Premium
Development of SH2 probes and pull‐down assays to detect pathogen‐induced, site‐specific tyrosine phosphorylation of the TLR adaptor SCIMP
Author(s) -
Luo Lin,
Tong Samuel J,
Wall Adam A,
Khromykh Tatiana,
Sweet Matthew J,
Stow Jennifer L
Publication year - 2017
Publication title -
immunology and cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.999
H-Index - 104
eISSN - 1440-1711
pISSN - 0818-9641
DOI - 10.1038/icb.2017.10
Subject(s) - signal transducing adaptor protein , phosphorylation , tyrosine phosphorylation , pathogen , tyrosine , sh2 domain , biology , microbiology and biotechnology , chemistry , computational biology , biochemistry
Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen‐activated Toll‐like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses. To analyze site‐specific phosphorylation events, we developed three probes based on the SH2 domains of known SCIMP effectors, and used them for pull‐downs from macrophage extracts. CRISPR‐mediated SCIMP‐deficient RAW264.7 macrophage‐like cells were reconstituted with various phosphorylation‐deficient (Y58F, Y96F, Y120F) SCIMPs, and used to demonstrate the specificity of LPS/TLR4‐induced, site‐specific phosphorylation of SCIMP for the temporal recruitment of the effectors Grb2, Csk and SLP65. Our findings reveal potential for differential SCIMP phosphorylation and specific effectors to influence TLR signaling and inflammatory programs. Furthermore, the use of Csk‐SH2 pull‐downs to identify additional known and new Csk targets in LPS‐activated macrophages reveals the wider utility of our SH2 probes.