c-ABL modulates MAP kinases activation downstream of VEGFR-2 signaling by direct phosphorylation of the adaptor proteins GRB2 and NCK1.
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.
Dipartimento di Biotecnologie, Università di Siena, Siena, Italy.
Francesca
F
Anselmi
Maurizio
M
Orlandini
Marina
M
Rocchigiani
Caterina
C
De Clemente
Ahmad
A
Salameh
Claudia
C
Lentucci
Salvatore
S
Oliviero
Federico
F
Galvagni
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D001549:Benzamides; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000068877:Imatinib Mesylate; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D009154:Mutation; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D015513:Oncogene Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D001549:Benzamides; D048049:Extracellular Signal-Regulated MAP Kinases; D051380:GRB2 Adaptor Protein; D000068877:Imatinib Mesylate; D015513:Oncogene Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D016315:Proto-Oncogene Proteins c-abl; D011743:Pyrimidines; D040301:Vascular Endothelial Growth Factor Receptor-2; C495948:GRB2 protein, human; C078332:Nck protein