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Directly Activating the Integrin ?IIb?3 Initiates Outside-In Signaling by Causing ?IIb?3 Clustering.

ABSTRACT: ?IIb?3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the ?3 cytosolic tail. Exogenous peptides designed to interact with the ?IIb transmembrane (TM) domain activate single ?IIb?3 molecules in platelets by binding to the ?IIb TM domain and causing separation of the ?IIb?3 TM domain heterodimer. Here we asked whether directly activating single ?IIb?3 molecules in platelets using the designed peptide anti-?IIb TM also initiates ?IIb?3-mediated outside-in signaling by causing activation of ?3-associated c-Src. Anti-?IIb TM caused activation of ?3-associated c-Src and the kinase Syk, but not the kinase FAK, under conditions that precluded extracellular ligand binding to ?IIb?3. c-Src and Syk are activated by trans-autophosphorylation, suggesting that activation of individual ?IIb?3 molecules can initiate ?IIb?3 clustering in the absence of ligand binding. Consistent with this possibility, incubating platelets with anti-?IIb TM resulted in the redistribution of ?IIb?3 from a homogenous ring located at the periphery of discoid platelets into nodular densities consistent with clustered ?IIb?3. Thus, these studies indicate that not only is resting ?IIb?3 poised to undergo a conformational change that exposes its ligand-binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well.

SUBMITTER: Fong KP

PROVIDER: S-EPMC4882439 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Directly Activating the Integrin αIIbβ3 Initiates Outside-In Signaling by Causing αIIbβ3 Clustering.

Fong Karen P KP Zhu Hua H Span Lisa M LM Moore David T DT Yoon Kyungchul K Tamura Ryo R Yin Hang H DeGrado William F WF Bennett Joel S JS

The Journal of biological chemistry 20160407 22

αIIbβ3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the β3 cytosolic tail. Exogenous peptides designed to interact with the αIIb transmembrane (TM) domain activate single αIIbβ3 molecules in platelets by binding to the αIIb TM domain and causing separation of the αIIbβ3 TM domain heterodimer. Here we asked whether directly activating single αIIbβ3 molecules in platelets using the designed peptide anti-αIIb TM also initiat ...[more]

PMID: 27056329

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Project description:BackgroundBidirectional integrin ?IIb?3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function.ResultsAn antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin ?IIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin ?IIb?3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling.ConclusionsThese findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin ?IIb?3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.

Dataset Information

Directly Activating the Integrin ?IIb?3 Initiates Outside-In Signaling by Causing ?IIb?3 Clustering.

Publications

Directly Activating the Integrin αIIbβ3 Initiates Outside-In Signaling by Causing αIIbβ3 Clustering.

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