Recruitment of Slp-76 to the Membrane and Glycolipid-Enriched Membrane Microdomains Replaces the Requirement for Linker for Activation of T Cells in T Cell Receptor Signaling

Abstract

Two hematopoietic-specific adapters, src homology 2 domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224–244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cγ1 phosphorylation, extracellular signal–regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224–244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.

In vivo disruption of T cell development by expression of a dominant‐negative polypeptide designed to abolish the SLP‐76/Gads interaction

“…Gads recruitment to LAT results in localization of SLP-76 and presumably its binding partners to the membrane. This model of SLP-76 recruitment by Gads is consistent with studies in which expression of membrane-targeted SLP-76 protein in LAT-deficient Jurkat cells restores TCR signaling in these cells [37,39].…”

“…Gads recruitment to LAT results in localization of SLP-76 and presumably its binding partners to the membrane. This model of SLP-76 recruitment by Gads is consistent with studies in which expression of membrane-targeted SLP-76 protein in LAT-deficient Jurkat cells restores TCR signaling in these cells [37,39].More recently, our laboratory generated a 50-amino acid polypeptide that corresponds to the region of SLP-76 that binds to Gads, termed the Gads-binding fragment (GBF) [39]. Expression of the GBF in Jurkat cells specifically disrupts the association of SLP-76 with Gads and functionally blocks SLP-76-dependent TCR signaling.…”

“…SLP-76 has also been implicated in TCR-induced activation of integrins [33]. To ascertain whether the GBF inhibits this SLP-76-mediated function, Jurkat cells were transfected with a murine stem cell virus-based vector that encodes DsRed2 (also referred to as DsRed) fused to GBF or DsRed2 alone as a control [37]. The transfection efficiency in all experiments was similar between DsRed2 and DsRed2-GBF cells.…”

“…After TCR ligation, SLP-76 is recruited to lipid raft microdomains, detergent-insoluble membrane fractions enriched for signaling components [37]. This recruitment is mediated through the constitutive association of SLP-76 with the SH3 domain of Gads.…”

70Z/3 Cbl induces PLCγ1 activation in T lymphocytes via an alternate Lat- and Slp-76-independent signaling mechanism

“…Furthermore, 70Z/3 Cbl can exploit a signaling mechanism distinct from that used by the TCR, resulting in PLCg1 activation in a manner that is independent of Lat and Slp-76. During TCR signaling, Lat, once phosphorylated, acts as a scaffold that recruits PLCg1 via a direct interaction with the SH2N domain of PLCg1 and indirectly via a ternary complex involving phospho-Lat binding to Gads, an interaction of Gads with Slp-76 Boerth et al, 2000), and a constitutive interaction of Slp-76 with the SH3 domain of PLCg1 (Yablonski et al, 2001). Slp-76 plays a role in TCR-induced PLCg1 activation (Yablonski et al, 1998), possibly by recruiting the Tec kinase Itk (Su et al, 1999).…”