Adaptor ShcA protein binds tyrosine kinase Tie2 receptor and regulates migration and sprouting but not survival of endothelial cells.
Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.
Audero
Enrica
E
Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment (IRCC), School of Medicine, University of Torino, 10060 Candiolo, Italy.
Cascone
Ilaria
I
Maniero
Fabrizio
F
Napione
Lucia
L
Arese
Marco
M
Lanfrancone
Luisa
L
Bussolino
Federico
F
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D042683:Angiopoietin-1 / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002465:Cell Movement; D002470:Cell Survival; D002478:Cells, Cultured; D002633:Chemotaxis; D004396:Coloring Agents / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D042787:Receptor, TIE-2 / Q000737:chemistry* / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D042683:Angiopoietin-1; D004396:Coloring Agents; D008024:Ligands; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013778:Tetrazolium Salts; D013844:Thiazoles; D014443:Tyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D042787:Receptor, TIE-2; C022616:thiazolyl blue