Multiple binding sites in the growth factor receptor Xmrk mediate binding to p59(fyn), 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-der ived cell line (PSM) resulting in constitutive Xmrk-mediated mitogenic sign aling. In order to define the underlying signaling pathway(s), triggered by the activated Xmrk receptor, we attempted to identify its physiological su bstrates. Examination of the Xmrk carboxyterminus for putative tyrosine aut ophosphorylation sites revealed the presence of potential binding motifs fo r GRB2 as well as for Shc. Binding of these adaptor proteins to the Xmrk; r eceptor was detected in vitro and in cells expressing the mrk kinase. The G RB2 and Shc interactions with the receptor could be disrupted individually by phosphotyrosine peptides containing putative Xmrk autophosphorylation si tes, 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 s ite for src-kinases (pYEDL) in the Xmrk carboxyterminus. Competition experi ments with phosphopeptides comprising this site confirmed that it is used f or high-affinity binding of Xiphoporus fyn (Xfyn) to Xmrk in melanoma cells . Thus, Xmrk can initiate different signaling pathways by using multiple su bstrate-binding sites to trigger proliferation of pigment cells.