Global analysis of dynamic changes in lipid raft proteins during T-cell activation.
Lipid rafts are considered as specialized microdomains within the plasma membrane with unique lipid compositions different from surrounding membranes. Following T-cell receptor (TCR) stimulation, lipid rafts assemble in T-cell/antigen-presenting cell (APC) contact site known as the immunological synapse, inner leaflets of which serve as activation or docking sites for downstream signaling components. To understand the signaling events occurring in lipid rafts, we globally analyzed dynamic changes in lipid raft proteins during TCR/CD28 costimulation using 2-D fluorescence difference gel electrophoresis. We detected multiple spots whose intensities were enhanced after costimulation, and identified proteins in these spots by PMF. Identified proteins include Src family tyrosine kinases, tyrosine phosphatase, phosphatidylinositol 3-kinase (PI3-kinase), actin-binding proteins, and regulators for small GTPases. Of particular interest, a number of pleckstrin homology (PH) domain-containing proteins were identified. Biochemical and histochemical analyses confirmed the translocation of these proteins from cytosol to lipid rafts. We also demonstrated that these proteins assembled at the T-cell/APC interface. These results indicate the efficacy of our system to systematically analyze dynamics of lipid raft proteins during extracellular stimulation.
Kobayashi
Michimoto
M
Division of Cellular Proteomics (BML), Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.
Katagiri
Takuya
T
Kosako
Hidetaka
H
Iida
Naoyuki
N
Hattori
Seisuke
S
D018106:CD28 Antigens / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D018106:CD28 Antigens; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell