title: Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway.
abstract: The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1's role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Reinardy
authors.affiliation: Department of Medicine, Division of Cardiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Corey
authors.affiliation: Center for Human Disease Modeling, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Christelle
authors.initials: C
authors.lastname: Golzio
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, United States of America; Duke University School of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Mueller
authors.affiliation: Center for Human Disease Modeling, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Katsanis
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, United States of America; Department of Medicine, Division of Cardiology, Duke University Medical Center, Durham, North Carolina, United States of America; Duke University School of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Kontos
mesh_terms: D042683:Angiopoietin-1 / Q000502:physiology; D000818:Animals; D001808:Blood Vessels / Q000196:embryology; D003094:Collagen; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007797:Laminin; D009024:Morphogenesis; D016297:Mutagenesis, Site-Directed; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011509:Proteoglycans; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D042810:Receptor, TIE-1 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D015027:Zebrafish / Q000196:embryology / Q000235:genetics; D029961:Zebrafish Proteins / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
chemical_list: C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C107953:RAC1 protein, human; D029961:Zebrafish Proteins; C582243:rac1a protein, zebrafish; D010769:Phosphothreonine; C059485:matrigel; D003094:Collagen; D042810:Receptor, TIE-1; C467973:tie1 protein, zebrafish; C515903:PAK1 protein, human; D051057:Proto-Oncogene Proteins c-akt; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein