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Redox-regulated pathway of tyrosine phosphorylation underlies NF-κB induction by an atypical pathway independent of the 26S proteasome.

Cullen S, Ponnappan S, Ponnappan U - Biomolecules (2015)

Bottom Line: Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported.Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB.Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. cullensarahjane@gmail.com.

ABSTRACT
Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

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Proteasome inhibition potentiates oxidative stress associated with PV-stimulation. (A) ILU-18 cells were either left untreated or treated with 0.25 µM Acla for 2 h. Following treatment with proteasome inhibitor, cells were washed and briefly incubated in 1X HBSS, prior to incubation with 10 μM H2DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular oxidation was detected following addition of PV (100 µM), as described in the methods section. (B) ILU-18 cells were either left untreated or pretreated with 0.25 µM Acla for 2 h, in the presence or absence of 20 mM NAC. Media was then replaced and cells were selectively treated with PV (100 µM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. (C) ILU-18 cells were treated with 100 µM pervanadate for 18 h, with or without pretreatment for 2 h with 0.25 µM Aclacinomycin. As controls, cells were either left untreated or subjected to treatment with 0.25 µM Aclacinomycin for 20 h. At the end of treatment, cells were lysed; 2 µL of MCB and GST were added to each sample and incubated at 37 °C for 15 min. Fluorescence was measured at an excitation wavelength of 340 nm and emission wavelength of 465 nm. Data obtained from duplicates of at least two independent experiments are presented; values represent mean fluorescence intensity (MFI) ± standard error.
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biomolecules-05-00095-f005: Proteasome inhibition potentiates oxidative stress associated with PV-stimulation. (A) ILU-18 cells were either left untreated or treated with 0.25 µM Acla for 2 h. Following treatment with proteasome inhibitor, cells were washed and briefly incubated in 1X HBSS, prior to incubation with 10 μM H2DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular oxidation was detected following addition of PV (100 µM), as described in the methods section. (B) ILU-18 cells were either left untreated or pretreated with 0.25 µM Acla for 2 h, in the presence or absence of 20 mM NAC. Media was then replaced and cells were selectively treated with PV (100 µM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. (C) ILU-18 cells were treated with 100 µM pervanadate for 18 h, with or without pretreatment for 2 h with 0.25 µM Aclacinomycin. As controls, cells were either left untreated or subjected to treatment with 0.25 µM Aclacinomycin for 20 h. At the end of treatment, cells were lysed; 2 µL of MCB and GST were added to each sample and incubated at 37 °C for 15 min. Fluorescence was measured at an excitation wavelength of 340 nm and emission wavelength of 465 nm. Data obtained from duplicates of at least two independent experiments are presented; values represent mean fluorescence intensity (MFI) ± standard error.

Mentions: While proteasome inhibition has no effect on the phospho-tyrosine dependent mechanisms that regulate PV-mediated induction of NF-κB (Figure 3), it remains unclear how proteasome inhibition impacts the intracellular redox balance, which is also critically necessary for PV-mediated induction of NF-κB. By measuring generation of ROS with the H2DCF-DA probe, we observed that pretreatment with proteasome inhibitors potentiated oxidative stress generated by pervanadate stimulation (Figure 5A). ILU-18 cells treated with proteasome inhibitor alone induced intracellular oxidation near background levels, thereby affirming that proteasome inhibition does not independently induce oxidative stress (Figure 5A). Despite the amplification in intracellular ROS generation, pretreatment with proteasome inhibitor did not intensify cellular tyrosine phosphorylation accompanying PV treatment (Figure 5B). Additionally, pretreatment with proteasome inhibitor did not interfere with redox-dependent tyrosine phosphorylation induced by PV (Figure 5B). Thus, while loss in proteasomal catalytic activity enhances PV-mediated oxidative stress, this effect appears to have no discernable impact on redox-dependent tyrosine phosphorylation by PV.


Redox-regulated pathway of tyrosine phosphorylation underlies NF-κB induction by an atypical pathway independent of the 26S proteasome.

Cullen S, Ponnappan S, Ponnappan U - Biomolecules (2015)

Proteasome inhibition potentiates oxidative stress associated with PV-stimulation. (A) ILU-18 cells were either left untreated or treated with 0.25 µM Acla for 2 h. Following treatment with proteasome inhibitor, cells were washed and briefly incubated in 1X HBSS, prior to incubation with 10 μM H2DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular oxidation was detected following addition of PV (100 µM), as described in the methods section. (B) ILU-18 cells were either left untreated or pretreated with 0.25 µM Acla for 2 h, in the presence or absence of 20 mM NAC. Media was then replaced and cells were selectively treated with PV (100 µM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. (C) ILU-18 cells were treated with 100 µM pervanadate for 18 h, with or without pretreatment for 2 h with 0.25 µM Aclacinomycin. As controls, cells were either left untreated or subjected to treatment with 0.25 µM Aclacinomycin for 20 h. At the end of treatment, cells were lysed; 2 µL of MCB and GST were added to each sample and incubated at 37 °C for 15 min. Fluorescence was measured at an excitation wavelength of 340 nm and emission wavelength of 465 nm. Data obtained from duplicates of at least two independent experiments are presented; values represent mean fluorescence intensity (MFI) ± standard error.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384113&req=5

biomolecules-05-00095-f005: Proteasome inhibition potentiates oxidative stress associated with PV-stimulation. (A) ILU-18 cells were either left untreated or treated with 0.25 µM Acla for 2 h. Following treatment with proteasome inhibitor, cells were washed and briefly incubated in 1X HBSS, prior to incubation with 10 μM H2DCF-DA for 30 min at 37 °C in the dark. At the end of the incubation, cells were washed and resuspended in 1X HBSS. Intracellular oxidation was detected following addition of PV (100 µM), as described in the methods section. (B) ILU-18 cells were either left untreated or pretreated with 0.25 µM Acla for 2 h, in the presence or absence of 20 mM NAC. Media was then replaced and cells were selectively treated with PV (100 µM) for 20 min. At the end of incubation, cells were washed and cytosolic lysates prepared. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using an antibody specific to Phospho-Tyrosine residues. Molecular weights derived from standards are indicated in kDa. (C) ILU-18 cells were treated with 100 µM pervanadate for 18 h, with or without pretreatment for 2 h with 0.25 µM Aclacinomycin. As controls, cells were either left untreated or subjected to treatment with 0.25 µM Aclacinomycin for 20 h. At the end of treatment, cells were lysed; 2 µL of MCB and GST were added to each sample and incubated at 37 °C for 15 min. Fluorescence was measured at an excitation wavelength of 340 nm and emission wavelength of 465 nm. Data obtained from duplicates of at least two independent experiments are presented; values represent mean fluorescence intensity (MFI) ± standard error.
Mentions: While proteasome inhibition has no effect on the phospho-tyrosine dependent mechanisms that regulate PV-mediated induction of NF-κB (Figure 3), it remains unclear how proteasome inhibition impacts the intracellular redox balance, which is also critically necessary for PV-mediated induction of NF-κB. By measuring generation of ROS with the H2DCF-DA probe, we observed that pretreatment with proteasome inhibitors potentiated oxidative stress generated by pervanadate stimulation (Figure 5A). ILU-18 cells treated with proteasome inhibitor alone induced intracellular oxidation near background levels, thereby affirming that proteasome inhibition does not independently induce oxidative stress (Figure 5A). Despite the amplification in intracellular ROS generation, pretreatment with proteasome inhibitor did not intensify cellular tyrosine phosphorylation accompanying PV treatment (Figure 5B). Additionally, pretreatment with proteasome inhibitor did not interfere with redox-dependent tyrosine phosphorylation induced by PV (Figure 5B). Thus, while loss in proteasomal catalytic activity enhances PV-mediated oxidative stress, this effect appears to have no discernable impact on redox-dependent tyrosine phosphorylation by PV.

Bottom Line: Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported.Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB.Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. cullensarahjane@gmail.com.

ABSTRACT
Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

Show MeSH
Related in: MedlinePlus