NDLI: Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2

Content Provider	Springer Nature : BioMed Central
Author	Tsai, Hui-Ju Cheng, Ju-Chien Kao, Man-Leng Chiu, Hung-Pin Chiang, Yi-Hsuan Chen, Ding-Ping Rau, Kun-Ming Liao, Hsiang-Ruei Tseng, Ching-Ping
Abstract	Background Bidirectional integrin αIIbβ3 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. Results An 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 αIIb 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 αIIbβ3 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. Conclusions These findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin αIIbβ3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.
Related Links	https://cellandbioscience.biomedcentral.com/counter/pdf/10.1186/s13578-021-00532-5.pdf
Ending Page	17
Page Count	17
Starting Page	1
File Format	HTM / HTML
ISSN	20453701
DOI	10.1186/s13578-021-00532-5
Journal	Cell & Bioscience
Issue Number	1
Volume Number	11
Language	English
Publisher	BioMed Central
Publisher Date	2021-02-08
Access Restriction	Open
Subject Keyword	Cell Biology Microbiology Stem Cells Neurobiology Proteomics Disabled-2 Phosphorylation Platelet activation Outside-in signaling
Content Type	Text
Resource Type	Article
Subject	Biochemistry, Genetics and Molecular Biology
Journal Impact Factor	6.1/2023
5-Year Journal Impact Factor	7/2023

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