c-ABL modulates MAP kinases activation downstream of VEGFR-2 signaling by direct phosphorylation of the adaptor proteins
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ORIGINAL PAPER
c-ABL modulates MAP kinases activation downstream of VEGFR-2 signaling by direct phosphorylation of the adaptor proteins GRB2 and NCK1 Francesca Anselmi • Maurizio Orlandini • Marina Rocchigiani Caterina De Clemente • Ahmad Salameh • Claudia Lentucci • Salvatore Oliviero • Federico Galvagni
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Received: 7 October 2011 / Accepted: 19 January 2012 / Published online: 11 February 2012 Ó Springer Science+Business Media B.V. 2012
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
Electronic supplementary material The online version of this article (doi:10.1007/s10456-012-9252-6) contains supplementary material, which is available to authorized users. F. Anselmi M. Orlandini M. Rocchigiani C. De Clemente A. Salameh C. Lentucci S. Oliviero (&) F. Galvagni (&) Dipartimento di Biotecnologie, Universita` di Siena, via Fiorentina 1, 53100 Siena, Italy e-mail: [email protected] F. Galvagni e-mail: [email protected]
c-ABL function is to establish a correct and tightly controlled response of endothelial cells to VEGF-A during the angiogenic process. Keywords Angiogenesis VEGF Signal transduction NCK ABL
Introduction The secreted Vascular Endothelial Growth Factor A (VEGF-A) is one of the most effective and specific signal driving the growth of new blood vessels during both physiological and pathological angiogenesis [1, 2]. In endothelial cells VEGF-A elicits its effects through binding two homologous Tyrosine Kinase receptors (TKRs): VEGF Receptor-1 (VEGFR-1) and VEGF Receptor-2 (VEGFR2). VEGFR-2 regulates endothelial cell differentiation, migration, proliferation and survival [3, 4], whereas VEGFR-1 acts as a negative regulator of cell proliferation during the assembly of the vascular endothelium [5]. Binding of VEGF-A to VEGFR-2 induces receptor autophosphorylation on tyrosine (Tyr) residues within its cytoplasmic domain [6]. The phosphorylated VEGFR-2 Tyr 1175 and 1214 mediate binding and activation/phosphorylation of PLCc and NCK1 respectively [7,
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