Two dimensional Blue Native-/SDS-PAGE analysis of SLP family adaptor protein complexes.
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.
Swamy
Mahima
M
Department of Molecular Immunology, Biologie III, University of Freiburg and Max Planck-Institut für Immunbiologie, Stübeweg 51, 79108 Freiburg, Germany.
Kulathu
Yogesh
Y
Ernst
Sandra
S
Reth
Michael
M
Schamel
Wolfgang W A
WW
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis; D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D011947:Receptors, Antigen, B-Cell / Q000819:agonists; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology*
D048868:Adaptor Proteins, Signal Transducing; C113939:B cell linker protein; D002352:Carrier Proteins; C409587:GRAP2 protein, human; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011947:Receptors, Antigen, B-Cell; C092947:SLP-76 signal Transducing adaptor proteins