Methylation is a post-translational modification (PTM) that governs protein–protein interactions and essential protein functions. Recently, Hartsough et al. evaluated the role methylation plays in the activation of vascular endothelial growth factor receptor-2 (VEGFR-2) and angiogenesis.1
VEGFR-2 is an endothelial receptor tyrosine kinase. It possesses an extracellular region and a cytoplasmic region with inherent tyrosine kinase activity that, when activated, triggers signal transduction pathways. VEGFR-2 mediates both normal embryonic development and pathological conditions, including ocular neovascularization and cancer-associated angiogenesis.2
Hartsough et al. used anti-methyl-lysine and -arginine antibodies to detect methylation of VEGFR-2 in human primary umbilical vein endothelial cells. For the experiment, they selected a chimeric VEGFR-2 chemokine receptor containing an altered extracellular domain to avoid interference. They observed that VEGFR-2 is directly methylated in the cytoplasmic region prior to ligand-mediated tyrosine phosphorylation.
The researchers also used an LTQ Orbitrap XL (Thermo Scientific) to perform liquid chromatography–tandem mass spectrometry (LC-MS/MS) on VEGFR-2 immunoprecipitated from porcine aortic endothelial (PAE) cells. They relied upon Proteome Discoverer (Thermo Scientific) for data processing.
LC-MS/MS isolated five methylated residues: Arg817, Lys856, Lys861, Lys1041 and Arg1115. In vitro assays of synthesized analogues of all but Arg817 revealed direct methylation of VEGFR-2. MS analysis also showed similarity between the stoichiometry of tyrosine phosphorylation of VEGFR-2 and methylation of Lys1041. This suggests that the latter PTM may occur as frequently as the phosphorylation of Tyr1057, which plays an essential role in triggering kinase activity in VEGFR-2.3
Further analysis with methylation mutant VEGFR-2 constructs revealed the vital nature of Lys1041 methylation. Using full-length mouse VEGFR-2, the researchers determined that the Lys1041 mutant (K1041R) inhibited both tyrosine phosphorylation and kinase activation. The researchers observed no difference, however, in ligand binding or dimerization between the Lys1041 mutant construct (K1041R) and the wild-type VEGFR-2.
Hartsough et al. treated PAE cells with pharmacological methylation reducers (Adox or DZNep). They determined that this significantly inhibited the kinase activity of affected proteins and also downregulated ligand-stimulated phosphorylation of tyrosine residues. Only the Lys1041 mutant (K1041R) demonstrated no inhibition of ligand-stimulated phosphorylation under these conditions. This indicates that, without an intact Lys1041 residue, Adox cannot inhibit tyrosine phosphorylation of VEGFR-2. The researchers also re-methylated VEGFR-2 and showed re-established kinase activity comparable to wild-type VEGFR-2.
Finally, the researchers determined that Lys1041 plays a crucial role in stimulating angiogenesis and tumor growth. To do this, they evaluated mutant VEGFR-2 (K1041R) for capillary tube formation, a necessary component of angiogenesis,4 and noted that this activity was absent in mutant samples but present in wild-type samples. They also observed that the expression of wild-type VEGFR-2 enhanced xenograft tumors composed of mouse melanomas, while mutant VEGFR-2 (K1041R) did not. Using a proven in vivo angiogenesis model,5,6 the researchers presented evidence that zebrafish expressing mutant VEGFR-2 (K1041R) demonstrate reduced angiogenesis, as compared to zebrafish expressing wild-type VEGFR-2.
Hartsough et al. note that angiogenesis is a pathological component of human disease. VEGFR-2-based research has already been used to develop therapeutic inhibitors to treat age-related macular degeneration and cancers of the lung, kidney, colorectal system and glioblastoma.7,8 They indicate that new information revealing the role methylation plays in VEGFR-2 activation and angiogenesis may lead to the development of novel treatment options.
1. Hartsough, E., et al. (2014) “Lysine Methylation Promotes VEGFR-2 Activation and Angiogenesis,” Cell Biology, 6(304) (p. ra104).
2. Rahimi, N. (2006) “Vascular endothelial growth factor receptors: Molecular mechanisms of activation and therapeutic potentials,” Experimental Eye Research, 83 (pp. 1005–16).
3. Meyer, R., et al. (2008) “IQGAP1-dependent signaling pathway regulates endothelial cell proliferation and angiogenesis,” PLOS ONE, 3 (p. e3848).
4. Carmeliet, P., and Jain, R.K. (2000) “Angiogenesis in cancer and other diseases,” Nature, 407 (pp. 249–57).
5. Serbedzija, G., et al. (1999) “Zebrafish angiogenesis: A new model for drug screening,” Angiogenesis, 3 (pp. 353–9).
6. Tobia, C., et al. (2011) “Zebrafish embryo, a tool to study tumor angiogenesis,” International Journal of Developmental Biology, 55 (pp. 505–9).
7. Carmeliet, P. (2003) “Angiogenesis in health and disease,” Nature Medicine, 9 (pp. 653–60).
8. Carmeliet, P. (2005) “Angiogenesis in life, disease and medicine,” Nature, 438 (pp. 932–36).
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.