title: The Vascular Endothelial Growth Factor-A phosphorylates Murine Double Minute-2 on its Serine 166 via the Extracellular Signal-Regulated Kinase 1/2 and p90 Ribosomal S6 Kinase in primary human endothelial cells.
abstract: Murine Double Minute-2 (Mdm2) has been identified as an essential regulator of skeletal muscle angiogenesis and the pro-angiogenic activity of endothelial cells. We have recently demonstrated that the pro-angiogenic Vascular Endothelial Growth Factor-A (VEGF-A) is a potent upstream regulator of Mdm2 phosphorylation on its Serine 166 (p-Ser166-Mdm2), a protein modification leading to an increase in endothelial cell migration. Here, we investigated the kinase signaling pathways that could be responsible for mediating VEGF-A-dependent Mdm2 phosphorylation. Incubation of primary human dermal microvascular endothelial cells with recombinant VEGF-A for 15 min led to increased phosphorylation levels of VEGF-receptor-2, Mdm2, Akt, Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), and p90 Ribosomal S6 Kinase (p90RSK) proteins. In addition to being linked to VEGF-A signaling, Akt, ERK1/2 and p90RSK have been shown to potentially lead to Mdm2 phosphorylation. We therefore next analyzed which of these kinases could be responsible for VEGF-A-dependent Mdm2 phosphorylation on Serine 166 by using kinase-specific pharmacological inhibitors. Inhibition of ERK1/2 phosphorylation by UO126 entirely abrogated the response of p-Ser166-Mdm2 to VEGF-A treatment, while Akt phosphorylation inhibition by wortmannin led to further elevations in p-Ser166-Mdm2. p90RSK has been identified as a potential candidate downstream of ERK1/2 that could induce Mdm2 Ser166 phosphorylation. Two independent p90RSK inhibitors, FMK and BI-D1870, each led to an entire loss of p-Ser166-Mdm2 responsiveness to VEGF-A. Taken together, our results demonstrate that VEGF-A driven Mdm2 phosphorylation on Ser166 is dependent on the ERK1/2/p90RSK signaling pathway in primary human endothelial cells, furthering our understanding of the complex relationship between Mdm2 and VEGF-A in a physiological context.
authors: Faculty of Health, School of Kinesiology and Health Science, York University, Toronto, ON, Canada.
authors: Julian
authors: J
authors: Aiken
authors: Faculty of Health, School of Kinesiology and Health Science, York University, Toronto, ON, Canada. Electronic address: birot@yorku.ca.
authors: Olivier
authors: O
authors: Birot
mesh_terms: D002070:Butadienes / Q000494:pharmacology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D011621:Pteridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: C516541:BI D1870; D002070:Butadienes; D009570:Nitriles; D011621:Pteridines; D011994:Recombinant Proteins; C113580:U 0126; D042461:Vascular Endothelial Growth Factor A; D010768:Phosphoserine; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D040301:Vascular Endothelial Growth Factor Receptor-2; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases
keywords: Angiogenesis; Endothelial cell; Skeletal muscle