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Negative Regulation of TCR Signaling by Linker for Activation of X Cells via Phosphotyrosine-Dependent and -Independent Mechanisms¹

Michael J. Shapiro^*,, Chau T. Nguyen^*, Haig Aghajanian^*, Weiguo Zhang and Virginia Smith Shapiro^2,*,

^* Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; Department of Immunology, Mayo Clinic, Rochester, MN 55905; and Department of Immunology, Duke University, Durham, NC 27710

The activation of T cells and the initiation of an immune response is tightly controlled through the crosstalk of both positive and negative regulators. Two adaptors that function as negative regulators of T cell activation are adaptor in lymphocytes of unknown function X (ALX) and linker for activation of X cell (LAX). Previously, we showed that T cells from mice deficient in ALX and LAX display similar hyperresponsiveness, with increased IL-2 production and proliferation upon TCR/CD28 stimulation, and that these adaptors physically associate. In this study, we analyze the nature of the association between ALX and LAX. We demonstrate that this association occurs in the absence of TCR/CD28 signaling via a mechanism independent of both tyrosine phosphorylation of LAX and the SH2 domain of ALX. Cotransfection of ALX with LAX resulted in LAX tyrosine phosphorylation in the absence of TCR/CD28 stimulation. ALX-mediated LAX phosphorylation depends upon the ALX SH2 domain, which functions to recruit Lck to LAX. We also show that LAX, like ALX, can inhibit RE/AP reporter activation. However, in contrast to its inhibition of NFAT, the inhibition of RE/AP by LAX is independent of its tyrosine phosphorylation. Therefore, it can be concluded that inhibition of signaling events involved in T cell activation by LAX occurs through mechanisms both dependent on and independent of its tyrosine phosphorylation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant R01 AI054974 (to V.S.S.).

² Address correspondence and reprint requests to Dr. Virginia Smith Shapiro, 401C Guggenheim Building, Department of Immunology, Mayo Clinic, Rochester, MN 55905. E-mail address: shapirov{at}mail.med.upenn.edu

³ Abbreviations used in this paper: LAT, linker for activation of T cells; ALX, adaptor in lymphocytes of unknown function X; LAX, linker for activation of X cells; YFP, yellow fluorescent protein; WT, wild type; WCE, whole cell extract.