Proteomic Analysis of the SH2Domain-containing Leukocyte Protein of 76 kDa (SLP76) Interactome

Abstract

We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn 2؉ -dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn 2؉ chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both 1 The abbreviations used are: FcRI, high-affinity receptors for the Fc portion of IgE; ADAP, adhesion-and degranulation-promoting adapter protein; Bcr, breakpoint cluster region protein; BMMC, Bone marrow-derived mast cells; DAG, diacyl-glycerol; DNP, Dinitrophenyl; ES, Embryonic stem; FDR, false discovery rate; Fyb, Fyn-binding protein; Grap2, Grb2-related adapter protein 2; Grb2, growth factor receptor-bound protein 2; HSA, human serum albumin; iBAQ, intensity-based absolute quantification; IP3, inositol tris-phosphate; ITAM, immunoreceptor tyrosine-based activation motif; Itk, IL-2-inducible T-cell kinase; KI, knock-in; LAT, linker of activation of T cells; LCP2, lymphocyte cytosolic protein 2, LM, laurylmaltoside; mAb, monoclonal antibody; MAP, mitogen-activated protein; MS/MS, tandem mass spectrometry; nanoLC, nano liquid chromatography; Nck, noncatalytic region of tyrosine kinase adapter protein; NTAL, non-T cell activation linker; OST, one-strep-tag; PAG, phosphoprotein associated with glycosphingolipid-enriched microdomains; PLC, phospholipase C; SH, Src homology; SHIP, SH2 domain-containing inositol 5Ј-phosphatase; SKAP, Src kinase-associated phopshoproteins; SLP76, SH2 domain-containing leukocyte protein of 76 kDa; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TCR, T cell receptor; WT, wild-type; ZAP70, zeta-associated protein of 70 kDa. Research

Decoding communication patterns of the innate immune system by quantitative proteomics

“…Further characterization detected proteolytic degradation of the coagulation factor by mast cell chymase for reduced activity during homeostatic and septic states. Earlier work supported this proteomic investigation of mast cell secretomes by profiling the SLP76 (cytosolic adaptor protein that nucleates signaling complexes from immunoreceptors) interactome in resting and activated primary mouse mast cells . Here, affinity purification of SLP76 binding partners detected a transmembrane adaptor LAT2 and a serine/threonine kinase as the most recruited molecules during mast cell activation.…”

Computational Modeling of the Main Signaling Pathways Involved in Mast Cell Activation

“…We illustrate this approach through the visualization of proteomic data on SLP-76 interactome published by Bounab et al (2013) for activated BMMCs (Figure 3). Such coloration facilitates the interpretation of expression and interaction data in the context of the known network.…”

“…Based on available data on co-aggregation of FcεRI with the inhibitory receptor FcγRIIB, we abstracted relevant information from the reaction map to define a regulatory graph. Beyond molecular interactions delineated using low-throughput approaches, we have used the proteomic data reported in Bounab et al (2013), which point to novel SLP76 interactants, some previously reported in T or B cell activation processes, but now specifically identified in mastocytes.…”

Automated inference of Boolean models from molecular interaction maps using CaSQ

“…The CaSQ-inferred model for mast cell activation comprises 73 nodes while the manually built, 47 nodes. The authors of the manually built extracted information from the molecular map, but they also used proteomic data from bone marrow mononuclear cells (BMMCs) reported in Bounab et al (2013) that focused on the SLP-76 protein and its partners. Node comparison revealed that 30 of these nodes are shared between the CaSQ inferred and the manually built models ( Supplementary Table S1 ).…”