title: SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling.
abstract: SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues.
authors.affiliation: From the ‡Department of Chemistry, Brown University Providence, RI 02903;
authors.forename: Qinqin
authors.initials: Q
authors.lastname: Ji
authors.affiliation: From the ‡Department of Chemistry, Brown University Providence, RI 02903;
authors.forename: Yiyuan
authors.initials: Y
authors.lastname: Ding
authors.affiliation: From the ‡Department of Chemistry, Brown University Providence, RI 02903; §Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University Providence, RI 02903 art@drsalomon.com.
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Salomon
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C092947:SLP-76 signal Transducing adaptor proteins; D014443:Tyrosine; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human