Activation of phosphatidylinositol 3-kinase beta by the platelet collagen receptors integrin alpha 2 beta 1 and GPVI: The role of Pyk2 and c-Cbl

作者：Manganaro Dania; Consonni Alessandra; Guidetti Gianni F; Canobbio Ilaria; Visconte Caterina; Kim Soochong; Okigaki Mitsuhiko; Falasca Marco; Hirsch Emilio; Kunapuli Satya P; Torti Mauro^*

来源：Biochimica et Biophysica Acta-Molecular Cell Research, 2015, 1853(8): 1879-1888.

DOI：10.1016/j.bbamcr.2015.05.004

• Platelets
• Signal transduction
• Collagen receptors
• Phosphatidylinositol 3-kinase
• Tyrosine kinase

Phosphatidylinositol 3-kinase beta (PI3K beta) 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 PI3K beta activation downstream of integrin alpha IIb beta 3, and promotes the phosphorylation of the PI3K beta-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3K beta upon recruitment of the two main platelet collagen receptors, integrin alpha 2 beta 1 and GPVI. PI3K beta-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin alpha 2 beta 1, but occurred normally upon GPVI ligation. Integrin 00131 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphotylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3K beta KD 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 PI3K beta KD, but not in PI3K gamma KD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3K beta activity also hampered GPVI-induced tyrosine-phosphorylation and activation of PLO gamma 2, 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 PI3K beta inhibitor TGX-221. These results outline important differences in the regulation of PI3K beta by GPVI and integrin alpha 2 beta 1 and suggest that inhibition of Pyk2 may target PI3K beta activation in a selective context of platelet stimulation.

• 出版日期2015-8