+, Gd3+, Ni2+, Pb2+, and neomycin activated the CaR, but Hg2+and Fe3+ did not. We analyzed the kinetics of activation of p42ERK and PLA2 by the CaR in response to Ca2+, Co2+, and Pb2+. The EC50 values ranged from ∼0.1 mM for Pb2+ to ∼4.0 mM for Ca2+. The Hill coefficients were >3, indicating multiple cooperative ligand binding sites or subunits. Submaximal concentrations of Ca2+ and Pb2+ were additive for activation of the CaR. The EC50 for Ca2+ or Pb2+ was reduced four- to fivefold by the presence of the other ion. These divalent cations also activated PLA2 via the CaR in Madin-Darby canine kidney cells that stably express the CaR. We conclude that many divalent cations activate the CaR and that their effects are additive. The facts that the CaR is a promiscuous polycation sensor and that the effects of these ions are additive to activate it suggest that the CaR may contribute to the toxicity of some heavy metals such as Pb2+, Cd2+, Co2+, and Fe2+ for the kidney and other tissues where it is expressed.

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543 Opioid Stimulation of Mitogen-Activated Protein Kinase (MAPK) in Enteric Neurons Is Mediated by Dynamin-Dependent μ Opioid Receptor (pOR) Endocytosis

Gastroenterology ◽

10.1016/s0016-5085(13)60363-7 ◽

2013 ◽

Vol 144 (5) ◽

pp. S-99

Author(s):

Celine C. Duraffourd ◽

Laura Anselmi ◽

Catia Sternini

Keyword(s):

Protein Kinase ◽

Opioid Receptor ◽

Mitogen Activated Protein Kinase ◽

Enteric Neurons ◽

Mitogen Activated Protein ◽

Μ Opioid Receptor ◽

Stimulation Of

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Phosphorylation and Agonist-Specific Intracellular Trafficking of an Epitope-Tagged μ-Opioid Receptor Expressed in HEK 293 Cells

Journal of Neurochemistry ◽

10.1046/j.1471-4159.1995.65041636.x ◽

2002 ◽

Vol 65 (4) ◽

pp. 1636-1645 ◽

Cited By ~ 213

Author(s):

James R. Arden ◽

Veronica Segredo ◽

Zaijie Wang ◽

Jelveh Lameh ◽

Wolfgang Sadée

Keyword(s):

Opioid Receptor ◽

Intracellular Trafficking ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Μ Opioid Receptor

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Proteasome Involvement in Agonist-induced Down-regulation of μ and δ Opioid Receptors

Journal of Biological Chemistry ◽

10.1074/jbc.m008054200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 12345-12355 ◽

Cited By ~ 102

Author(s):

Kirti Chaturvedi ◽

Persis Bandari ◽

Norihiro Chinen ◽

Richard D. Howells

Keyword(s):

Opioid Receptor ◽

Opioid Receptors ◽

Proteasome Inhibitors ◽

Metabolic Labeling ◽

Down Regulation ◽

Hek 293 ◽

293 Cells ◽

Ubiquitin Proteasome ◽

Μ Opioid Receptor ◽

Δ Opioid Receptor

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged δ and μ receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [35S]methionine metabolic labeling indicated that the turnover rate of δ receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional Giand Goproteins by pertussis toxin-attenuated down-regulation of the μ opioid receptor, while down-regulation of the δ opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on μ and δ opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced μ and δ receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state μ and δ opioid receptor levels. Immunoprecipitation of μ and δ opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.

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Phosphorylation of the ARE-binding protein DAZAP1 by ERK2 induces its dissociation from DAZ

Biochemical Journal ◽

10.1042/bj20060681 ◽

2006 ◽

Vol 399 (2) ◽

pp. 265-273 ◽

Cited By ~ 23

Author(s):

Simon Morton ◽

Huei-Ting Yang ◽

Ntsane Moleleki ◽

David G. Campbell ◽

Philip Cohen ◽

...

Keyword(s):

Protein Kinase ◽

Rna Binding ◽

Binding Protein ◽

Mitogen Activated Protein Kinase ◽

Raw 264.7 ◽

Raw 264.7 Macrophages ◽

Hek 293 ◽

Extracellular Signal ◽

Mitogen Activated Protein

A protein in RAW 264.7 macrophages, which became phosphorylated in response to LPS (lipopolysaccharide), was identified as the RNA-binding protein called DAZAP1 [DAZ (deleted in azoospermia)-associated protein 1]. The phosphorylation of this protein was prevented by specific inhibition of MKK1 [MAPK (mitogen-activated protein kinase) kinase 1], indicating that it was phosphorylated via the classical MAPK cascade. Further experiments showed that DAZAP1 was phosphorylated stoichiometrically in vitro by ERK2 (extracellular-signal-regulated protein kinase 2) at two Thr-Pro sequences (Thr269 and Thr315), and that both sites became phosphorylated in HEK-293 (human embryonic kidney 293) cells in response to PMA or EGF (epidermal growth factor), or RAW 264.7 macrophages in response to LPS. Phosphorylation induced by each stimulus was prevented by two structurally distinct inhibitors of MKK1 (PD184352 and U0126), demonstrating that DAZAP1 is a physiological substrate for ERK1/ERK2. The mutation of Thr269 and Thr315 to aspartate or the phosphorylation of these residues caused DAZAP1 to dissociate from its binding partner DAZ. DAZ interacts with PABP [poly(A)-binding protein] and thereby stimulates the translation of mRNAs containing short poly(A) tails [Collier, Gorgoni, Loveridge, Cooke and Gray (2005) EMBO J. 24, 2656–2666]. In the present study we have shown that DAZ cannot bind simultaneously to DAZAP1 and PABP, and suggest that the phosphorylation-induced dissociation of DAZ and DAZAP1 may allow the former to stimulate translation by interacting with PABP.

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

Biochemical Journal ◽

10.1042/bj20070231 ◽

2007 ◽

Vol 405 (3) ◽

pp. 591-596 ◽

Cited By ~ 31

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.

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