Sp3-mediated VEGF regulation is dependent on phosphorylation by extra-cellular signals regulated kinases (Erk).
We have previously demonstrated that phosphorylation by Erk of Sp1 was essential for its full activity in the context of the VEGF promoter. Here, we show that Sp3, which, as Sp1, belongs to the GC-rich binding transcription factor family, is also phosphorylated by Erk in vitro on serine 73. We have established cell lines in which expression of wild-type Sp3 or a serine 73 to alanine (S73A) mutant is controlled by tetracycline. One of these cells lines also express the Raf:ER chimera which permits stimulation of Erk by tamoxifen. Difference in electrophoretic mobility and antibody directed against the phosphorylated serine 73 demonstrate that it is phosphorylated in vivo. Wild-type Sp3 half-life is increased upon Erk activation but the S73 is poorly implicated in this mechanism suggesting that Erk-dependent Sp3 stability depends on other(s) domain(s) of the protein. Electro-mobility shift assays and utilization of Gal4/Sp3 chimeric proteins show that Erk does not alter Sp3 DNA binding capacity but enhances its transcriptional activity. The S73A mutant Sp3 posses a reduced activity in Erk-stimulated cells. In the inducible cell lines, expression of wild-type form of Sp3 increases VEGF production whereas the S73A form has a reduced potential reflecting its lower transcriptional activity. Altogether our results described a new link between constitutive Erk activity and the regulation of VEGF expression two common denominators implicated in tumor angiogenesis.
Pagès
Gilles
G
Institute of Signalling, Developmental Biology and Cancer Research UMR CNRS 6543, University of Nice-Sophia Antipolis Equipe labellisée Ligue Nationale contre le Cancer, France. gpages@unice.fr
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D042461:Vascular Endothelial Growth Factor A; D051705:Sp3 Transcription Factor; D012694:Serine; D048049:Extracellular Signal-Regulated MAP Kinases