Ancient ubiquitous protein 1 and Syk link cytoplasmic tails of the integrin alpha(IIb)beta(3).
It is currently accepted that activity of the integrin alpha(IIb)beta(3) 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 alpha(IIb)beta(3) (inside-out signaling). We previously reported that ancient ubiquitous protein (Aup1) binds to the membrane-proximal sequence of integrin alphaCTs 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 alpha(IIb)beta(3), Src, Syk, and Aup1 are constitutively complexed. In vitro binding study with recombinant Syk and glutathione (GSH) S-transferase (GST) - Src, -Aup1, and -alpha(IIb) and - beta(3) CTs that are immobilized to GSH- beads revealed direct binding of Syk to Aup1 as well as the beta(3) CT. Dot blot analysis with synthetic peptides for alpha(IIb) and beta(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 alpha(IIb) CT, and suggest that the alpha(IIb) -Aup1- Syk- beta(3) complex formation links alpha(IIb) and beta(3) CTs to sustain alpha(IIb)beta(3) in an inactive state and Syk dissociates from Aup1 after activation.
Kato
Atsushi
A
Division of Hematology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan. akato@musashino.jrc.or.jp
Oshimi
Kazuo
K
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D000072377:Syk Kinase
C464942:AUP1 protein, human; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase