PI3-kinase is involved in mitogenic signaling by the oncogenic receptor tyrosine kinase Xiphophorus melanoma receptor kinase in fish melanoma

Overexpression of the mutationally activated receptor tyrosine kinase Xipho phorus melanoma receptor kinase (Xmrk) initiates formation of hereditary ma lignant melanoma in the fish Xiphophorus. In melanoma as well as in a melan oma-derived cell line (PSM) this receptor is highly activated resulting in constitutive Xmrk-mediated mitogenic signaling. In order to analyze mitogen ic signaling triggered by Xmrk a possible involvement of phosphatidylinosit ol 3 (PI3)-kinase in Xmrk signal transduction was examined. Constitutive bi nding of the p85 adapter subunit of PI3-kinase to the Xmrk receptor was det ected in PSM melanoma cells. Further analyses in BHK. cells expressing a Xm rk chimera (HER-mrk) showed that p85 association with the intracellular par t of Xmrk was dependent on autophosphorylation of the receptor. In vitro bi nding studies revealed that the interaction is mediated mainly through the N-terminal SH2 domain of p85 which directly binds to a sequence motif aroun d phosphorylated Tyr-983 in the Xmrk carboxy-terminus. In accordance with r ecruitment of p85 by Xmrk in PSM cells, the PI3-kinase downstream target Ak t was found to be highly phosphorylated on Ser-473, indicating efficient PI 3-kinase signaling in melanoma cells. PI3-kinase activation was also detect ed in Xiphophorus melanoma. Moreover, malignant melanomas exhibited an incr eased level of PI3-kinase activity which was about three times higher than that in benign pigmented lesions. Inhibition of PI3-kinase activity in PSM melanoma cells by both Wortmannin and LY294002 blocked entry into S-phase. Together these data demonstrate that PI3-kinase is a substrate of the oncog enic Xmrk receptor and plays a significant role in mitogenic signaling of m elanoma cells and the formation of malignant melanoma in Xiphophorus. (C) 1 999 Academic Press.