Characterization of the B cell-specific adaptor SLP-65 and other protein tyrosine kinase substrates by two-dimensional gel electrophoresis.
The identification of substrates for protein tyrosine kinases in B cells is a critical step to a better understanding of the molecular mechanism(s) of lymphocyte activation through the antigen receptor. The substrate proteins were immunopurified from stimulated B cells and separated by two-dimensional gel electrophoresis techniques using either the isoelectric focussing (IEF)/SDS-PAGE or the non-equilibrium PH gradient electrophoresis (NEPHGE)/SDS-PAGE method. The biochemical characteristics of the proteins (isoelectric point and relative molecular mass) obtained and the subsequent use of antibodies that are specific for different cellular proteins confirmed the participation of HS1, Vav, Ig-alpha, Lyn and Btk in antigen receptor-mediated signal transduction. The heat shock cognate protein HSC70 was identified as a novel substrate protein in activated B cells. An important signaling function has previously been suggested for a 65-kDa protein (p65), whose phosphorylation can be detected before that of other substrate proteins. The analysis identified p65 as a so far unknown protein. Based on p65 peptide sequences, the full length cDNA was isolated and found to encode a B cell-specific adaptor protein, called SLP-65.
Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck-Institute for Immunobiology, Germany.
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Wollscheid
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Reth
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Wienands
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D016393:Lymphoma, B-Cell; D051379:Mice; D009101:Multiple Myeloma; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D000954:Antigens, Surface; C113939:B cell linker protein; D002352:Carrier Proteins; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases