|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
Recent studies suggest a functional role for lipid rafts in TCR signaling. Upon TCR engagement, various signaling molecules are shown to be enriched in rafts. Although the fact that molecules involved in TCR signal transduction migrate to rafts following T cell stimulation strongly suggests a functional role of rafts in TCR signaling, it is still possible that the molecules migrate to rafts for purposes other than signal transduction. To test the necessity of rafts in TCR signal transduction, experiments in which the structure of rafts is destroyed by reagents such as methyl-b-cyclodextrin have been performed. This reagent is known to selectively extract cholesterol from cell membranes. It was shown that disruption of raft structure with these reagents impairs early TCR signaling events, indicating that rafts act as functional compartments for the transduction of the TCR signal. However, since cholesterol is also a component of non-raft areas of cell membranes, treatment of cells with
these reagents may have caused disruptions in lymphocyte function that were unrelated to the changes rendered to raft structure.
In this study, we have developed an experimental method where activated phosphatase is targeted to rafts and the subsequent effect on TCR signal transduction is analyzed. SHP-1, a phosphatase with two SH2 domains, is expressed mainly in haematopoietic cells and is generally regarded as a negative regulator of cell signaling. We have constructed a chimeric molecule that localizes the activated form of SHP-1 to rafts. Activated SHP-1 was able to inhibit TCR-mediated T cell activation when it was localized to rafts, whereas the plasma membrane targeting of activated SHP-1 did not induce this inhibition. These results clearly demonstrate that regulated phosphorylation of signaling molecules in rafts is indispensable for progression of the TCR signal. Our results also indicate that raft targeting of activated SHP-1 impairs LAT activation and subsequent downstream signaling events without affecting proximal steps such as tyrosine phosphorylation of TCRzeta and ZAP-70, and the kinase activity of Lck. Moreover, we observed that endogenous SHP-1 recruited to rafts and associated with LAT after TCR engagement, suggesting that SHP-1 is involved in raft-mediated T cell activation. Less