Multiple binding sites in the growth factor receptor Xmrk mediate binding to p59fyn, GRB2 and Shc.
Melanoma formation in Xiphoporus is initiated by overexpression of the EGFR-related receptor tyrosine kinase Xmrk (Xiphoporus melanoma receptor kinase). This receptor is activated in fish melanoma as well as in a melanoma-derived cell line (PSM) resulting in constitutive Xmrk-mediated mitogenic signaling. In order to define the underlying signaling pathway(s), triggered by the activated Xmrk receptor, we attempted to identify its physiological substrates. Examination of the Xmrk carboxyterminus for putative tyrosine autophosphorylation sites revealed the presence of potential binding motifs for GRB2 as well as for Shc. Binding of these adaptor proteins to the Xmrk receptor was detected in vitro and in cells expressing the mrk kinase. The GRB2 and Shc interactions with the receptor could be disrupted individually by phosphotyrosine peptides containing putative Xmrk autophosphorylation sites, indicating direct binding of both proteins. Recruitment of GRB2 by the constitutively activated Xmrk receptor led to strong MAP kinase activation in Xiphoporus melanoma cells. We also identified a high-affinity binding site for src-kinases (pYEDL) in the Xmrk carboxyterminus. Competition experiments with phosphopeptides comprising this site confirmed that it is used for high-affinity binding of Xiphoporus fyn (Xfyn) to Xmrk in melanoma cells. Thus, Xmrk can initiate different signaling pathways by using multiple substrate-binding sites to trigger proliferation of pigment cells.
Wellbrock
C
C
Department of Physiological Chemistry I, University of Würzburg, Germany. wellbrock@biozentrum.uni.wuerzburg.de
Schartl
M
M
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003532:Cyprinodontiformes; D004789:Enzyme Activation; D029941:Fish Proteins; D051380:GRB2 Adaptor Protein; D008545:Melanoma; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000502:physiology; D014407:Tumor Cells, Cultured; D018909:src Homology Domains / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D029941:Fish Proteins; D051380:GRB2 Adaptor Protein; D010748:Phosphopeptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D055765:Shc Signaling Adaptor Proteins; D020794:Receptor Protein-Tyrosine Kinases; C077101:Xmrk protein, Xiphophorus; D051076:Proto-Oncogene Proteins c-fyn; D017871:Calcium-Calmodulin-Dependent Protein Kinases