T cell receptor-induced activation of phospholipase C-gamma1 depends on a sequence-independent function of the P-I region of SLP-76.
SLP-76 forms part of a hematopoietic-specific adaptor protein complex, and is absolutely required for T cell development and activation. T cell receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) depends on three features of SLP-76: the N-terminal tyrosine phosphorylation sites, the Gads-binding site, and an intervening sequence, denoted the P-I region, which binds to the SH3 domain of PLC-gamma1 (SH3(PLC)) via a low affinity interaction. Despite extensive research, the mechanism whereby SLP-76 regulates PLC-gamma1 remains uncertain. In this study, we uncover and explore an apparent paradox: whereas the P-I region as a whole is essential for TCR-induced activation of PLC-gamma1 and nuclear factor of activated T cells (NFAT), no particular part of this region is absolutely required. To better understand the contribution of the P-I region to PLC-gamma1 activation, we mapped the PLC-gamma1-binding site within the region, and created a SLP-76 mutant that fails to bind SH3(PLC), but is fully functional, mediating TCR-induced phosphorylation of PLC-gamma1 at tyrosine 783, calcium flux, and nuclear factor of activated T cells activation. Unexpectedly, full functionality of this mutant was maintained even under less than optimal stimulation conditions, such as a low concentration of the anti-TCR antibody. Another SLP-76 mutant, in which the P-I region was scrambled to abolish any sequence-dependent protein-binding motifs, also retained significant functionality. Our results demonstrate that SLP-76 need not interact with SH3(PLC) to activate PLC-gamma1, and further suggest that the P-I region of SLP-76 serves a structural role that is sequence-independent and is not directly related to protein-protein interactions.
Gonen
Ronnie
R
Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Bat Galim, Haifa 31096, Israel.
Beach
Dvora
D
Ainey
Carmit
C
Yablonski
Deborah
D
D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D020816:Amino Acid Motifs; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry; D017353:Gene Deletion; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000737:chemistry* / Q000502:physiology*; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D000939:Epitopes; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C092947:SLP-76 signal Transducing adaptor proteins; D011392:Proline; D008156:Luciferases; D005982:Glutathione Transferase; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium