A cell-free binding assay maps the LSP1 cytoskeletal binding site to the COOH-terminal 30 amino acids.
The leukocyte specific protein 1 or LSP1 is a multi functional protein involved in such divers biological processes as the regulation of neutrophil motility, chemotaxis, adhesion and membrane immunoglobulin M (mIgM) mediated apoptosis of B-lymphocytes. The 330-amino-acid mouse LSP1 protein contains a high-affinity F-actin binding site and in intact cells localizes to the F-actin filament containing cytoskeleton. Here we use a high-speed F-actin co sedimentation assay and transfection experiments in the LSP1- T-lymphoma cell line BW5147 to show that LSP1 interacts with F-actin and the cytoskeleton through residues downstream of amino acid residue 230. We then designed a novel cell-free cytoskeleton binding assay in which a set of GST-LSP1 fusion proteins are allowed to bind to the cytoskeleton in NP-40 soluble lysates of BW5147 cells and are recovered in the low-speed detergent insoluble pellet. Using this assay the cytoskeleton binding site of mouse LSP1 maps to the 300-330 interval. These results will allow the design of LSP1 mutants that do not bind to the cytoskeleton to determine the importance of LSP1 cytoskeleton binding for the diverse functions of LSP1.
Wong
Michael J
MJ
Toronto Western Research Institute, Cell and Molecular Biology Division and the Department of Immunology, University of Toronto, Toronto, ON, Canada M5T 2S8.
Malapitan
Irish A
IA
Sikorski
Barbara A
BA
Jongstra
Jan
J
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D001681:Biological Assay / Q000379:methods; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002474:Cell-Free System / Q000378:metabolism*; D003599:Cytoskeleton / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured
D000199:Actins; D002135:Calcium-Binding Proteins; C489183:LSP1 protein, human; C057701:Lsp1 protein, mouse; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins