期刊
PLATELETS
卷 20, 期 2, 页码 105-110出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/09537100802641507
关键词
Ancient ubiquitous protein 1(Aup1); integrin IIb3; Src; Syk; inside-out signaling; cytoplasmic tails
资金
- Juntendo University
It is currently accepted that activity of the integrin IIb3 is modulated by direct interaction between its cytoplasmic tails (CTs) and association of cytoplasmic proteins with them, and disruption of the close linkage between CTs leads to activation of IIb3 (inside-out signaling). We previously reported that ancient ubiquitous protein (Aup1) binds to the membrane-proximal sequence of integrin CTs that plays a pivotal role in the inside-out signaling. To explore biological function of Aup1, we examined in this study interaction of Aup1 with Src and Syk that are quickly activated in platelets before fibrinogen binding following thrombin stimulation. By immunoprecipitation assay with resting platelets, we first found that IIb3, Src, Syk, and Aup1 are constitutively complexed. In vitro binding study with recombinant Syk and glutathione (GSH) S-transferase (GST) - Src, -Aup1, and -IIb and - 3 CTs that are immobilized to GSH- beads revealed direct binding of Syk to Aup1 as well as the 3 CT. Dot blot analysis with synthetic peptides for IIb and 3 CTs, and GST-Aup1 and -Src immobilized to PVDF membrane exhibited concordant result with the GST pull-down assay. Immunoprecipitation of platelet lysates 10 seconds after thrombin stimulation, when activity and tyrosine phosphorylation of Syk are maimum, exhibited that active Syk does not coprecipitate with Aup1. In vitro kinase assay with GST-Syk and -Aup1 proteins at the presence or absence of active Src, a potent activator of Syk, revealed that Aup1 does not directly influence activation of Syk by autophosphorylation or tyrosine phosphorylation by Src. These results indicate that Aup1 is an adaptor recruiting Syk to the IIb CT, and suggest that the IIb -Aup1- Syk- 3 complex formation links IIb and 3 CTs to sustain IIb3 in an inactive state and Syk dissociates from Aup1 after activation.
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