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

Additional file 1: Table S1. of Partial uniparental isodisomy of chromosome 16 unmasks a deleterious biallelic mutation in IFT140 that causes Mainzer-Saldino syndrome

Clinical genetic testing summary. Table S2. Whole exome sequencing capture statistics. Table S3. Final variant list following trio-based exome analysis (<1% minor allele frequency (MAF), homozygous, compound heterozygous, de novo, or X-linked). Figure S1. Fundus imaging and full field electroretinograms indicate retinal dystrophy. a. Fundus imaging of the proband at 5 years 6 months in the right (top) and left (bottom) eyes show mottling of the retinal pigmentary epithelium. b. Photopic (light adapted) testing of cone function demonstrated reduced a-wave implicit time and amplitudes with slowed implicit times for the b-wave. c. Scotopic (dark adapted) testing of rod function demonstrated slower implicit time and diminished amplitudes for both a- and b-waves. The a-wave was substantially delayed with reduced incremental increases in a-wave and b-wave. Figure S2. Validation of the ift140 splice-blocking (sb) morpholino (MO). a. Schematic of the D. rerio ift140 locus at chr24:37,937,737-38,013,596 (GRCz10; top) and translated protein (bottom). Exons are depicted as green boxes; untranslated regions are shown as white boxes (ENSDART00000129889.4). The sb-MO targets the splice donor of exon (Ex) 2 (red box). Protein schematic (blue) indicates predicted WD40 repeat (WD40) and tetratricopeptide-like helical domains (TPR). b. ift140 sb-MO induces aberrant splicing of endogenous transcript. ift140 transcript was evaluated by RT-PCR; embryos were injected with 9 ng MO at the one-to-four-cell stage and harvested for RNA extraction at the mid-somitic stage. Resulting cDNA was amplified with primers flanking the MO target site (arrows in panel a), and migrated on a 2.5% agarose gel. β–actin was used as a control to ensure RNA integrity. c. Chromatograms indicate splicing defects in ift140 mRNA in morphants. Sequence analysis of purified PCR product indicates that the majority of ift140 message is missing exon 2 (bottom), which contains the translation initiation codon. Modest amounts of wild type (wt; center) and mRNA containing a 33 nucleotide in-frame intronic insertion (top) are also detectable. d. Representative dose curve of the ift140 sb-MO. Embryo batches were injected at the one-to-four-cell stage with increasing amounts of ift140 sb-MO and scored for gastrulation defects at the mid-somitic stage (eight to ten somites). Embryos were scored live according to previously established phenotypic criteria: class I, modest shortening of the body axis and reduction in size of anterior structures; class II, severe shortening of the body axis and decreased anterior structures accompanied by broadening and/or kinking of the notochord and thinning of the somites (See Figure 3; n = 59–70 embryos/injection; repeated twice). Figure S3. Read depth and variant calls of the homozygous variants called on 16p13. Plot of the 76 maternally inherited homozygous variants identified in the 16p13 region of the proband. Red indicates alternate (alt) reads; blue indicates reference (ref) reads; 49/76 (64%) are comprised only of alternate reads; 27/76 (36%) had one to three reference reads at positions annotated as homozygous. (PDF 20018 kb

Expression of <i>ISL1</i> and <i>GATA4</i> transcripts in the human heart between 26 and 38 days of gestation.

<p><b>A–H</b>: <i>ISL1 in situ</i> at Carnegie stages (CS)12 (26–28 days post fertilization [dpf]), CS13 (28–31 dpf), CS14 (32–33 dpf) and CS15 (34–36 dpf) respectively. <b>E–H</b> are magnifications of <b>A–D</b> respectively. <b>I–K</b> show <i>GATA4</i> expression in adjacent sections to <b>B–D</b>. <b>A</b>: <i>ISL1</i> is expressed at CS12 in foregut endoderm, splanchnic mesoderm, and early motoneurons. <b>B, F</b>: At CS13, <i>ISL1</i> is transcribed by mesenchyme around the cardiac OFT and pharyngeal arches. <i>ISL1</i> expression continues in the splanchnic mesoderm between the trachea and OFT, and is visible in dorsal root ganglia, at CS14 (<b>C, G</b>) and CS15 (<b>D, H</b>). <b>I–K</b>: <i>GATA4</i> is expressed in the endocardium and myocardium of the arterial pole at CS13, CS14 and CS15 (<b>I, J, K</b> respectively). <b>Inset</b>: RT-PCR of <i>ISL1</i>, <i>GATA4</i>, <i>GATA5</i>, <i>GATA6</i>, <i>FGF10</i> and positive control <i>ACTB</i> mRNAs in embryonic human hearts at stages CS13-16 (to 40 dpf). Abbreviations: drg, dorsal root ganglia; es, esophagus; fb, forebrain; fg, foregut; ph, pharynx; nt, neural tube; oft, OFT; ra, right atrium; t, trachea. Arrows, motoneurons. Bar: 110 µm (A–D, I) and 55 µm (E–H, J, K).</p

<i>In vitro</i> reporter assays support an additive combinatorial effect of transcription factors upon the FGF10 intronic enhancer.

<p>LUC-<i>FGF10</i>-Int1, which construct placed the luciferase gene under the control of the FGF10-Int1 element, was transfected alone or together with <i>ISL1</i>, <i>GATA4</i> and <i>TBX20</i> expression vectors into 10T1/2 cells. Each factor alone potentiated luciferase expression and these effects were additive in combination.</p

Bioinformatics analyses of the human <i>FGF10</i> locus surrounding the first exon.

<p><b>A</b>: Alignment of genomic regions around and within the human [hg18] <i>FGF10</i> locus to those of frog [xenTro2], chicken [galGal3], opossum [monDom4], mouse [mm9], dog [canFam2] and rhesus macaque [rheMac2] with colored regions >90% identical and the vertical scale ranging from 50% (bottom) to 100% (top). Color code for genomic features at <a href="http://ecrbrowser.dcode.org/ecrInstructions/ecrInstructions.html" target="_blank">http://ecrbrowser.dcode.org/ecrInstructions/ecrInstructions.html</a>. The <i>FGF10</i>-Pr1, <i>FGF10</i>-Pr2 and FGF10-Int1 regions examined in this study are boxed. <b>B</b>: A non-canonical predicted site for GATA-type transcription factors is 52 nucleotides 5′ to the ISL1 cognate sequence in <i>FGF10</i>-Int1 in the direction of transcription on the – strand in humans, mice and (not shown) macaque and opossum. <b>C</b>: Nucleotide sequence of the <i>FGF10</i>-Int1 enhancer module and position of conserved putative transcription factor binding sites as predicted by rVista (<a href="http://rvista.dcode.org" target="_blank">http://rvista.dcode.org</a>). All indicated human sites are identical to those of the macaque and mouse except for the SMAD prediction, only found in mouse; the ISL1, GATA and HOXA7 sites are also identical to the opossum, and the ISL1, NKX2-5 and TBX sites are also identical to the dog.</p

Sites of β-galactosidase activity in transgenic mouse embryos.

<p>All sites showed only selective cells positive for enhancer activation. DRGs = dorsal root ganglia; E = embryonic day of gestation; MN = motoneurons; OFT = cardiac outflow tract; PA = pharyngeal arch; PSM = pre-somitic mesoderm.</p