title: Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP.
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.
authors.affiliation: Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Lin
authors.initials: L
authors.lastname: Luo
authors.affiliation: Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Tong
authors.affiliation: Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Adam A
authors.initials: AA
authors.lastname: Wall
authors.affiliation: Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Khromykh
authors.affiliation: Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Sweet
authors.affiliation: Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Stow
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D054884:Host-Pathogen Interactions; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D000067996:RAW 264.7 Cells; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000378:metabolism; D051193:Toll-Like Receptors / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014443:Tyrosine; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases