Original contributionT cell receptor-stimulated generation of hydrogen peroxide inhibits MEK-ERK activation and lck serine phosphorylation
Introduction
Contrary to the view of reactive oxygen species (ROS) as damaging, toxic agents, it has been established that multiple cell surface receptors promote the intracellular generation of ROS that serve as key second messengers. Thus, stimulation of receptors by agonists such as angiotensin II, PDGF, or TGFβ induces production of ROS, which are required for control of protein kinase activation, gene expression, or cell proliferation. In lymphocytes, stimulation with lectins (conA or PHA) 1, 2, 3, mitogens (PMA or superantigens) 1, 4, 5, anti-CD28 [6], or the anti-CD3 4, 6, 7, have been proposed to induce oxidant generation and were suggested to regulate cell activation, proliferation and/or survival. While a regulatory role for ROS in these systems has been proposed, the mechanism(s) by which they regulate signal transduction and subsequent biological processes has not been well described.
Exposure to exogenous oxidants, sometimes at nontoxic concentrations, has been suggested to activate a number of signaling pathways, such as src-family kinases 8, 9, NF-κB dependent transcription 10, 11, Ras 12, 13, Akt [14], or the JAK-STAT pathway 15, 16. An important redox-sensitive signaling pathway is the MAPK family of protein kinases, including ERK, JNK, and p38. Addition of oxidants to cells has been shown to activate each of these kinases and they have been characterized as stress responsive elements (reviewed in [17]).
The MAPK family of protein kinases represent a group (ERK1/2, JNK, p38, ERK5) of serine/threonine protein kinases that have the unusual characteristic of requiring phosphorylation for activation on both threonine and tyrosine in a T-x-Y motif that is present on an activation loop. This phosphorylation is catalyzed by a group of relatively specific dual specificity kinases (MAPK kinases) that phosphorylate both Thr and Tyr of the respective MAPK and lead to activation of the kinase. The relative specificity of these MAPK kinases has led to a model in which modules exist for the selective activation of MAPKs. Despite this selective interaction, there is significant redundancy in the activation of the MAPK kinases (MEK, MKK) by the upstream MAPK kinase kinase (MEKK). Multiple MEKK enzymes have been identified, including Raf, MEKK1-4, Tpl-2, and ASK, and in many cases they are capable of activating different MEK or MKK. Further diversity is encountered when one considers the number of signals, both intracellular and extracellular, that can lead to activation of different MEKK. For example, the Raf family of protein kinases can be activated by inputs from proteins such as Ras, PKC family members, Src, PAK, or Akt [18]. Thus, in the case of ERK activation in T cells, signals proceed through Raf and MEK, but the upstream activation signals may be derived from multiple upstream pathways [19].
As exogenous oxidants have targeted activation of MAPK pathways, it was not completely unexpected that receptor-stimulated, endogenously generated ROS would also regulate MAPK activation. In fact, PDGF-stimulated ROS production is required for activation of ERK in VSMC or fibroblasts [20]. Also in VSMC, stimulation with angiotensin II-induced production of ROS that were necessary for JNK or p38 MAPK activation 21, 22. Similarly, serotonin (5-HT) exposure induced ROS generation in mesangial cells, fibroblasts and smooth muscle cells that, in all cases, controlled ERK activation 23, 24. Thus, many studies on receptor-stimulated production of ROS have also focused upon their ability to activate members of the MAPK family of enzymes. This family of enzymes is not the only signaling target of oxidants in the cell, however. Receptor-stimulated production of ROS has also been shown to target activation of key signaling molecules such as Ras, Src, STAT1, and Akt (reviewed in 25, 26).
Our previous data has indicated that cross-linking the T cell receptor (TcR) also leads to the rapid production of both superoxide anion and hydrogen peroxide [7]. The goal of the current study was to investigate whether TcR-stimulated production of ROS would regulate certain signaling pathways. The data extend previous results, indicating that TcR cross-linking induces production of hydrogen peroxide within 2–4 min in a MEK1-dependent manner in both Jurkat T cells and primary human T blasts. Furthermore, detailed kinetic analysis of MAPK activation indicated that the rapid generation of hydrogen peroxide served to selectively inhibit TcR-stimulated phosphorylation of MEK and ERK in both cell models. Using pharmacologic antioxidants and overexpression of the thioredoxin-dependent peroxidase, peroxiredoxin II, TcR-induced activation of MEK and ERK was enhanced and sustained with little or no effect on the activation of JNK or Akt. Further analysis of potential ERK substrates suggested that enhanced ERK activation was associated with increased serine phosphorylation of lck. Thus, the data suggest that TcR cross-linking elicits generation of hydrogen peroxide in a MEK-dependent pathway that then acts in a negative feedback loop to limit MEK-ERK activation and subsequent phosphorylation of important regulatory proteins in T cell function.
Section snippets
Reagents
Dihydroethidium (DHE) and dichlorodihydrofluorescein diacetate (DCFDA) were obtained from Molecular Probes (Eugene, OR, USA). Manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) was from Alexis (San Diego, CA, USA), while ebselen, PD98059, U0126, and diphenylene iodonium (DPI) were from Calbiochem (San Diego, CA, USA). N-acetyl-l-cysteine, ionomycin, PMA, rabbit anti-hamster IgG, goat anti-mouse IgG, and all other chemicals were obtained from Sigma (St. Louis, MO, USA). All cell
TcR cross-linking induces MAPK phosphorylation
Our previous data have shown that TcR cross-linking induced rapid generation of both hydrogen peroxide and superoxide anion in mature T cells [7]. To study whether TcR signal transduction pathways are sensitive to reactive oxygen species (ROS), the kinetics of MAPK and Akt activation in Jurkat T cells was determined. The phosphorylation status of these kinases was determined by Western blot analyses using corresponding phospho-specific antibodies, which are proposed to recognize only the
Discussion
Our previous studies have established that TcR cross-linking in mature T cells also induces rapid generation of intracellular ROS [7]. However the molecular signaling mechanisms that are targeted by TcR-stimulated production of oxidants in T cells are poorly understood. The current study extends our previous observations 4, 7 to show that H2O2 production occurs as early as 2–4 min after TcR cross-linking in both cultured cell lines and primary T cells. Using detailed kinetic analysis of MAPK
Abbreviations
DCFDA—dichlorodihydrofluorescein diacetate
DHE—dihydroethidium
MnTBAP—Manganese (III) tetrakis (4-benzoic acid) porphyrin
NAC—N-acetyl-l-cysteine
Prx II—peroxiredoxin II
ROS—reactive oxygen species
TcR—T cell receptor
Acknowledgements
The authors would like to acknowledge Dr. Sue Goo Rhee for expression plasmids and antibodies for Prx II. We thank Cheng-Kui Qu, Greg Carey and Pierre Henkart for critical reading of the manuscript. This project was supported by a Scientist Development Grant (#0030033N) from the American Heart Association and by funds from the American Red Cross.
References (64)
- et al.
Increased oxidative metabolism in PMA-activated lymphocytesa flow cytometric study
Int. J. Immunopharmacol
(1992) - et al.
A signaling pathway coupled to T cell receptor ligation by MMTV superantigen leading to transient activation and programmed cell death
Immunity
(1995) - et al.
Reactive oxygen intermediates activate NF-kappa B in a tyrosine kinase-dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes
Blood
(1993) - et al.
c-Src is required for oxidative stress-mediated activation of big mitogen-activated protein kinase 1
J. Biol. Chem
(1997) - et al.
A redox-triggered ras-effector interaction. Recruitment of phosphatidylinositol 3′-kinase to Ras by redox stress
J. Biol. Chem.
(1998) - et al.
p21ras as a common signaling target of reactive free radicals and cellular redox stress
J. Biol. Chem
(1995) - et al.
Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells
J. Biol. Chem.
(1999) - et al.
p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy
J. Biol. Chem
(1998) - et al.
Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in Jurkat T cells
Arch. Biochem. Biophys
(1995) - et al.
Manganic porphyrins possess catalase activity and protect endothelial cells against hydrogen peroxide-mediated injury
Arch. Biochem. Biophys
(1997)