Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2
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-seri...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2021-02-01
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| Series: | Cell & Bioscience |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13578-021-00532-5 |
| _version_ | 1818459033174016000 |
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| author | Hui-Ju Tsai Ju-Chien Cheng Man-Leng Kao Hung-Pin Chiu Yi-Hsuan Chiang Ding-Ping Chen Kun-Ming Rau Hsiang-Ruei Liao Ching-Ping Tseng |
| author_facet | Hui-Ju Tsai Ju-Chien Cheng Man-Leng Kao Hung-Pin Chiu Yi-Hsuan Chiang Ding-Ping Chen Kun-Ming Rau Hsiang-Ruei Liao Ching-Ping Tseng |
| author_sort | Hui-Ju Tsai |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T23:07:55Z |
| format | Article |
| id | doaj.art-5a1e6dd35ef346cba8f30b10bf7c7740 |
| institution | Directory Open Access Journal |
| issn | 2045-3701 |
| language | English |
| last_indexed | 2024-12-14T23:07:55Z |
| publishDate | 2021-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell & Bioscience |
| spelling | doaj.art-5a1e6dd35ef346cba8f30b10bf7c77402022-12-21T22:44:17ZengBMCCell & Bioscience2045-37012021-02-0111111710.1186/s13578-021-00532-5Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2Hui-Ju Tsai0Ju-Chien Cheng1Man-Leng Kao2Hung-Pin Chiu3Yi-Hsuan Chiang4Ding-Ping Chen5Kun-Ming Rau6Hsiang-Ruei Liao7Ching-Ping Tseng8Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung UniversityDepartment of Medical Laboratory Science and Biotechnology, China Medical UniversityDepartment of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung UniversityDepartment of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung UniversityDepartment of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung UniversityDepartment of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung UniversityDepartment of Hematology-Oncology, E-Da Cancer HospitalGraduate institute of Natural Products, College of Medicine, Chang-Gung UniversityDepartment of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung UniversityAbstract 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.https://doi.org/10.1186/s13578-021-00532-5Disabled-2PhosphorylationPlatelet activationOutside-in signaling |
| spellingShingle | Hui-Ju Tsai Ju-Chien Cheng Man-Leng Kao Hung-Pin Chiu Yi-Hsuan Chiang Ding-Ping Chen Kun-Ming Rau Hsiang-Ruei Liao Ching-Ping Tseng Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2 Cell & Bioscience Disabled-2 Phosphorylation Platelet activation Outside-in signaling |
| title | Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2 |
| title_full | Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2 |
| title_fullStr | Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2 |
| title_full_unstemmed | Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2 |
| title_short | Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2 |
| title_sort | integrin αiibβ3 outside in signaling activates human platelets through serine 24 phosphorylation of disabled 2 |
| topic | Disabled-2 Phosphorylation Platelet activation Outside-in signaling |
| url | https://doi.org/10.1186/s13578-021-00532-5 |
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