T細胞の活性化に関与する脂質ラフト内シグナル伝達因子の網羅的解析

抄録

Lipid rafts are membrane microdomains enriched in sphingolipid and cholesterol, which regulate numerous cellular events including signal transduction, membrane traffic and viral entry. During T-cell activation, many signaling molecules are shown to be activated in lipid rafts, or to be associated to their components. Following TCR stimulation, Src family tyrosine kinases including Lck and Fyn are activated in lipid rafts, and some types of molecules such as Zap70, PKC-theta and PLC-gamma are recruited onto lipid rafts. To understand the signaling events taking place in lipid raft, alteration of protein constituents in lipid rafts were investigated using proteomic techniques. [Methods] Jurkat T-cells are stimulated with anti-CD3 and anti-CD28 antibodies. Proteins of lipid rafts were isolated by sucrose density gradients using non-ionic detergents. To analyze the difference between the stimulated and unstimulated cells, we employed two-dimensional difference gel electrophoresis (DIGE) system. In this system, the samples from stimulated and unstimulated cells were labeled with different CyDyes, mixed and subjected to 2D electrophoresis. The gel was scanned at the appropriate wavelength for the fluor, and the spot intensities were quantified using imaging analysis software. The protein spots were identified by combination of protease digestion and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. [Results] Several protein spots were observed on 2D-gels, which increased or decreased upon stimulation. By mass-spectrometric analysis, we identified one of them as Lck, which is phosphorylated upon TCR stimulation and undergoes an electrophoretic and isoelectrophoretic mobility shift. Furthermore, several raft-associated proteins were also identified. These results demonstrate that the approach using a combination of DIGE system and mass-spectrometric analysis is a powerful tool for understanding the signaling events on lipid rafts.