title: VEGFR2 functions as an H2S-targeting receptor protein kinase with its novel Cys1045-Cys1024 disulfide bond serving as a specific molecular switch for hydrogen sulfide actions in vascular endothelial cells.
abstract: OBJECTIVE
  The potential receptor for hydrogen sulfide (H2S) remains unknown.
  
  
  RESULTS
  H2S could directly activate vascular endothelial growth factor receptor 2 (VEGFR2) and that a small interfering RNA (siRNA)-mediated knockdown of VEGFR2 inhibited H2S-induced migration of human vascular endothelial cells. H2S promoted angiogenesis in Matrigel plug assay in mice and this effect was attenuated by a VEGF receptor inhibitor. Using tandem mass spectrometry (MS), we identified a new disulfide complex located between Cys1045 and Cys1024 within VEGFR2 that was labile to H2S-mediated modification. Kinase activity of the mutant VEGFR2 (C1045A) devoid of the Cys1045-Cys1024 disulfide bond was significantly higher than wild-type VEGFR2. Transfection with vectors expressing VEGFR2 (C1045A) caused a significant increase in cell migration, while the migration-promoting effect of H2S disappeared in the cells transfected with VEGFR2 (C1045A). Therefore, the Cys1045-Cys1024 disulfide bond serves as an intrinsic inhibitory motif and functions as a molecular switch for H2S. The formation of the Cys1045-Cys1024 disulfide bond disrupted the integrity of the active conformation of VEGFR2. Breaking the Cys1045-Cys1024 disulfide bond recovered the active conformation of VEGFR2. This motif was prone to a nucleophilic attack by H2S via an interaction of their frontier molecular orbitals. siRNA-mediated knockdown of cystathionine γ-lyase attenuated migration of vascular endothelial cells induced by VEGF or moderate hypoxia.
  
  
  CONCLUSIONS
  The study provides the first piece of evidence of a molecular switch in H2S-targeting receptor protein kinase in H2S-induced angiogenesis and that may be applicable to additional kinases containing functionally important disulfide bonds in mediating various H2S actions.
authors.affiliation: Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, Shanghai, China.
authors.forename: Bei-Bei
authors.initials: BB
authors.lastname: Tao
authors.forename: Shu-Yuan
authors.initials: SY
authors.lastname: Liu
authors.forename: Cai-Cai
authors.initials: CC
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Fu
authors.forename: Wen-Jie
authors.initials: WJ
authors.lastname: Cai
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Qing
authors.initials: Q
authors.lastname: Shen
authors.forename: Ming-Jie
authors.initials: MJ
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Li-Jia
authors.initials: LJ
authors.lastname: Zhang
authors.forename: Yi-Zhun
authors.initials: YZ
authors.lastname: Zhu
authors.forename: Yi-Chun
authors.initials: YC
authors.lastname: Zhu
mesh_terms: D000818:Animals; D003542:Cystathionine gamma-Lyase / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D006862:Hydrogen Sulfide / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D056004:Molecular Dynamics Simulation; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; D019869:Phosphatidylinositol 3-Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; D003542:Cystathionine gamma-Lyase; D006862:Hydrogen Sulfide