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P4‐216: Fyn kinase regulates phosphorylation and localization of APP and Dab1 to lipid rafts
Author(s) -
Minami S. Sakura,
Hoe Hyang Sook,
Burns Mark P.,
Matsuoka Yasuji,
Rebeck G. William
Publication year - 2009
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2009.04.682
Subject(s) - fyn , lipid raft , dab1 , phosphorylation , chemistry , microbiology and biotechnology , signal transducing adaptor protein , src family kinase , tyrosine phosphorylation , immunoprecipitation , tyrosine kinase , biochemistry , proto oncogene tyrosine protein kinase src , signal transduction , biology , reelin , receptor , gene
Background: We have recently shown that Fyn, a Src family tyrosine kinase, phosphorylates APP, increases cell surface APP, and increases alpha-cleavage of APP. These effects were mediated through Dab1, an adaptor protein whose interaction with APP alters its trafficking and decreases its amyloidogenic processing. However, it is unknown where and how these interactions between APP, Fyn, and Dab1 take place. Fyn is highly enriched in lipid rafts, suggesting that its effect on APP and Dab1 may be compartmentally regulated. Methods: We isolated lipid rafts from wild-type and Fyn knock-out mouse brains in 1% Triton-X using a sucrose gradient and ultracentrifuged for 12 hrs at 4 C at 33,000rpm (Beckman S4w40Ti). Co-immunoprecipitations were conducted with 200mg lysate, 20mL Sepharose beads, and 1mL antibody. Primary neurons (DIV14) were treated with 100x concentrated Reelin or control media for 20min, 1hr, or 4hrs. Results: A large fraction of Dab1 and APP was found in lipid rafts of wild-type mice, but significantly lower fractions were found in lipid rafts of knock-out mice. Co-immunoprecipitation revealed similar levels of interaction between APP and Dab1 in and out of lipid rafts in wild-type mice. Surprisingly, despite the decreased fraction of APP and Dab1 in lipid rafts of Fyn knock-out mice, interaction between APP and Dab1 occurred predominantly in lipid rafts. We found that tyrosine-phosphorylated Dab1 and APP were found only in lipid rafts of wild-type mice; however, pDab1 was distributed equally in and out of lipid rafts isolated from Fyn knock-out mice. To determine whether Dab1 phosphorylation by Fyn regulated the APP-Dab1 interaction, we treated primary neurons with Reelin, an extracellular protein that activates Dab1 through Fyn, and measured co-precipitation of APP and Dab1 at various time points. We found that the APPDab1 interaction decreased as Dab1 phosphorylation increasedan effect that recovered over time as levels of p-Dab1 returned to baseline levels. Conclusions: These data suggest that Fyn localizes APP and Dab1 to lipid rafts, and that phosphorylation of Dab1 by Fyn in lipid rafts reduces the interaction between APP and Dab1. Fyn regulation of APP and Dab1 phosphorylation and localization may have significant effects on the trafficking and processing of APP.