Ubiquitination of Lyn-kinase in rat basophilic leukemia RBL-2H3 cells.
Receptor-mediated signal transduction pathways of cells involved in allergy and inflammations are extremely significant. Lyn is a member of the Src family of non-receptor protein tyrosine kinases and is associated with a number of cell surface receptors, including the B-cell antigen receptor and immunoglobulin E receptor (FcepsilonRI). Lyn is necessary for FcepsilonRI-mediated mast cell activation. To investigate how the level of Lyn is maintained in mast cell activation, it was studied whether Lyn binds to ubiquitin and is ubiquitinated for proteasomal degradation in cells. In the yeast two hybrid system, Lyn specifically interacted with ubiquitin in vivo. Furthermore, Lyn bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. Pulse-chase experiments indicated intracellular degradation of Lyn was associated with the generation of a high molecular weight complex in the presence of proteasome-specific inhibitor, lactacystin. This high molecular weight complex cross-reacted with anti-Lyn and anti-ubiquitin demonstrating the ubiquitination Lyn. Overexpression of Lyn and ubiquitin in COS 7.2 cells also resulted in the ubiquitination of Lyn in the presence of lactacystin, supporting the ubiquitination of Lyn by a proteasome specific pathway.
Bhattacharyya
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Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, NIH Campus, Building 29, Room 304, 29 Lincoln Drive, Bethesda, MD 20892, USA. bhattacharyya@cber.fda.gov
D000818:Animals; D001491:Basophils / Q000276:immunology / Q000378:metabolism*; D019556:COS Cells; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D014452:Ubiquitins / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014452:Ubiquitins; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases