Association of the X-linked Lymphoproliferative Disease Gene Product SAP/SH2D1A with 2B4, a Natural Killer Cell-activating Molecule, Is Dependent on Phosphoinositide 3-Kinase

Rusung Tan,Ala Aoukaty

doi:10.1074/jbc.m112029200

Abstract

Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.

Highlights

Natural killer (NK)1 cells are an integral component of the antiviral immune response acting to limit viral replication through both cellular cytotoxic mechanisms and the secretion of cytokines [1, 2]
SAP/SH2D1A is expressed in both NK cells and T-lymphocytes, and the mutations of SAP/SH2D1A are associated with the immunodeficiency syndrome, X-linked lymphoproliferative disease (XLP) (19 –21)
NK Cell Activation by Anti-2B4 Results in 2B4 Phosphorylation and the Recruitment of SAP/SH2D1A in Normal but Not XLP NK Cells—Previous reports indicate that the activation of NK cell lines by the ligation of 2B4 is followed by 2B4 phosphorylation and the recruitment of SAP/SH2D1A [12, 17]

Summary

Introduction

Natural killer (NK) cells are an integral component of the antiviral immune response acting to limit viral replication through both cellular cytotoxic mechanisms and the secretion of cytokines [1, 2]. Following stimulation, human 2B4 has been shown to be phosphorylated [12, 13] and associate with a number of SH2 proteins including the SHP-1 and SHP-2 phosphatases [12, 14, 15] and p62dok (Dok1) [16] as well as with the linker for the activation of T-lymphocytes (LAT), an adaptor protein that acts as a substrate for SH2 proteins [17]. Consistent with the expression of SAP/ SH2D1A in NK cells and its intracellular association with 2B4, XLP patients manifest defects in NK cytotoxic function, following 2B4 activation [18, 22,23,24].

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