Proteomic analysis of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) interactome in resting and activated primary mast cells [corrected]

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 partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells.

Fig. 1.

Expression and function of SLP76^OST in BMMC. A, Diagram of the generation of Slp76^OST/OST BMMC. B, Schematic representation of SLP76^OST molecules. The One-Strep-tag (OST) is composed of two 8-amino acid peptides (in red) separated by a 12-amino acid linker. C, SLP76 expression in Slp76^+/+ and Slp76^OST/OST BMMC. BMMC were solubilized in SDS lysis buffer. Increasing amounts (1×, 2×, and 3×) of lysates were electrophoresed and Western blotted with anti-SLP76 and anti-actin antibodies. D, β-hexosaminidase release. IgE anti-DNP sensitized Slp76^+/+ and Slp76^OST/OST BMMC were challenged with the indicated concentrations of DNP-HSA. β-hexosaminidase was measured in supernatant by an enzymatic assay. E, Intracellular signaling. IgE anti-DNP-sensitized Slp76^+/+ and Slp76^OST/OST BMMC were challenged with or without DNP-HSA for 2 min and lysed in SDS lysis buffer. Equal amounts of proteins were electrophoresed and Western blotted with indicated antibodies.

Fig. 2.

Proteolysis of SLP76 molecules in BMMC. (A, B) BMMC (A) and thymocytes (B) were solubilized in LM lysis buffer or in SDS lysis buffer. Equal amounts of cell lysates were electrophoresed and Western blotted with the indicated antibodies. C, IgE anti-DNP-sensitized BMMC were challenged with DNP-HSA for the indicated times and solubilized in LM lysis buffer. Equal amounts of BMMC lysates were electrophoresed and Western blotted with anti-SLP76 and anti-actin antibodies. D, Equal amounts of proteins from a thymocyte LM lysate, a BMMC LM lysate or a mixture of both were incubated for 10 min on ice, electrophoresed and Western blotted with anti-SLP76 antibodies. E, BMMC were lysed in SDS lysis buffer or in LM lysis buffer containing no inhibitors (1), Roche mixture inhibitors 1× (2), Roche mixture inhibitors 10× (3), Sigma-Adlrich mixture 1× (4) or custom-made mixture inhibitors (21) (5). Lysates were electrophoresed and Western blotted with anti-SLP76 and anti-actin antibodies.

Fig. 3.

A new protease inhibitor mixture prevents SLP76 degradation in mast cells. A, Families of proteases detected in BMMC by transcriptomic analysis. B, Families of metal-binding protease encoded by BMMC metalloprotease genes. C, Screening protease inhibitors that can protect SLP76 in BMMC lysates. BMMC were solubilized in LM lysis buffer containing specific protease inhibitors. Equal amounts of lysates were electrophoresed and Western blotted with anti-SLP76 and anti-actin antibodies. D, Total protection of SLP76 degradation. BMMC were solubilized in LM lysis buffer supplemented with a new custom-made inhibitor mixture for 10 min or 90 min. Equal amounts of lysates were electrophoresed and Western blotted with anti-SLP76 and anti-actin antibodies.

Fig. 4.

Strep-Tactin affinity purification of functional SLP76 protein complexes from BMMC lysates. A, StrepTactin affinity-purification in LM lysates of BMMC from Slp76^+/+ (upper panel) and Slp76^OST/OST (lower panel) mice. Aliquots from each step of the purification procedure were Western blotted with anti-SLP76 antibodies (Input 1%, FT; flow-through 1%, washes 1%; eluates 10%). The upper panels show a schematic of the Strep-Tactin affinity-purification from each cell. B, Copurification of known interacting proteins of SLP76. IgE anti-DNP-sensitized Slp76^+/+ and Slp76^OST/OST BMMC were challenged with DNP-HSA or without for 2 min and lysed. Lysates were subjected to affinity-purification with Strep-Tactin. Eluates were electrophoresed and Western blotted with anti-phosphotyrosine (pY), anti-SLP76, anti-PLCγ-1, anti-Grap2, and anti-Grb2 antibodies.

Fig. 5.

SLP76 protein-protein interaction network in resting and activated mast cells. A, Network view of the SLP76 interactome in mast cells. White nodes: partners identified in resting cells; red nodes: additional partners identified in activated cells. Only proteins significantly associated with SLP76 were included in the network. B, Comparison using Venn diagram of SLP76 partners identified in mast cells in the present study and of previously published SLP76 partners. (C–E) Interactome networks of LAT2 (C), Bcr (D), and Dok-3 (E). Networks were obtained from the STRING database.

Fig. 6.

Validation of the SLP76-Dok-3 interaction in activated mast cells. A, Co-purification of Bcr with SLP76^OST. Slp76^OST/OST BMMC sensitized with IgE anti-DNP were challenged with DNP-HSA (+) or without (−) for 2 min at 37 °C and lysed. Samples of whole cell lysates (WCL) were Western blotted with anti-Dok-3 antibodies (upper panel). Remaining lysates were subjected to affinity-purification with Strep-Tactin. Eluates were electrophoresed and Western blotted with anti-SLP76, anti-phosphotyrosine (pY), and anti-Dok-3 antibodies (lower panel). B, Co-immunoprecipitation of SLP76 and SHIP1 with phosphorylated Dok-3. Slp76^+/+ BMMC sensitized with IgE anti-DNP were challenged with DNP-HSA (+) or without (−) for 2 min at 37 °C and lysed. Samples of whole cell lysates (WCL) were Western blotted with anti-Dok-3 or anti-phospho-PLCγ-1 antibodies (upper panel). Remaining lysates were immunoprecipitated with anti-Dok-3 antibody. Eluates were electrophoresed and Western blotted with anti-Dok-3, anti-phosphotyrosine (pY), anti-SLP76 or anti-SHIP1 antibodies (lower panel). (C, D) Lack of genetic evidence that Dok-3 is involved in FcεRI signaling. Aliquots of BMMC from Dok-3^−/− and from Dok-3^+/+ mice were lysed in SDS. whole cell lysates (WCL) were Western blotted with anti-Dok-3 antibodies and, as positive controls, with anti-Bcr or anti-Grb2 antibodies (C). Aliquots of the same cells were sensitized with IgE anti-DNP, and challenged with the indicated concentrations of DNP-HSA. β-hexosaminidase was measured in supernatant 10 min later (D).

Fig. 7.

Validation of the SLP76-Bcr interaction in activated mast cells. A, Co-purification of Bcr with SLP76^OST. Slp76^OST/OST BMMC sensitized with IgE anti-DNP were challenged with DNP-HSA (+) or without (−) for 2 min at 37 °C and lysed. Samples of whole cell lysates (WCL) were Western blotted with anti-Bcr antibodies. Remaining lysates were subjected to affinity-purification with Strep-Tactin. Eluates were electrophoresed and Western blotted with anti-SLP76, anti-phosphotyrosine (pY), and anti-Bcr antibodies. B, Co-immunoprecipitation of SLP76 with Bcr. Slp76^+/+ BMMC sensitized with IgE anti-DNP were challenged with DNP-HSA (+) or without (−) for 2 min at 37 °C and lysed. Samples of whole cell lysates (WCL) were Western blotted with anti-Bcr or anti-phospho-PLCγ-1 antibodies. Remaining lysates were immunoprecipitated with anti-Bcr antibody. Eluates were electrophoresed and Western blotted with anti-Bcr, or anti-SLP76 antibodies. C, Inducible Bcr phosphorylation in activated mast cells. BMMC sensitized with IgE anti-DNP were challenged with DNP-HSA (+) or without (−) for 2 min at 37 °C and lysed. Samples of whole cell lysates (WCL) were Western blotted with anti-Bcr, anti-pY178 Bcr, anti-phospho-SLP76 or anti-Grb2 antibodies. (D, E) Genetic evidence that Bcr is involved in FcεRI signaling. Aliquots of BMMC from Bcr^−/− and from Bcr^+/+ littermate control mice were lysed in SDS. whole cell lysates (WCL) were Western blotted with anti-Bcr antibodies and, as positive controls, with anti-Dok-3 or anti-Grb2 antibodies (D). Aliquots of the same cells were sensitized with IgE anti-DNP, and challenged with the indicated concentrations of DNP-HSA. β-hexosaminidase was measured in supernatant 10 min later (E).