| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
We have examined the molecular mechanisms how SHP-1, a cytosolic protein tyrosine phoshatase, regulates B cell receptor-mediated signaling and obtained the following results.
1)By using substrate trapping approach, we identified SLP-76 as a new substrates of SHP-1 in WEHI-231 cells, a murine immature B cell line. In contrast to the previous reports, our data demonstrated that SLP-76 is expressed in all murine B cell lines tested and in normal splenic B cells. We next examined whether SLP-76 expression in B cell is regulated during differentiation of B cells and found that immature B cells expressed relatively low amount of SLP-76, which showed 〜20% increase in transitional type 1 B cells and 〜40% increase in transitional type 2 and mature B cells, indicating the regulated expression of SLP-76 during B cell development.
2)Dephosphorylation of SLP-76 by SHP-1 inhibits its association with Nck, downregulating JNK activation and exerting positive effect on apoptosis. Knock down of SLP-76 in
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WEHI-231 cells by small interfering RNA attenuated JNK activation but showed little effects on ERK or p38 activation.
3)It has been shown that for T cell activation, SLP-76/Gads complex needs to be translocated to tyrosine-phosphorylated LAT and that both SLP-76 and LAT are required for the restoration of BCR-signaling in BLNK-deficient cells. Contrally, we found the expression of SLP-76 alone is sufficient at least for BCR-induced JNK activation, although WEHI-231 lacks LAT expression. To clarify mechanisms by which SLP-76 functions in the absence of LAT, we first determined the subcellular localization of SLP-76. Although WEHI-231 does not express LAT, SLP-76 localizes in membrane fraction, which increases following B cell receptor (BCR) crosslinking. Further analyses reveals that SLP-76 complexed with Gads is associated with tyrosine-phosphorylated CD22 through the SH2 domains of SLP-76 and Gads. Given that SHP-1 binds to CD22 upon BCR ligation, our findings suggest that dephosphorylation of SLP-76 recruited to CD22 by SHP-1 inhibits BCR-induced JNK activation, dictating apoptosis. Less
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