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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 does not impact PV-mediated activation of Src or MEK kinases. ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin (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 left untreated or treated with 0.25 μM Aclacinomycin alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using antibodies to phospho-Src (Tyr-416) and Src (A); or antibodies to phospho-p44/p42 ERK1/2 (Thr-202/Tyr-204) and ERK1/2 (B).
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biomolecules-05-00095-f003: Proteasome inhibition does not impact PV-mediated activation of Src or MEK kinases. ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin (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 left untreated or treated with 0.25 μM Aclacinomycin alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using antibodies to phospho-Src (Tyr-416) and Src (A); or antibodies to phospho-p44/p42 ERK1/2 (Thr-202/Tyr-204) and ERK1/2 (B).

Mentions: To elucidate which family of tyrosine kinases is principally involved in PV-dependent NF-κB activation, we subjected ILU-18 cells to short-term activation with pervanadate (20 min) and then examined Syk and phospho-Syk by immunoblotting with antibodies recognizing Syk & tyrosine-phosphorylated Syk. Though Syk was detected, tyrosine phosphorylation of Syk was not observed following PV treatment in ILU-18 cells (Figure 2B). In contrast, we confirmed that c-Src is indeed phosphorylated following PV stimulation in this cell line by employing an antibody to phospho-Src (Figure 3). Given the PV-mediated phosphorylation of c-Src, we sought to determine if Src kinase was necessary in PV-dependent NF-κB activity. Employing a selective inhibitor of Src, we pretreated cells with Src Kinase Inhibitor I, before subjecting them to PV-stimulation. As depicted in Figure 2C, pretreatment with Src Kinase Inhibitor I significantly inhibited PV-induced NF-κB activity, thus demonstrating the involvement of Src kinase in PV-induced activation of NF-κB. Since c-Src kinase has been implicated in the tyrosine phosphorylation of IκBα, we evaluated if in the ILU-18 cell line, Src kinase plays a role in the tyrosine phosphorylation of IκBα at tyrosine 42. Pretreatment with Src Kinase Inhibitor I abrogated PV-dependent, tyrosine phosphorylation of IκBα (Tyr-42), as determined by immunoblotting with an antibody recognizing phospho-IκBα (Tyr-42) (Figure 2D). Taken together, these results demonstrate that c-Src contributes to PV-mediated NF-κB activation by phosphorylating IκBα at tyrosine 42.


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 does not impact PV-mediated activation of Src or MEK kinases. ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin (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 left untreated or treated with 0.25 μM Aclacinomycin alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using antibodies to phospho-Src (Tyr-416) and Src (A); or antibodies to phospho-p44/p42 ERK1/2 (Thr-202/Tyr-204) and ERK1/2 (B).
© Copyright Policy
Related In: Results  -  Collection

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

biomolecules-05-00095-f003: Proteasome inhibition does not impact PV-mediated activation of Src or MEK kinases. ILU-18 cells were treated with PV (100 μM) for 20 min, with or without prior treatment with Aclacinomycin (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 left untreated or treated with 0.25 μM Aclacinomycin alone for 140 min. Lysates, equalized for 30 μg protein, were resolved using SDS-PAGE, followed by Western blotting using antibodies to phospho-Src (Tyr-416) and Src (A); or antibodies to phospho-p44/p42 ERK1/2 (Thr-202/Tyr-204) and ERK1/2 (B).
Mentions: To elucidate which family of tyrosine kinases is principally involved in PV-dependent NF-κB activation, we subjected ILU-18 cells to short-term activation with pervanadate (20 min) and then examined Syk and phospho-Syk by immunoblotting with antibodies recognizing Syk & tyrosine-phosphorylated Syk. Though Syk was detected, tyrosine phosphorylation of Syk was not observed following PV treatment in ILU-18 cells (Figure 2B). In contrast, we confirmed that c-Src is indeed phosphorylated following PV stimulation in this cell line by employing an antibody to phospho-Src (Figure 3). Given the PV-mediated phosphorylation of c-Src, we sought to determine if Src kinase was necessary in PV-dependent NF-κB activity. Employing a selective inhibitor of Src, we pretreated cells with Src Kinase Inhibitor I, before subjecting them to PV-stimulation. As depicted in Figure 2C, pretreatment with Src Kinase Inhibitor I significantly inhibited PV-induced NF-κB activity, thus demonstrating the involvement of Src kinase in PV-induced activation of NF-κB. Since c-Src kinase has been implicated in the tyrosine phosphorylation of IκBα, we evaluated if in the ILU-18 cell line, Src kinase plays a role in the tyrosine phosphorylation of IκBα at tyrosine 42. Pretreatment with Src Kinase Inhibitor I abrogated PV-dependent, tyrosine phosphorylation of IκBα (Tyr-42), as determined by immunoblotting with an antibody recognizing phospho-IκBα (Tyr-42) (Figure 2D). Taken together, these results demonstrate that c-Src contributes to PV-mediated NF-κB activation by phosphorylating IκBα at tyrosine 42.

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