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Identification of a Novel Lipid Raft-Targeting Motif in Src Homology 2-Containing Phosphatase 1¹

Mohan Sankarshanan^*, Zhong Ma, Tessy Iype^* and Ulrike Lorenz^2,*,

^* Department of Microbiology and Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, VA 22908

The tyrosine phosphatase Src homology 2-containing phosphatase 1 (SHP-1) is a key negative regulator of TCR-mediated signaling. Previous studies have shown that in T cells a fraction of SHP-1 constitutively localizes to membrane microdomains, commonly referred to as lipid rafts. Although this localization of SHP-1 is required for its functional regulation of T cell activation events, how SHP-1 is targeted to the lipid rafts was unclear. In this study, we identify a novel, six-amino acid, lipid raft-targeting motif within the C terminus of SHP-1 based on several biochemical and functional observations. First, mutations of this motif in the context of full-length SHP-1 result in the loss of lipid raft localization of SHP-1. Second, this motif alone restores raft localization when fused to a mutant of SHP-1 (SHP-1 C) that fails to localize to rafts. Third, a peptide encompassing the 6-mer motif directly binds to phospholipids whereas a mutation of this motif abolishes lipid binding. Fourth, whereas full-length SHP-1 potently inhibits TCR-induced tyrosine phosphorylation of specific proteins, expression of a SHP-1-carrying mutation within the 6-mer motif does not. Additionally, although SHP-1 C was functionally inactive, the addition of the 6-mer motif restored its functionality in inhibiting TCR-induced tyrosine phosphorylation. Finally, this 6-mer mediated targeting of SHP-1 lipid rafts was essential for the function of this phosphatase in regulating IL-2 production downstream of TCR. Taken together, these data define a novel 6-mer motif within SHP-1 that is necessary and sufficient for lipid raft localization and for the function of SHP-1 as a negative regulator of TCR signaling.

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 AI48672 and an award from the Thomas F. and Kate Miller Jeffress Memorial Trust.

² Address correspondence and reprint requests to Dr. Ulrike Lorenz, Department of Microbiology, Jordan Hall 7212, University of Virginia Health System, P.O. Box 800734, Charlottesville, VA 22908. E-mail address: ulorenz{at}virginia.edu

³ Abbreviations used in this paper: LAT, linker for activation of T cells; EGFP, enhanced GFP; HA, hemagglutinin; SH2, Src homology 2; SHP-1, SH2-containing phosphatase 1.