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.
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 Cgamma1 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.
Boerth
N J
NJ
Signal Transduction Program, The Leonard and Madlyn Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104-6160, USA.
Sadler
J J
JJ
Bauer
D E
DE
Clements
J L
JL
Gheith
S M
SM
Koretzky
G A
GA
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D006017:Glycolipids; C110810:LAT protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C092947:SLP-76 signal Transducing adaptor proteins; D014443:Tyrosine