Lysine Methylation Activates VEGFR-2 and Mediates Angiogenesis

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.

 

References

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.