Elsevier

Clinical Immunology

Volume 106, Issue 1, January 2003, Pages 41-49
Clinical Immunology

Regular article
Defective activity of ERK-1 and ERK-2 mitogen-activated protein kinases in peripheral blood T lymphocytes from patients with systemic lupus erythematosus: potential role of altered coupling of Ras guanine nucleotide exchange factor hSos to adapter protein Grb2 in lupus T cells

https://doi.org/10.1016/S1521-6616(02)00052-9Get rights and content

Abstract

The integrity of the Ras/Raf/mitogen-activated protein kinase (MAPK) cascade is critical for maintenance of T cell tolerance, a process that fails in patients with systemic lupus erythematosus (SLE). In this study we have examined the activity of mitogen-activated protein kinases ERK-1 and ERK-2 in resting and TCR-activated peripheral blood T lymphocytes from patients with SLE. We also examined the binding of Ras guanine nucleotide exchange factor, human Son of Sevenless (hSos), to cytosolic adapter protein growth factor receptor-bound protein 2. T cells from lupus patients showed diminished catalytic activity and TCR-driven dual phosphorylation of ERK-1 and ERK-2 upon stimulation through the TCR/CD3 receptor, a defect that may be related to altered translocation of hSos to the Ras/Raf membrane complex and diminished nuclear translocation of trans-acting factor AP-1. Defective MAPK activity triggered by TCR/ CD3 activation may alter the coordination of signals needed for normal interleukin-2 production and maintenance of tolerance in lupus T cells.

Introduction

Patients with systemic lupus erythematosus (SLE) exhibit evidences of humoral and cellular self-responses to a wide array of autoantigens, indicating a serious defect in the mechanisms responsible for maintaining peripheral tolerance. The mitogen-activated protein kinase (MAPK) cascade is essential for signaling of various extracellular stimuli from the membrane to the nucleus [1]. Recent studies suggest that the small GTP-binding protein Ras participates in signaling, leading to induction of tolerance in T cells [2], [3]. A blockade of Ras activation [2], [3] and AP-1 nuclear translocation [4] results in an unresponsive or anergic state in CD4+ cells. The Ras/Raf/MAPK pathway is initially triggered by activation of Ras, a step requiring upstream coupling of signaling molecules, including the guanine nucleotide exchange factors human Son of Sevenless (hSos) and Vav, to the growth factor receptor-bound protein 2 (Grb2) and Shc adapter proteins. The formation of these intermolecular complexes allows Grb2 to position hSos in proximity to Ras, allowing the exchange of GDP for GTP, and switching Ras from an inactive to an active form [5]. Ras engagement induces activation of the serine kinase, Raf, which in turn activates the dual specific kinase, MEK-1-2. MEK-1-2 then activates ERK-1 and ERK-2, leading to nuclear translocation of various transcription factors involved in enhancing interleukin 2 (IL-2) gene promoter activity and cell proliferation [5].

Key biochemical events triggered by TCR/CD3 ligation are required for activation of the Ras/Raf complex. The most proximal known event is activation of a number of protein tyrosine kinases (PTKs), including the src family PTK, Lck, and the syk family PTK, ZAP-70. Lck phosphorylates tyrosines within the immunoreceptor tyrosine-based activation motifs (ITAMS) of the CD3 chains. These phosphotyrosines are excellent docking sites for the src homology 2 (SH2) domains of ZAP-70, which is then recruited to the various CD3 chains. ZAP-70 is itself activated further by Lck, leading to the phosphorylation of numerous substrates and the creation of a larger signaling complex nucleated by membrane-associated adapter protein linker of activated T cells (LAT). Importantly, guanine nucleotide exchange factors (such as RasGrp, hSos, and Vav) for several low-molecular-weight GTP binding proteins are brought into this larger signaling complex linking PTK activation with the Ras/Raf/MAPK pathway [6], [7]. Thus, it is clear that defects in postreceptor tyrosine phosphorylation events may alter the coupling of early and more distal signaling steps needed for full Ras activation in T lymphocytes.

We and others have previously shown abnormal patterns of protein tyrosine phosphorylation [8], [9], [10], [11] and expression of key scaffold molecules such as the TCR ζ chain [10], [12], [13]in human lupus T lymphocytes. The goal of this study was to extend these observations to examine the integrity of the Ras/Raf/MAPK pathway in T lymphocytes activated via the TCR/CD3 complex in patients with SLE. To this end, we examined the protein expression, catalytic activity, and tyrosine/threonine phosphorylation of ERK-1 and ERK-2 in immunoprecipitates obtained from resting and activated T lymphocytes from a group of SLE patients and compared this response to that of a group of non-SLE patients with various autoimmune chronic inflammatory conditions and to that of healthy donors. We also measured the coupling to cytosolic adapter protein Grb2 of Ras guanine nucleotide exchange factor hSos, as well as AP-1 nuclear translocation, a distal outcome of MAPK activation in T lymphocytes.

Section snippets

Patient and control populations

Forty-one Hispanic SLE patients, mean age 32 (range 15 to 57), 37 females and 4 males, were classified according to the 1982 revised criteria by the American College of Rheumatology (formerly the American Rheumatism Association) [14]. The patients had a mean disease duration of 94 months (range 4 to 312) and a mean SLEDAI score [15] of 9 (range 0 to 39). At the time of the study 30 patients were receiving variable doses of prednisone (mean dose 28.5 mg daily, range 5–60) or a combination of

Diminished ERK activity in SLE T cells

We first tested TCR-inducible phosphorylation of ERK-1 and ERK-2 in T cell lysates from patients and controls. Increasing concentrations of OKT3 MAb induced augmented dual phosphorylation of ERK-1–2, but the effect was more prominent in control cells (Fig. 1A). We next tested the catalytic activity of ERK-1 and ERK-2 immunoprecipitates in patients and controls by an in vitro kinase assay. With the number of T cells available from our frequently leukopenic patients, we were not consistently

Discussion

In this report we have demonstrated a defect in MAPK activation in T cells from SLE patients in response to stimulation via the TCR/CD3 complex. Time course studies revealed that the diminished MAPK activity by lupus T lymphocytes was not due to altered kinetics of response of ERK-1 and ERK-2, following TCR/CD3 ligation, nor to differences in ERK protein levels between groups. The defective MAPK activation could be circumvented by stimulation with a phorbolester, indicating intrinsic normal

Acknowledgements

We thank Dr. Gary Koretzky (University of Pennsylvania) for donating plasmids containing GST-Grb2 fusion proteins and for critical review of the manuscript. We are grateful to the following colleagues for helping with the clinical study of SLE patients: Consuelo Lopez, M.D., Wilfredo Rodriguez, M.D., Frankie Perez, M.D., Ana J. Rodriguez, M.D., Esther Ettedgui, M.D., and Arelys Diaz, M.D. We thank Marcel Erminy for helping with the art work. We appreciate the diligent work of our secretary,

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    Supported by Grant S1-200000440 from Fondo Nacional de Ciencia, Innovación y Tecnologia (FONACIT), Ministerio de Ciencia y Tecnologia.

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