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

<div><p>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 (<i>Danio rerio</i>) 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 <i>in vitro</i>, 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.</p></div

Tie1 contains an Akt consensus phosphorylation site and can be phosphorylated <i>in vitro</i> by Akt.

<p><b>a</b> The juxtamembrane (JM) domain of Tie1 is highly conserved from zebrafish through humans (light gray), including a high probability Akt phosphorylation consensus sequence (RRRTFTY) within the JM region (dark gray). The predicted phosphorylation site at Tie1-Thr794 is not present in Tie2. <b>b</b> GST-Tie1 fusion protein was incubated with EC lysates that had been infected with AdEmpty (-) or AdmyrAkt (+) virus in an <i>in vitro</i> kinase reaction. Phosphorylated Tie1 (arrow) was detected with a phospho-Akt substrate antibody. <b>c</b> HUVECs expressing Tie1WT, -T794A, or Tie2 were infected with AdEmpty (-) or AdmyrAkt (+) and Tie1 or Tie2 was immunoprecipitated (IP) and probed with a phospho-specific Tie1-pT794 antibody.</p

The Tie1 T794 mutant disrupts endothelial tube formation.

<p><b>a</b> Representative images of HUVECs left uninfected or infected with adenoviruses encoding GFP, Tie1-WT, or Tie1-T794A, plated on Matrigel and allowed to form tubes for 6 hours. <b>b, c</b> Quantification of endothelial tube networks <i>in vitro</i>. Cells expressing the T794A mutant formed significantly shorter tubes (<b>b</b>; *, multiple comparisons P≤ 0.02) with fewer nodes and branches (<b>c</b>; *, multiple comparisons P< 0.01) (n = 11 independent experiments with 4 or more images per experiment).</p

Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment

Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy