Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway

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.

内皮受体酪氨酸激酶（receptor tyrosine kinase, RTK）Tie1已被发现二十余年，但其确切功能与作用机制仍不甚明晰。为阐明Tie1在内皮细胞生物学中的功能，我们针对Tie1近膜结构域内一处潜在的苏氨酸磷酸化位点开展了研究。将该位点的非磷酸化突变体（T794A）在斑马鱼（Danio rerio）中表达后，可显著干扰血管发育，导致斑马鱼的体节间血管发育迟缓、分支稀疏。同理，在三维基质胶（Matrigel）培养体系中，表达T794A的人脐静脉内皮细胞所形成的管腔显著更短，分支也更少。然而，T794的突变并未在可检测范围内改变Tie1或Tie2的酪氨酸磷酸化水平及下游信号通路，这提示T794的磷酸化可能通过不依赖于其RTK活性的方式调控Tie1的功能。尽管T794位于Akt共识磷酸化位点内，但我们未能找到可诱导T794磷酸化的生理性Akt激活剂，这表明Akt并非生理性的Tie1-T794激酶。不过，研究发现小GTP酶Ras相关肉毒杆菌毒素底物1（Ras-related C3 botulinum toxin substrate 1, Rac1）——其在血管生成与毛细血管形态发生中发挥不可或缺的作用——可与磷酸化T794位点结合，却无法结合非磷酸化的T794A突变体。体外实验中，Rac1的药理学激活可诱导p21活化激酶（PAK1）的下游激活以及T794的磷酸化，而PAK1的抑制则会阻断T794的磷酸化。本研究首次证实了内皮细胞中由Tie1介导的信号通路，并且提示涉及Rac1/PAK1介导的Tie1 T794位点磷酸化的新型反馈环路，对于正常血管生成是必需的。