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. 2022 May;298(5):101857.
doi: 10.1016/j.jbc.2022.101857. Epub 2022 Mar 22.

The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs

Liping Liu  1 , Richard M Lucas  1 , Jeffrey D Nanson  2 , Yan Li  2 , Jason Whitfield  1 , James E B Curson  1 , Neeraj Tuladhar  1 , Kirill Alexandrov  3 , Mehdi Mobli  4 , Matthew J Sweet  1 , Bostjan Kobe  5 , Jennifer L Stow  6 , Lin Luo  7
Affiliations

The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs

Liping Liu et al. J Biol Chem. 2022 May.

Abstract

Innate immune signaling by Toll-like receptors (TLRs) involves receptor phosphorylation, which helps to shape and drive key inflammatory outputs, yet our understanding of the kinases and mechanisms that mediate TLR phosphorylation is incomplete. Spleen tyrosine kinase (Syk) is a nonreceptor protein tyrosine kinase, which is known to relay adaptive and innate immune signaling, including from TLRs. However, TLRs do not contain the conserved dual immunoreceptor tyrosine-based activation motifs that typically recruit Syk to many other receptors. One possibility is that the Syk-TLR association is indirect, relying on an intermediary scaffolding protein. We previously identified a role for the palmitoylated transmembrane adapter protein SCIMP in scaffolding the Src tyrosine kinase Lyn, for TLR phosphorylation, but the role of SCIMP in mediating the interaction between Syk and TLRs has not yet been investigated. Here, we show that SCIMP recruits Syk in response to lipopolysaccharide-mediated TLR4 activation. We also show that Syk contributes to the phosphorylation of SCIMP and TLR4 to enhance their binding. Further evidence pinpoints two specific phosphorylation sites in SCIMP critical for its interaction with Syk-SH2 domains in the absence of immunoreceptor tyrosine-based activation motifs. Finally, using inhibitors and primary macrophages from SCIMP-/- mice, we confirm a functional role for SCIMP-mediated Syk interaction in modulating TLR4 phosphorylation, signaling, and cytokine outputs. In conclusion, we identify SCIMP as a novel, immune-specific Syk scaffold, which can contribute to inflammation through selective TLR-driven inflammatory responses.

Keywords: SCIMP; Syk recruitment; TLR4 phosphorylation; inflammation; macrophage.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Syk is a novel binding partner of SCIMP.A, pull-downs from LPS-activated RAW264.7 cell extracts. GST-SCIMP-T1 was immobilized to GSH-Sepharose to capture its binding partners in macrophage lysates. Bound proteins were eluted by a protease cleavage elution (PCE) method (28) and separated by SDS–PAGE. Excised bands were identified by LC/MS/MS. B, list of the top hits from the LC/MS/MS analysis of GST-SCIMP-T1 pull-downs. C, GST-SCIMP-T1 pull-down from cell extracts of LPS activated, SCIMP-deficient RAW264.7 cells stably reconstituted with WT-V5-SCIMP. Syk and other binding partners are detected in the pull-down by immunoblotting. D, immunoprecipitation of myc-SCIMP-WT expressed in human PMA-differentiated THP-1 cells (56). Cells were treated with LPS over a time course and lysates made at each time point were used for IP with a Myc antibody and Syk was detected by immunoblotting. Immunoblotting of Lyn was used as a control. E and F, RAW264.7 cells cotransfected with Halo-SCIMP (red) and GFP-Syk (green). Cells were treated with Halo-549 ligand and LPS ligand for 15 min and 10 min, respectively, prior to imaging. The scale bar represents 10 μm or 4 μm. Intensity profiles of surface ruffles across the membrane (dash white line) is quantified for individual channels. Panels C–E are representative of three independent experiments.IP, immunoprecipitation; LPS, lipopolysaccharides; PMA, phorbol 12-myristate 13-acetate; Syk, Spleen tyrosine kinase.
Figure 2
Figure 2
Syk binds to specific tyrosine residues of SCIMP via its dual SH2 domains.A, schematic diagram of Syk domains and its truncation constructs representing the two tandem SH2 domains with or without linker regions. B, GST-Syk-N-SH2 and Syk-C-SH2 proteins were used to pull down SCIMP from LPS-induced primary BMMs and immunoblotted with SCIMP antibody. C, GST-Syk-N-SH2 and Syk-C-SH2 proteins were used to pull down V5-SCIMP in SCIMP-deficient RAW264.7 cell lines reconstituted with wild type WT, Y58F, Y96F, or Y120F V5-SCIMP. D, coimmunoprecipitation of V5-SCIMP WT and Y58F, Y96F, or Y120F V5-SCIMP from RAW264.7 cell lysates with a V5 antibody, followed by immunoblotting for Syk. Panels B–D are representative of three independent experiments. BMMs, bone marrow–derived macrophages; LPS, lipopolysaccharides; SH2, Src homology domain 2; Syk, Spleen tyrosine kinase.
Figure 3
Figure 3
Syk acts downstream of Lyn for SCIMP and TLR4 phosphorylation.A, RAW246.7 cells were pretreated with the Lyn inhibitor SU6656 for 1 h before LPS stimulation. Phospho-Syk was detected by immunoblotting. Syk phosphorylation at the peak time point is presented as mean with SEM (n = 3) and was quantified by student’s t test, ∗p < 0.05. B, V5-SCIMP was immunoprecipitated using the V5 antibody from the extracts of CRISPR-mediated SCIMP-deficient, V5-SCIMP reconstituted RAW264.7 cells. Note, the same eluate was used here (left panel) to probe for SCIMP phosphorylation and in Figure 5B. Cells were pretreated for 1 h with Lyn (SU6656) or Syk (SykIV) inhibitors before addition of LPS followed by harvesting for immunoprecipitation. C, GST-TLR4-TIR protein was immobilized on GSH beads and coincubated with LPS activated macrophage lysates for pull-down. Phospho-tyrosine was detected by immunoblotting in bead eluates. Data for panels A and B are representative of three independent experiments, and panel C is representative of two experiments. LPS, lipopolysaccharide; Syk, Spleen tyrosine kinase; TLR, Toll-like receptor.
Figure 4
Figure 4
SCIMP-TLR interaction in macrophages. Primary BMMs were treated with the TLR2 ligand Pam3CSK4 (A) or the TLR4 ligand LPS (B), cell extracts were then used for immunoprecipitation with a SCIMP antibody, and immunoblotting detected endogenous TLR2 or TLR4, Lyn and a phospho-tyrosine antibody detected phospho-SCIMP. All data are representative of three experiments. C, GST-SCIMP effector-based pull-downs were performed in LPS-treated BMMs using site-specific phosphotyrosine probes (GST-Grb2-SH2, GST-Csk-SH2, and GST-SLP65-SH2) to detect SCIMP phosphorylated at sites at Y58, Y96, and Y120, respectively, shown on three separate gels. BMM lysates represent the input samples used for these pull downs immunoblotted to detect proteins of interest. BMMs, bone marrow–derived macrophages; LPS, lipopolysaccharides; TLR, Toll-like receptor.
Figure 5
Figure 5
Phospho-tyrosine kinase activity enhances TLR4-SCIMP binding. CRISPR-mediated SCIMP-deficient RAW264.7 cells reconstituted with V5-SCIMP were untreated or pretreated with Lyn or Syk inhibitors (SU6656, SykIV) added to cells 1 h before the addition of LPS. Cell extracts were used for (A) pull-down by GST-SCIMP-T1; TLR4 is detected in eluates by immunoblotting and (B) immunoprecipitation of V5-SCIMP with a V5 antibody with TLR4 is detected by immunoblotting. Note the same eluate was used in Figure 3B. C, WT or SCIMP-deficient BMMs were treated with LPS for 30 min. GST-NC-Syk -SH2 was used as a bait to capture endogenous TLR4 detected by immunoblotting. D, GST-TLR4-TIR used to pull down Syk from LPS-induced WT or SCIMP-deficient BMMs; Syk detected by immunoblotting. Data are shown from three independent experiments (mean + SEM, n = 3). Quantification of TLR4-Syk binding was performed by student’s t test, ∗∗p < 0.01. E, GST-NC-Syk SH2 was used to pull down TLR4 in extracts of immortalized WT-, MAL-, TRIF-, or TRIF-/TRAM-deficient BMMs with TLR4 immunoblotted. F, GST-SCIMP-T1 was used to pull down Syk from extracts of immortalized WT-, MAL-, TRIF-, or TRIF-/TRAM-deficient BMMs. All data are representative of three experiments. BMMs, bone marrow–derived macrophages; LPS, lipopolysaccharides; Syk, Spleen tyrosine kinase; TLRs, Toll-like receptors.
Figure 6
Figure 6
Regulation of TLR-induced inflammatory cytokine expression by SCIMP and Syk.A, BMMs were obtained from femurs and tibias of WT and SCIMP-deficient mice and treated with LPS for 4 h before analysis. mRNA levels of different types of cytokines were detected by RT-PCR and normalized relative to a house keeping gene (Hprt). B, WT BMMs were preincubated with Syk inhibitor SykIV for 1 h before LPS treatment for 4 h. Cytokines were detected at the mRNA level by RT-PCR. All data are combined from three independent experiments (n = 3) and are shown as mean + SEM. Statistical analysis was performed using student’s t test via GraphPad Prism (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). LPS, lipopolysaccharides; Syk, Spleen tyrosine kinase; TLRs, Toll-like receptors.
Figure 7
Figure 7
Model of Syk recruitment by SCIMP to TLRs. The transmembrane adapter SCIMP constitutively binds Lyn via a PRD-SH3 interaction. Upon ligand activation of TLR4, activated Lyn kinase phosphorylates SCIMP at three tyrosine sites. The Y96 and Y120 residues then function as two docking sites for the tandem SH2 domains of Syk, which triggers a conformational change of Syk to expose its kinase domain for amplifying SCIMP and TLR4 phosphorylation and enhancing their interaction. SCIMP-scaffolded Syk helps to propagate TLR4 signal transduction to drive proinflammatory cytokine secretion. Syk, Spleen tyrosine kinase; TLRs, Toll-like receptors; SH2, Src homology 2.
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