Molecular regulation of VEGFR-2 function and expression through post-translational modifications
Hartsough, Edward J
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Vascular endothelial growth factor receptor-2 (VEGFR-2) is an endothelial cell receptor tyrosine kinase (RTK) whose activity is an obligate requirement for both normal development and pathological angiogenesis. A critical post-translational modification (PTM) of RTKs such as VEGFR-2 is tyrosine phosphorylation, which regulates these proteins at multiple levels including, tyrosine kinase activation, signaling, trafficking, and degradation. Similarly, growing evidence now suggests that protein methylation is another important type of PTM that plays a role in protein-protein interaction and signal transduction. In an effort to explore the possibility that methylation plays a role in regulation of VEGFR-2 function, we have employed mass spectrometry analysis coupled with pharmacological inhibitors of the methylation pathway. Our analysis revealed the presence of five methylated residues, three lysine and two arginine. Particularly, methylation of lysine 1041 (K1041), which is proximal to the conserved activation loop of the kinase domain, significantly contributed to VEGFR-2 kinase activation. Mutation of K1041 to multiple different amino acids rendered VEGFR-2 inactive and inhibited the activation of key downstream signaling proteins. Moreover, these mutations reduced VEGFR-2 mediated cell proliferation and capillary tube formation. Single mutations of R817, K856, K861 and R1115 yielded no apparent effect on tyrosine phosphorylation of VEGFR-2, however compounding the methyl deficiencies with triple and quadruple mutations markedly weakened tyrosine phosphorylation and the ligand-mediated downregulation of VEGFR-2. Furthermore, treatment of endothelial cells with global methylation inhibitors including adenosine dialdehyde (AdOx) and 3-deazaneplanocin A (DZNep) decreased ligand mediated tyrosine phosphorylation of VEGFR-2. The study presented here provides evidence that arginine and lysine methylation of VEGFR-2 through both combinatorial and non-combinatorial mechanisms regulate VEGFR-2 phosphorylation and function. This study also demonstrates that RNF121, an endoplasmic reticulum (ER) resident ubiquitin E3 ligase, binds to nascent VEGFR-2 protein and controls the abundance of cell surface VEGFR-2. Taken together, our data describes a novel role for arginine and lysine methylation in the regulation of VEGFR-2 functions and identifies a link between RNF121 ubiquitin E3 ligase and cell surface expression of VEGFR-2.
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