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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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Pervanadate (PV) stimulation induces tyrosine phosphorylation of IκBα but not its proteolytic degradation. (A) ILU-18 cells were either left untreated or treated with Pervanadate (100 μM) or TNF-α (20 ng/mL) for 20 min. At the end of treatment, cytosolic lysates were obtained and 30μg protein from each lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were detected by Western blotting using antibody to nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and enhanced chemiluminescence (ECL). The blot was stripped and re-probed with antibody to β-actin to ensure equal protein loading. (B) ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin [Acla] (0.25 μM) for 2 h. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells either left untreated or treated for 140 min with 0.25 μM Aclacinomycin alone. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42) or (Tyr-305). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading.
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biomolecules-05-00095-f001: Pervanadate (PV) stimulation induces tyrosine phosphorylation of IκBα but not its proteolytic degradation. (A) ILU-18 cells were either left untreated or treated with Pervanadate (100 μM) or TNF-α (20 ng/mL) for 20 min. At the end of treatment, cytosolic lysates were obtained and 30μg protein from each lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were detected by Western blotting using antibody to nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and enhanced chemiluminescence (ECL). The blot was stripped and re-probed with antibody to β-actin to ensure equal protein loading. (B) ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin [Acla] (0.25 μM) for 2 h. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells either left untreated or treated for 140 min with 0.25 μM Aclacinomycin alone. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42) or (Tyr-305). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading.

Mentions: TNFα-mediated activation of NF-κB induction has been demonstrated to invoke serine phosphorylation of the inhibitory IκB proteins followed by ubiquitination and degradation via the 26S proteasome pathway [5]. In contrast, NF-κB activation by pervanadate involves tyrosine phosphorylation of IκBα and is not contigent upon proteasomal degradation of IκBα [6,7]. To test whether PV-mediated activation of NF-κB occurs by a proteasomal-independent mechanism in a murine stromal cell line, we subjected ILU-18 cells to short-term activation with TNFα or PV and then tested cytosolic lysates by immunoblotting with an antibody recognizing IκBα. While IκBα is no longer detected in response to TNFα treatment, IκBα remains in the cytosol following short-term PV treatment, indicating absence of IκBα degradation in PV-induced NF-κB (Figure 1A).


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)

Pervanadate (PV) stimulation induces tyrosine phosphorylation of IκBα but not its proteolytic degradation. (A) ILU-18 cells were either left untreated or treated with Pervanadate (100 μM) or TNF-α (20 ng/mL) for 20 min. At the end of treatment, cytosolic lysates were obtained and 30μg protein from each lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were detected by Western blotting using antibody to nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and enhanced chemiluminescence (ECL). The blot was stripped and re-probed with antibody to β-actin to ensure equal protein loading. (B) ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin [Acla] (0.25 μM) for 2 h. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells either left untreated or treated for 140 min with 0.25 μM Aclacinomycin alone. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42) or (Tyr-305). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4384113&req=5

biomolecules-05-00095-f001: Pervanadate (PV) stimulation induces tyrosine phosphorylation of IκBα but not its proteolytic degradation. (A) ILU-18 cells were either left untreated or treated with Pervanadate (100 μM) or TNF-α (20 ng/mL) for 20 min. At the end of treatment, cytosolic lysates were obtained and 30μg protein from each lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were detected by Western blotting using antibody to nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and enhanced chemiluminescence (ECL). The blot was stripped and re-probed with antibody to β-actin to ensure equal protein loading. (B) ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin [Acla] (0.25 μM) for 2 h. At the end of incubation, cells were washed and cytosolic lysates prepared. As controls, cell lysates were made from ILU-18 cells either left untreated or treated for 140 min with 0.25 μM Aclacinomycin alone. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using a phospho-specific antibody recognizing IκBα (Tyr-42) or (Tyr-305). After stripping, the blot was re-probed with antibody to β-actin to demonstrate equal protein loading.
Mentions: TNFα-mediated activation of NF-κB induction has been demonstrated to invoke serine phosphorylation of the inhibitory IκB proteins followed by ubiquitination and degradation via the 26S proteasome pathway [5]. In contrast, NF-κB activation by pervanadate involves tyrosine phosphorylation of IκBα and is not contigent upon proteasomal degradation of IκBα [6,7]. To test whether PV-mediated activation of NF-κB occurs by a proteasomal-independent mechanism in a murine stromal cell line, we subjected ILU-18 cells to short-term activation with TNFα or PV and then tested cytosolic lysates by immunoblotting with an antibody recognizing IκBα. While IκBα is no longer detected in response to TNFα treatment, IκBα remains in the cytosol following short-term PV treatment, indicating absence of IκBα degradation in PV-induced NF-κB (Figure 1A).

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