Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP

Luo, Lin, Tong, Samuel J., Wall, Adam A., Khromykh, Tatiana, Sweet, Matthew J. and Stow, Jennifer L. (2017) Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP. Immunology and Cell Biology, . doi:10.1038/icb.2017.10


Author Luo, Lin
Tong, Samuel J.
Wall, Adam A.
Khromykh, Tatiana
Sweet, Matthew J.
Stow, Jennifer L.
Title Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP
Journal name Immunology and Cell Biology   Check publisher's open access policy
ISSN 1440-1711
0818-9641
Publication date 2017-03-14
Sub-type Article (original research)
DOI 10.1038/icb.2017.10
Open Access Status Not yet assessed
Total pages 7
Place of publication London, United Kingdom
Publisher Nature Publishing Group
Collection year 2018
Language eng
Abstract 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.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
Institute for Molecular Bioscience - Publications
 
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