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c-ABL modulates MAP kinases activation downstream of VEGFR-2 signaling by direct phosphorylation of the adaptor proteins GRB2 and NCK1

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Abstract

Vascular Endothelial Growth Factor-A (VEGF-A) is a key molecule in normal and tumor angiogenesis. This study addresses the role of c-ABL as a novel downstream target of VEGF-A in primary Human Umbilical Vein Endothelial Cells (HUVEC). On the basis of immunoprecipitation experiments, in vitro kinase assay and RNA interference, we demonstrate that VEGF-A induces the c-ABL kinase activity through the VEGF Receptor-2/Phosphatidylinositol-3-Kinase pathway. By treating HUVEC with the specific tyrosine kinase inhibitor STI571 and over-expressing a dominant negative c-ABL mutant, we show that the VEGF-A-activated c-ABL reduces the amplitude of Mitogen-Activated Protein Kinases (ERK1/2, JNKs and p38) activation in a dose-dependent manner by a negative feedback mechanism. By analysis of the adaptor proteins NCK1 and GRB2 mutants we further show that the negative loop on p38 is mediated by c-ABL phosphorylation at tyrosine 105 of the adaptor protein NCK1, while the phosphorylation at tyrosine 209 of GRB2 down-modulates ERK1/2 and JNKs signaling. These findings suggest that c-ABL function is to establish a correct and tightly controlled response of endothelial cells to VEGF-A during the angiogenic process.

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Acknowledgments

We thank Beatrice Grandi for technical support, Dr. Daniela Barilà (University of Rome “Tor Vergata”) for c-ABL constructs, Dr. Michiyuki Matsuda (Department of Pathology and Biology of Diseases, Kyoto University, Japan) for CRKII constructs and Dr. Shaoguang Li (The Jackson Laboratory, Bar Harbor, ME, USA) for GRB2 constructs. This study was supported by grants from Associazione Italiana Ricerca sul Cancro (AIRC), Ministero Italiano dell’Istruzione, dell’Università e della Ricerca (MIUR), Fondazione Monte dei Paschi di Siena (MPS) and Istituto Toscano Tumori (ITT).

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Authors and Affiliations

  1. Dipartimento di Biotecnologie, Università di Siena, via Fiorentina 1, 53100, Siena, Italy

    Francesca Anselmi, Maurizio Orlandini, Marina Rocchigiani, Caterina De Clemente, Ahmad Salameh, Claudia Lentucci, Salvatore Oliviero & Federico Galvagni

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  1. Francesca Anselmi
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  2. Maurizio Orlandini
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  3. Marina Rocchigiani
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  4. Caterina De Clemente
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  5. Ahmad Salameh
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  6. Claudia Lentucci
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  7. Salvatore Oliviero
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  8. Federico Galvagni
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Correspondence to Salvatore Oliviero or Federico Galvagni.

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Anselmi, F., Orlandini, M., Rocchigiani, M. et al. c-ABL modulates MAP kinases activation downstream of VEGFR-2 signaling by direct phosphorylation of the adaptor proteins GRB2 and NCK1. Angiogenesis 15, 187–197 (2012). https://doi.org/10.1007/s10456-012-9252-6

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