Reciprocal Regulation of SH3 and SH2 Domain Binding via Tyrosine Phosphorylation of a Common Site in CD3ε

Abstract

Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3ε. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3ε to Eps8L1 and Nck SH3 domains. CD3ε bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3ε. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3ε in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3ε to interact with either SH3 or SH2 domain-containing proteins.

Cells diversify transmembrane signaling through the controlled chaos of protein disorder

“…Another MIRR, FcεRI, generates the activation signal using 2 ITAMs of the FcεRIβ and FcRγ chains. Most of experimental data in the literature reported to date strongly support the distinct rather than redundant functions for the ITAM signaling modules including those located on the different signaling chains 39,[44][45][46][47][48][49][50][51][52][53][54][55] and those located on the same signaling module (e.g., 3 different ITAMs on TCR ζ chain). 56 Within the SCHOOL model, 10,11,42,43 cytoplasmic homooligomerization controls the ITAM signaling through the different patterns of MIRR signaling subunit homooligomerization 11,43 that produce distinct activation signals provided by different ITAMs.…”

Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands

“…The phage library that we used has previously proven its value in identifying high-affinity SH3 partners for a number of cellular and pathogen-encoded ligand proteins (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). These studies have established the positive predictive value of the hits generated by this screening method to be remarkably high, i.e.…”

“…Because these target proteins are expressed in their native form, this system has the potential to explore binding affinity and specificity contributed by contacts provided by the peptide binding interface, as well as more complex and atypical interactions. The binding affinity required for positive identification of a specific interaction in this discovery system is relatively high (estimated to be in the range of 2 to 5 M), as interactions with dissociation constants higher than 5 M are rarely found (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and unpublished observations). Although this may be seen as a technical limitation when considering that many SH3 interactions with established roles in cell biology are weak such an affinity threshold is also a major experimental advantage by filtering out nonspecific background caused by promiscuous low affinity binding that most SH3 domains exhibit toward a variety of proline-rich sequences.…”

“…Our earlier work on a variety of individual ligand proteins of interest has established the value of this phage library in identifying SH3-mediated interactions that involve distinct affinity and selectivity (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In this study we apply recombination-mediated cloning for expression of hundreds of potential SH3 ligand proteins in cultured human cells to identify preferred SH3-mediated interactions among the human proteome in a high throughput manner.…”

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor

“…Intriguingly, mutating BRS causes augmented preTCR signaling but attenuated TCR signaling, demonstrating the complicated roles of BRS in different T cells. The major function of PRS is to recruit Nck via an atypical PxxDY motif that includes the first tyrosine of the ITAM [23,72,73]. CD3ε-Nck binding inhibits the phosphorylation of both ITAM tyrosines, and conversely the tyrosine phosphorylation inhibits CD3ε-Nck interaction.…”