Tyrosine phosphorylation of DEP-1/CD148 as a mechanism controlling Src kinase activation, endothelial cell permeability, invasion, and capillary formation

• Published in: Blood 2012-09, Vol.120 (13), p.2745-2756
• Main Authors: Spring, Kathleen, Chabot, Catherine, Langlois, Simon, Lapointe, Line, Trinh, Nguyen Thu Ngan, Caron, Christine, Hebda, Jagoda K., Gavard, Julie, Elchebly, Mounib, Royal, Isabelle
• Format: Article
• Language: English
• Subjects: Antigens, CD - metabolism; Biological and medical sciences; Blotting, Western; Cadherins - metabolism; Capillaries - metabolism; Cell Adhesion; Cell Membrane Permeability; Cell Movement; Cell Proliferation; Cells, Cultured; Cortactin - metabolism; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Fluorescent Antibody Technique; Hematologic and hematopoietic diseases; Human Umbilical Vein Endothelial Cells - cytology; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Immunoprecipitation; Medical sciences; Mutation - genetics; Neoplasm Invasiveness; Neovascularization, Pathologic; Phosphorylation; Receptor-Like Protein Tyrosine Phosphatases, Class 3 - genetics; Receptor-Like Protein Tyrosine Phosphatases, Class 3 - metabolism; Signal Transduction; src-Family Kinases - metabolism; Tyrosine - metabolism; Vascular Endothelial Growth Factor A - metabolism
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2011-12-398040

DEP-1/CD148 is a receptor-like protein tyrosine phosphatase with antiproliferative and tumor-suppressive functions. Interestingly, it also positively regulates Src family kinases in hematopoietic and endothelial cells, where we showed it promotes VE-cadherin–associated Src activation and endothelial cell survival upon VEGF stimulation. However, the molecular mechanism involved and its biologic functions in endothelial cells remain ill-defined. We demonstrate here that DEP-1 is phosphorylated in a Src- and Fyn-dependent manner on Y1311 and Y1320, which bind the Src SH2 domain. This allows DEP-1–catalyzed dephosphorylation of Src inhibitory Y529 and favors the VEGF-induced phosphorylation of Src substrates VE-cadherin and Cortactin. Accordingly, RNA interference (RNAi)–mediated knockdown of DEP-1 or expression of DEP-1 Y1311F/Y1320F impairs Src-dependent biologic responses mediated by VEGF including permeability, invasion, and branching capillary formation. In addition, our work further reveals that above a threshold expression level, DEP-1 can also dephosphorylate Src Y418 and attenuate downstream signaling and biologic responses, consistent with the quiescent behavior of confluent endothelial cells that express the highest levels of endogenous DEP-1. Collectively, our findings identify the VEGF-dependent phosphorylation of DEP-1 as a novel mechanism controlling Src activation, and show this is essential for the proper regulation of permeability and the promotion of the angiogenic response.