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

  • Cell Biosci. 2021 Feb 8;11(1):32. doi: 10.1186/s13578-021-00532-5.
Hui-Ju Tsai  1 ,  Ju-Chien Cheng  2 ,  Man-Leng Kao  1 ,  Hung-Pin Chiu  1 ,  Yi-Hsuan Chiang  1 ,  Ding-Ping Chen  1  3 ,  Kun-Ming Rau  4  5 ,  Hsiang-Ruei Liao  6  7  8 ,  Ching-Ping Tseng  9  10  11  12
Affiliations
  • 1. Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, 333, Taiwan, Republic of China.
  • 2. Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, 404, Taiwan, Republic of China.
  • 3. Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, 333, Taiwan, Republic of China.
  • 4. Department of Hematology-Oncology, E-Da Cancer Hospital, Kaohsiung, 824, Taiwan, Republic of China.
  • 5. School of Medicine, College of Medicine, I-Shou University, Kaohsiung, 824, Taiwan, Republic of China.
  • 6. Graduate institute of Natural Products, College of Medicine, Chang-Gung University, Taoyuan, 333, Taiwan, Republic of China.
  • 7. Graduate institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, 333, Taiwan, Republic of China.
  • 8. Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan, 333, Taiwan, Republic of China.
  • 9. Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, 333, Taiwan, Republic of China. [email protected].
  • 10. Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, 333, Taiwan, Republic of China. [email protected].
  • 11. Graduate institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, 333, Taiwan, Republic of China. [email protected].
  • 12. Molecular Medicine Research Center, Chang Gung University, Taoyuan, 333, Taiwan, Republic of China. [email protected].
PMID: 33557943 DOI: 10.1186/s13578-021-00532-5
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.

Keywords
Disabled-2; Outside-in signaling; Phosphorylation; Platelet activation.
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