Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and GPVI: The role of Pyk2 and c-Cbl
Phosphatidylinositol 3-kinaseβ (PI3Kβ) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kβ activation downstream of integrin αIIbβ3, and promotes the phosphorylation of the PI3K-associated...
| Main Authors: | , , , , , , , , , , |
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| Format: | Journal Article |
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Elsevier
2015
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| Online Access: | http://hdl.handle.net/20.500.11937/19479 |
| _version_ | 1848750045041524736 |
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| author | Manganaro, D. Consonni, A. Guidetti, G. Canobbio, I. Visconte, C. Kim, S. Okigaki, M. Falasca, Marco Hirsch, E. Kunapuli, S. Torti, M. |
| author_facet | Manganaro, D. Consonni, A. Guidetti, G. Canobbio, I. Visconte, C. Kim, S. Okigaki, M. Falasca, Marco Hirsch, E. Kunapuli, S. Torti, M. |
| author_sort | Manganaro, D. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | Phosphatidylinositol 3-kinaseβ (PI3Kβ) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kβ activation downstream of integrin αIIbβ3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kβ upon recruitment of the two main platelet collagen receptors, integrin α2β1 and GPVI. PI3Kβ-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2β1, but occurred normally upon GPVI ligation. Integrin α2β1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kβ activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KβKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kβ activity also hampered GPVI-induced tyrosine–phosphorylation and activation of PLCγ2, preventing intracellular Ca2 + increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2 + increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kβ inhibitor TGX-221. These results outline important differences in the regulation of PI3Kβ by GPVI and integrin α2β1 and suggest that inhibition of Pyk2 may target PI3Kβ activation in a selective context of platelet stimulation. |
| first_indexed | 2025-11-14T07:30:35Z |
| format | Journal Article |
| id | curtin-20.500.11937-19479 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T07:30:35Z |
| publishDate | 2015 |
| publisher | Elsevier |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-194792017-10-02T02:28:05Z Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and GPVI: The role of Pyk2 and c-Cbl Manganaro, D. Consonni, A. Guidetti, G. Canobbio, I. Visconte, C. Kim, S. Okigaki, M. Falasca, Marco Hirsch, E. Kunapuli, S. Torti, M. Phosphatidylinositol 3-kinaseβ (PI3Kβ) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kβ activation downstream of integrin αIIbβ3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kβ upon recruitment of the two main platelet collagen receptors, integrin α2β1 and GPVI. PI3Kβ-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2β1, but occurred normally upon GPVI ligation. Integrin α2β1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kβ activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KβKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kβ activity also hampered GPVI-induced tyrosine–phosphorylation and activation of PLCγ2, preventing intracellular Ca2 + increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2 + increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kβ inhibitor TGX-221. These results outline important differences in the regulation of PI3Kβ by GPVI and integrin α2β1 and suggest that inhibition of Pyk2 may target PI3Kβ activation in a selective context of platelet stimulation. 2015 Journal Article http://hdl.handle.net/20.500.11937/19479 10.1016/j.bbamcr.2015.05.004 Elsevier unknown |
| spellingShingle | Manganaro, D. Consonni, A. Guidetti, G. Canobbio, I. Visconte, C. Kim, S. Okigaki, M. Falasca, Marco Hirsch, E. Kunapuli, S. Torti, M. Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and GPVI: The role of Pyk2 and c-Cbl |
| title | Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and GPVI: The role of Pyk2 and c-Cbl |
| title_full | Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and GPVI: The role of Pyk2 and c-Cbl |
| title_fullStr | Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and GPVI: The role of Pyk2 and c-Cbl |
| title_full_unstemmed | Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and GPVI: The role of Pyk2 and c-Cbl |
| title_short | Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and GPVI: The role of Pyk2 and c-Cbl |
| title_sort | activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin a2ß1 and gpvi: the role of pyk2 and c-cbl |
| url | http://hdl.handle.net/20.500.11937/19479 |