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Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2
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 ph...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869483/ https://www.ncbi.nlm.nih.gov/pubmed/33557943 http://dx.doi.org/10.1186/s13578-021-00532-5 |
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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 |
author_facet | 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 |
author_sort | Tsai, Hui-Ju |
collection | PubMed |
description | 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 (RRRRRRRRRRR(19)APKAPSKKEKK(29)) 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. |
format | Online Article Text |
id | pubmed-7869483 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-78694832021-02-08 Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2 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 Cell Biosci Research 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 (RRRRRRRRRRR(19)APKAPSKKEKK(29)) 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. BioMed Central 2021-02-08 /pmc/articles/PMC7869483/ /pubmed/33557943 http://dx.doi.org/10.1186/s13578-021-00532-5 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research 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 Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2 |
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 | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869483/ https://www.ncbi.nlm.nih.gov/pubmed/33557943 http://dx.doi.org/10.1186/s13578-021-00532-5 |
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