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.
Highlights
Development and maintenance of the mature vasculature requires a delicate balance between vessel growth and quiescence regulated by constant integration of signals from the bloodstream and surrounding tissues [1,2,3]
These findings demonstrate a novel signaling pathway involving Tie1 that is required for angiogenesis
To test whether Akt could phosphorylate Tie1, we performed in vitro kinase reactions using a fusion protein comprised of the Tie1 JM and kinase domains fused to GST (GST-Tie1) incubated with endothelial cell lysates that had been infected with either a control, empty adenovirus (AdEmpty) or constitutively active, myristoylated Akt adenovirus (AdmyrAkt)
Summary
Development and maintenance of the mature vasculature requires a delicate balance between vessel growth (angiogenesis) and quiescence regulated by constant integration of signals from the bloodstream and surrounding tissues [1,2,3]. The Tyrosine kinases with Immunoglobulin and Epidermal growth factor homology domains (Tie) receptors, Tie and Tie, are known to play important roles in vascular growth, remodeling, and maintenance, as knockout of either Tie or Tie results in embryonic lethality due to vascular defects. While Tie and its ligands, the angiopoietins, have been fairly well characterized for their effects on vascular morphogenesis, Tie1’s role in the developing and adult vasculature has been difficult to characterize, in part because it does not directly bind any identified ligand and has weak endogenous kinase activity, making investigation of its signaling pathways difficult [6]. Tie1’s overall function and mechanisms of action remain poorly understood
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