| Research Abstract |
MIST is a mast cell-specific signaling protein structurally related to SLP-76 and BLNK/BASH/SLP-65 hematopoietic cell-specific adaptor proteins. By using the BLNK-deficient DT40 chicken B cell system, we constructed the system for analysing the function of MIST in immunoreceptor signaling and also for screening the MIST inhibitors to develop anti-allergic drugs. In this reconstitution system, MIST can partially restore the B cell antigen receptor (BCR) signaling in the BLNK-deficient cells, which requires phosphorylation of the two N-terminal tyrosine residues. Co-expression of LAT with MIST fully restored the BCR signaling, and dispenses with the requirement of the two tyrosines in MIST for BCR signaling. However, some other tyrosine( s ), as well as the SH2 domain and the two proline-rich regions in MIST are still required for full reconstitution of the BCR signaling, in cooperation with LAT. The C-terminal proline-rich region of MIST is dispensable for the LAT-aided full restoration
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of MAP kinase activation, although it is responsible for the interaction with LAT and for the localization in glycolipidenriched microdomains. On the other hand, the N-terminal proline-rich region, which is a binding site of the SH3 domain of PLCg, is essential for BCR signaling. We also demonstrated that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to with the SH2 domain of Src-family protein tyrosine kinases (PTKs), Mist directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP-55 was required for the recruitment of MIST to Lyn. Farthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Taken together, these informations on the function of MIST in immunoreceptor signaling obtained in the present study have established the basis on the future development of antiallergic drugs using this MIST-reconstituted BLNK-deficient DT40 system Less
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