Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1.
Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.
Beach
Dvora
D
Rappaport Family Institute for Research in the Medical Sciences, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Gonen
Ronnie
R
Bogin
Yaron
Y
Reischl
Ilona G
IG
Yablonski
Deborah
D
D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D017353:Gene Deletion; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D021962:Membrane Microdomains / Q000502:physiology; D051966:Phospholipase C gamma / Q000378:metabolism*; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C092947:SLP-76 signal Transducing adaptor proteins; D051966:Phospholipase C gamma