Figure 7
Figure 7. Binding of a phosphorylated VE-PTP substrate induces VE-cadherin/VE-PTP dissociation. (A) VE-cadherin/VE-PTP association is severely reduced in CHO cells expressing a VE-PTP substrate-trapping (C/S) mutant compared with wild-type (WT) VE-PTP. CHO cells triple-transfected with VE-cadherin, VEGFR-2, and either the WT form or the C/S trapping mutant of VE-PTP was immunoprecipitated for VE-PTP, and the precipitated material was analyzed in immunoblots for VE-cadherin (WB: VE-cadherin) or for VE-PTP (WB: VE-PTP). Bottom panel (WB: VE-cadherin total lysate) shows a VE-cadherin immunoblot of total cell lysates. (B) The cytoplasmic domain of VE-PTP is necessary for its T-cell–induced dissociation from VE-cadherin. Endothelioma cells generated from embryos of either WT mice (VE-PTP+/+) or from VE-PTPmut/mut mice lacking the cytoplasmic domain and the transmembrane domain of VE-PTP were incubated with or without T cells. Also, VE-PTP immunoprecipitates were analyzed in immunoblots for VE-cadherin (WB: VE-cadherin) or for VE-PTP (WB: VE-PTP). Bottom panel (WB: VE-cadherin total lysate) shows a VE-cadherin immunoblot of total cell lysates. (C) A model substrate of VE-PTP can dissociate VE-PTP from VE-cadherin. bEnd.5 cells were incubated without or with a cell-penetrating, phosphorylated Tie2 peptide (phospho-Tyr 992, Tat-pTyr) or the same nonphosphorylated peptide (Tat-Tyr), followed by VE-PTP/VE-cadherin coimmunoprecipitation analysis. (D) Proposed signaling mechanism for the lymphocyte-induced dissociation of VE-PTP from VE-cadherin. Lymphocyte binding to VCAM-1 or stimulation by VEGF triggers the production of ROS via Rac1-mediated activation of NOX. This leads to activation of the redox-sensitive kinase Pyk2 that triggers, directly or indirectly, the phosphorylation of a VE-PTP substrate that, in turn, binds to VE-PTP. This binding may cause structural or conformational changes across the membrane that lead to detachment of the extracellular domain of VE-PTP from VE-cadherin. This facilitates phosphorylation of components or associated factors of the VE-cadherin-catenin complex, which participates in the destabilization of endothelial cell contacts.

Binding of a phosphorylated VE-PTP substrate induces VE-cadherin/VE-PTP dissociation. (A) VE-cadherin/VE-PTP association is severely reduced in CHO cells expressing a VE-PTP substrate-trapping (C/S) mutant compared with wild-type (WT) VE-PTP. CHO cells triple-transfected with VE-cadherin, VEGFR-2, and either the WT form or the C/S trapping mutant of VE-PTP was immunoprecipitated for VE-PTP, and the precipitated material was analyzed in immunoblots for VE-cadherin (WB: VE-cadherin) or for VE-PTP (WB: VE-PTP). Bottom panel (WB: VE-cadherin total lysate) shows a VE-cadherin immunoblot of total cell lysates. (B) The cytoplasmic domain of VE-PTP is necessary for its T-cell–induced dissociation from VE-cadherin. Endothelioma cells generated from embryos of either WT mice (VE-PTP+/+) or from VE-PTPmut/mut mice lacking the cytoplasmic domain and the transmembrane domain of VE-PTP were incubated with or without T cells. Also, VE-PTP immunoprecipitates were analyzed in immunoblots for VE-cadherin (WB: VE-cadherin) or for VE-PTP (WB: VE-PTP). Bottom panel (WB: VE-cadherin total lysate) shows a VE-cadherin immunoblot of total cell lysates. (C) A model substrate of VE-PTP can dissociate VE-PTP from VE-cadherin. bEnd.5 cells were incubated without or with a cell-penetrating, phosphorylated Tie2 peptide (phospho-Tyr 992, Tat-pTyr) or the same nonphosphorylated peptide (Tat-Tyr), followed by VE-PTP/VE-cadherin coimmunoprecipitation analysis. (D) Proposed signaling mechanism for the lymphocyte-induced dissociation of VE-PTP from VE-cadherin. Lymphocyte binding to VCAM-1 or stimulation by VEGF triggers the production of ROS via Rac1-mediated activation of NOX. This leads to activation of the redox-sensitive kinase Pyk2 that triggers, directly or indirectly, the phosphorylation of a VE-PTP substrate that, in turn, binds to VE-PTP. This binding may cause structural or conformational changes across the membrane that lead to detachment of the extracellular domain of VE-PTP from VE-cadherin. This facilitates phosphorylation of components or associated factors of the VE-cadherin-catenin complex, which participates in the destabilization of endothelial cell contacts.

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