title: Activation of phosphatidylinositol 3-kinase β by the platelet collagen receptors integrin α2β1 and GPVI: The role of Pyk2 and c-Cbl.
abstract: 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.
authors.affiliation: Department of Biology and Biotechnology, Division of Biochemistry, University of Pavia, Italy.
authors.forename: Daria
authors.initials: D
authors.lastname: Manganaro
authors.affiliation: Department of Biology and Biotechnology, Division of Biochemistry, University of Pavia, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Consonni
authors.affiliation: Department of Biology and Biotechnology, Division of Biochemistry, University of Pavia, Italy.
authors.forename: Gianni F
authors.initials: GF
authors.lastname: Guidetti
authors.affiliation: Department of Biology and Biotechnology, Division of Biochemistry, University of Pavia, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Canobbio
authors.affiliation: Department of Biology and Biotechnology, Division of Biochemistry, University of Pavia, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Visconte
authors.affiliation: Department of Physiology, Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, United States.
authors.forename: Soochong
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Cardiovascular Medicine, Kyoto Prefectural University, Japan.
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Okigaki
authors.affiliation: Metabolic Signalling Group, School of Biomedical Sciences, CHIRI Biosciences, Curtin University, Perth, Western Australia, Australia.
authors.forename: Marco
authors.initials: M
authors.lastname: Falasca
authors.affiliation: Molecular Biotechnology Center, University of Turin, Italy.
authors.forename: Emilio
authors.initials: E
authors.lastname: Hirsch
authors.affiliation: Department of Physiology, Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, United States.
authors.forename: Satya P
authors.initials: SP
authors.lastname: Kunapuli
authors.affiliation: Department of Biology and Biotechnology, Division of Biochemistry, University of Pavia, Italy. Electronic address: mtorti@unipv.it.
authors.forename: Mauro
authors.initials: M
authors.lastname: Torti
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2 / Q000502:physiology*; D006801:Humans; D038982:Integrin alpha2beta1 / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl / Q000502:physiology*; D015398:Signal Transduction
chemical_list: D038982:Integrin alpha2beta1; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D050721:Proto-Oncogene Proteins c-cbl; D019869:Phosphatidylinositol 3-Kinases; D051418:Focal Adhesion Kinase 2
keywords: Collagen receptors; Phosphatidylinositol 3-kinase; Platelets; Signal transduction; Tyrosine kinase