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Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
Published on: February 25, 2011
Two dimensional Blue Native-/SDS-PAGE analysis of SLP family adaptor protein complexes
Mahima Swamy1, Yogesh Kulathu, Sandra Ernst
1Department of Molecular Immunology, Biologie III, University of Freiburg and Max Planck-Institut für Immunbiologie, Stübeweg 51, 79108 Freiburg, Germany.
Insights
SH2 domain-containing adaptor proteins like SLP-76 and SLP-65 form protein complexes crucial for lymphocyte activation. These complexes, particularly SLP-76/Gads, are constitutively present, with SLP-65 complexes being phosphorylated upon stimulation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- SH2 domain-containing leukocyte protein (SLP) adaptor proteins are key in antigen-mediated lymphocyte activation.
- These proteins organize multiprotein signaling complexes essential for immune responses.
Purpose of the Study:
- To investigate the number, size, and abundance of protein complexes containing SLP family proteins.
- To understand the role of these complexes in T cell and B cell signaling pathways.
Main Methods:
- Utilized two-dimensional native-/SDS-gel electrophoresis to analyze protein complexes.
- Employed Drosophila S2 cell expression system to reconstitute and study protein complexes.
Main Results:
- In non-stimulated T cells, SLP-76 forms a constitutive ~400 kDa complex with Gads.
- In B cells, SLP-65 exists in a 180 kDa complex and as a monomer; only complexed SLP-65 is phosphorylated upon stimulation.
- Stimulation-induced alterations in SLP complexes were not detected, suggesting transient and low-abundance active signaling complexes.
Conclusions:
- Constitutive SLP-76/Gads complex formation is essential for T cell signaling.
- SLP-65 complex phosphorylation is critical for B cell activation.
- Active signaling complexes in lymphocytes are transient and present in low abundance.
Abstract:
SH2 domain containing leukocyte protein (SLP) adaptor proteins serve a central role in the antigen-mediated activation of lymphocytes by organizing multiprotein signaling complexes. Here, we use two dimensional native-/SDS-gel electrophoresis to study the number, size and relative abundance of protein complexes containing SLP family proteins. In non-stimulated T cells all SLP-76 proteins are in a approximately 400 kDa complex with the small adaptor protein Grb2-like adaptor protein downstream of Shc (Gads), whereas half of Gads is monomeric. This constitutive SLP-76/Gads complex could be reconstituted in Drosophila S2 cells expressing both components, suggesting that it might not contain additional subunits. In contrast, in B cells SLP-65 exists in a 180 kDa complex as well as in monomeric form. Since the complex was not found in S2 cells expressing only SLP-65, it was not di/trimeric SLP-65. Upon antigen-stimulation only the complexed SLP-65 was phosphorylated. Surprisingly, stimulation-induced alteration of SLP complexes could not be detected, suggesting that active signaling complexes form only transiently, and are of low abundance.
