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.

Fig 1. Tie1 contains an Akt consensus phosphorylation site and can be phosphorylated in vitro by Akt.

a 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 GST-Tie1 fusion protein was incubated with EC lysates that had been infected with AdEmpty (-) or AdmyrAkt (+) virus in an in vitro kinase reaction. Phosphorylated Tie1 (arrow) was detected with a phospho-Akt substrate antibody. c 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.

Fig 2. Tie1 and Thr794 are required for proper embryonic vascular development in zebrafish.

a-e Zebrafish embryos expressing Fli1:EGFP were injected with a control or Tie1 morpholino (MO) with or without mRNA encoding WT or T794A (TA) mutant Tie1 and effects on vascular development were assessed. b Zebrafish injected with a control MO develop normally and resemble uninjected (Control) embryos. c Tie1 MO-injected embryos display vascular abnormalities. d Co-injection of Tie1 MO and WT mouse Tie1 mRNA rescued the MO phenotype. e Injection of T794A mutant mouse Tie1 mRNA alone resulted in vascular abnormalities similar to those of MO-injected embryos. Arrows indicate blunted intersomitic vessel formation. f Quantification of mutant embryos after injection of Tie1 MO with WT or T794A mutant mRNA, P = 3.0x10^-19 by Chi squared analysis. g Quantification of mutant embryos after injection of Tie1-WT or -TA mRNA alone, P = 1.3x10^-18 by Chi squared analysis.

Fig 3. The Tie1 T794 mutant disrupts endothelial tube formation.

a 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, c Quantification of endothelial tube networks in vitro. Cells expressing the T794A mutant formed significantly shorter tubes (b; *, multiple comparisons P≤ 0.02) with fewer nodes and branches (c; *, multiple comparisons P< 0.01) (n = 11 independent experiments with 4 or more images per experiment).

Fig 4. Rac1 association with Tie1 is enhanced by T794 phosphorylation.

HUVECs were infected with adenoviruses encoding Tie1-WT and -T794A, with or without AdAkt, and Tie1 was immunoprecipitated (IP) from cell lysates and western blotted as indicated. Whole cell lysates were blotted for total Tie1 and Rac1.

Fig 5. Rac1-mediated PAK1 activation results in Tie1-T794 phosphorylation.

a Rac1 was activated using CN04 in HUVECs infected with AdTie1 WT or T794A. Tie1 was immunoprecipitated (IP) and western blotted as indicated. CN04-mediated Rac1 activation resulted in T794 phosphorylation which was ablated by the T794A mutant. b CN04-mediated T794 phosphorylation was reduced by PAK1 inhibition (IPA3, 10μM) but not ROCK1 inhibition (Y–27632, 10μM). c HEK-293T cells transfected with Tie1-WT alone or together with PAK1 were treated with or without CN04, and Tie1 IPs and whole cell lysates were western blotted as indicated. Co-expression of PAK1 induced Tie1-T794 phosphorylation, which was augmented by CN04-mediated activation of Rac1 and PAK1.

Fig 6. Proposed model for Tie1-T794 phosphorylation by Rac1/PAK1 and modulation of Rac1 signaling.

1) Angiogenic stimuli (e.g., extracellular matrix (ECM)-mediated integrin activation, VEGF stimulation, etc.) induce Rac1 activation (Rac1-GTP). 2) Rac1-GTP activates PAK1, resulting in PAK1 autophosphorylation. 3) PAK1 phosphorylates Tie1 on T794. 4) Rac1 associates with Tie1-pT794. 5) Rac1 association with Tie1 facilitates interaction with effector molecules that either stabilize it in its active GTP-bound form or mediate GTP hydrolysis and subsequent Rac1 inactivation. Mutation of Tie1-T794 therefore disrupts Rac1 signaling and inhibits proper angiogenesis and capillary morphogenesis.