Quantitative analysis of HGF and EGF-dependent phosphotyrosine signaling networks.
We have used stable isotope labeling by amino acids in cell culture (SILAC), in combination with high-resolution mass spectrometry, to identify common and discrete components of the respective receptor tyrosine kinase-dependent phosphotyrosine-associated networks induced by acute stimulation of A549 lung adenocarcinoma cells with EGF or HGF. In total, we obtained quantitative information for 274 proteins, which respond to either or both stimuli by >1.5 fold changes in enrichment, following immuno-precipitation with antiphosphotyrosine antibodies. The data reveal a high degree of overlap between the respective signaling networks but also clear points of departure. A small number of HGF specific effectors were identified including myosin-X, galectin-1, ELMO2 and EphrinB1, while a larger set of EGF specific effectors (39 proteins) includes both novel (e.g., MAP4K3) and established components of receptor tyrosine kinase receptor signaling pathways. Using available protein-interaction data the identified proteins have been assembled into a highly connected network that can be visualized using the Cytoscape tool.
Hammond
Dean E
DE
Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, United Kingdom.
Hyde
Russell
R
Kratchmarova
Irina
I
Beynon
Robert J
RJ
Blagoev
Blagoy
B
Clague
Michael J
MJ
D015153:Blotting, Western; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D019000:Phosphotyrosine / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D015398:Signal Transduction
C066855:HGF protein, human; D020543:Proteome; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor; D011505:Protein-Tyrosine Kinases