MKK6 phosphorylation regulates production of superoxide by enhancing Rac GTPase activity

Maged Harraz, Andrea Park, Duane Abbott, Weihong Zhou, Yulong Zhang, John F. Engelhardt

Research output: Contribution to journalArticle

Abstract

Rac-dependent NADPH oxidases generate reactive oxygen species used in cell signaling and microbial killing or both. Whereas the mechanisms leading to NADPH oxidase activation are fairly well studied, the mechanisms that control downregulation of this enzyme complex remain unclear. We hypothesized that reactive oxygen species produced by NADPH oxidase may autoregulate the complex by inhibiting Rac activity. To this end, we searched for binding partners of Rac1 and identified a tyrosine-phosphorylated fragment of MKK6 that bound to Rac1 under redox-stress conditions. Constitutively active MKK6 interacted directly with Rac1 in vitro, and this interaction was enhanced when MKK6 was phosphorylated on tyrosine 219. Both Rac1 and Rac2 immunoprecipitated an MKK6 fragment under conditions that elevate cellular peroxide levels in 293 and RAW cells, respectively. Constitutively active and wild-type MKK6 enhanced Rac-GTPase activity in vitro, and their overexpression inhibited PMA-induced NADPH oxidase activation in RAW cells. In contrast, a Y219F mutant of MKK6 only partially enhanced Rac1 GTPase activity, and its overexpression did not alter PMA-induced NADPH oxidase activation in RAW cells. Last, MKK6 deficiency led to an increase in Rac1-GTP levels in brain tissue. Our findings suggest that MKK6 downregulates NADPH oxidase activity by enhancing Rac-GTPase activity.

Original languageEnglish (US)
Pages (from-to)1803-1813
Number of pages11
JournalAntioxidants and Redox Signaling
Volume9
Issue number11
DOIs
StatePublished - Oct 1 2007
Externally publishedYes

Fingerprint

Phosphorylation
NADPH Oxidase
GTP Phosphohydrolases
Superoxides
Chemical activation
Tyrosine
Reactive Oxygen Species
Down-Regulation
Cell signaling
Peroxides
Guanosine Triphosphate
Oxidation-Reduction
Brain
Tissue
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

MKK6 phosphorylation regulates production of superoxide by enhancing Rac GTPase activity. / Harraz, Maged; Park, Andrea; Abbott, Duane; Zhou, Weihong; Zhang, Yulong; Engelhardt, John F.

In: Antioxidants and Redox Signaling, Vol. 9, No. 11, 01.10.2007, p. 1803-1813.

Research output: Contribution to journalArticle

Harraz, Maged ; Park, Andrea ; Abbott, Duane ; Zhou, Weihong ; Zhang, Yulong ; Engelhardt, John F. / MKK6 phosphorylation regulates production of superoxide by enhancing Rac GTPase activity. In: Antioxidants and Redox Signaling. 2007 ; Vol. 9, No. 11. pp. 1803-1813.
@article{da2fe53722c049a6ba31dfcb60bf6c09,
title = "MKK6 phosphorylation regulates production of superoxide by enhancing Rac GTPase activity",
abstract = "Rac-dependent NADPH oxidases generate reactive oxygen species used in cell signaling and microbial killing or both. Whereas the mechanisms leading to NADPH oxidase activation are fairly well studied, the mechanisms that control downregulation of this enzyme complex remain unclear. We hypothesized that reactive oxygen species produced by NADPH oxidase may autoregulate the complex by inhibiting Rac activity. To this end, we searched for binding partners of Rac1 and identified a tyrosine-phosphorylated fragment of MKK6 that bound to Rac1 under redox-stress conditions. Constitutively active MKK6 interacted directly with Rac1 in vitro, and this interaction was enhanced when MKK6 was phosphorylated on tyrosine 219. Both Rac1 and Rac2 immunoprecipitated an MKK6 fragment under conditions that elevate cellular peroxide levels in 293 and RAW cells, respectively. Constitutively active and wild-type MKK6 enhanced Rac-GTPase activity in vitro, and their overexpression inhibited PMA-induced NADPH oxidase activation in RAW cells. In contrast, a Y219F mutant of MKK6 only partially enhanced Rac1 GTPase activity, and its overexpression did not alter PMA-induced NADPH oxidase activation in RAW cells. Last, MKK6 deficiency led to an increase in Rac1-GTP levels in brain tissue. Our findings suggest that MKK6 downregulates NADPH oxidase activity by enhancing Rac-GTPase activity.",
author = "Maged Harraz and Andrea Park and Duane Abbott and Weihong Zhou and Yulong Zhang and Engelhardt, {John F.}",
year = "2007",
month = "10",
day = "1",
doi = "10.1089/ars.2007.1579",
language = "English (US)",
volume = "9",
pages = "1803--1813",
journal = "Antioxidants and Redox Signaling",
issn = "1523-0864",
publisher = "Mary Ann Liebert Inc.",
number = "11",

}

TY - JOUR

T1 - MKK6 phosphorylation regulates production of superoxide by enhancing Rac GTPase activity

AU - Harraz, Maged

AU - Park, Andrea

AU - Abbott, Duane

AU - Zhou, Weihong

AU - Zhang, Yulong

AU - Engelhardt, John F.

PY - 2007/10/1

Y1 - 2007/10/1

N2 - Rac-dependent NADPH oxidases generate reactive oxygen species used in cell signaling and microbial killing or both. Whereas the mechanisms leading to NADPH oxidase activation are fairly well studied, the mechanisms that control downregulation of this enzyme complex remain unclear. We hypothesized that reactive oxygen species produced by NADPH oxidase may autoregulate the complex by inhibiting Rac activity. To this end, we searched for binding partners of Rac1 and identified a tyrosine-phosphorylated fragment of MKK6 that bound to Rac1 under redox-stress conditions. Constitutively active MKK6 interacted directly with Rac1 in vitro, and this interaction was enhanced when MKK6 was phosphorylated on tyrosine 219. Both Rac1 and Rac2 immunoprecipitated an MKK6 fragment under conditions that elevate cellular peroxide levels in 293 and RAW cells, respectively. Constitutively active and wild-type MKK6 enhanced Rac-GTPase activity in vitro, and their overexpression inhibited PMA-induced NADPH oxidase activation in RAW cells. In contrast, a Y219F mutant of MKK6 only partially enhanced Rac1 GTPase activity, and its overexpression did not alter PMA-induced NADPH oxidase activation in RAW cells. Last, MKK6 deficiency led to an increase in Rac1-GTP levels in brain tissue. Our findings suggest that MKK6 downregulates NADPH oxidase activity by enhancing Rac-GTPase activity.

AB - Rac-dependent NADPH oxidases generate reactive oxygen species used in cell signaling and microbial killing or both. Whereas the mechanisms leading to NADPH oxidase activation are fairly well studied, the mechanisms that control downregulation of this enzyme complex remain unclear. We hypothesized that reactive oxygen species produced by NADPH oxidase may autoregulate the complex by inhibiting Rac activity. To this end, we searched for binding partners of Rac1 and identified a tyrosine-phosphorylated fragment of MKK6 that bound to Rac1 under redox-stress conditions. Constitutively active MKK6 interacted directly with Rac1 in vitro, and this interaction was enhanced when MKK6 was phosphorylated on tyrosine 219. Both Rac1 and Rac2 immunoprecipitated an MKK6 fragment under conditions that elevate cellular peroxide levels in 293 and RAW cells, respectively. Constitutively active and wild-type MKK6 enhanced Rac-GTPase activity in vitro, and their overexpression inhibited PMA-induced NADPH oxidase activation in RAW cells. In contrast, a Y219F mutant of MKK6 only partially enhanced Rac1 GTPase activity, and its overexpression did not alter PMA-induced NADPH oxidase activation in RAW cells. Last, MKK6 deficiency led to an increase in Rac1-GTP levels in brain tissue. Our findings suggest that MKK6 downregulates NADPH oxidase activity by enhancing Rac-GTPase activity.

UR - http://www.scopus.com/inward/record.url?scp=35448962057&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=35448962057&partnerID=8YFLogxK

U2 - 10.1089/ars.2007.1579

DO - 10.1089/ars.2007.1579

M3 - Article

C2 - 17854274

AN - SCOPUS:35448962057

VL - 9

SP - 1803

EP - 1813

JO - Antioxidants and Redox Signaling

JF - Antioxidants and Redox Signaling

SN - 1523-0864

IS - 11

ER -