Signalling by the oncogenic receptor tyrosine kinase Xmrk leads to activation of STAT5 in Xiphophorus melanoma

Abstract

Overexpression of the mutationally activated Xmrk receptor initiates the formation of hereditary malignant melanoma in the ®sh Xiphophorus. In addition to transcriptional overexpression a cell-type speci®c signal transduction is essential for Xmrk mediated tumor formation. To elucidate the consequence of Xmrk signalling and to identify target proteins that characterize the tumor phenotype, we analysed proteins that are strongly tyrosine phosphorylated in the ®sh melanoma cell line PSM. One of the most prominent phosphotyrosine proteins was found to be the signal transducer and activator of transcription STAT5. In a heterologous cell system (murine pro B-cells), activation of the Xmrk kinase in a chimeric receptor induced tyrosine phosphorylation, nuclear translocation and DNA binding of STAT5. Following receptor stimulation, expression of the STAT5 speci®c target genes cis, osm and pim-1 was induced. In Xiphophorus PSM cells STAT5 was found to be preferentially localized in the nucleus, but treatment with tyrphostin AG555, a speci®c Xmrk kinase-inhibitor, blocked nuclear localization. In these cells as well as in Xiphophorus melanoma expression of pim-1 and constitutive DNA-binding activity of STAT5 was detectable. This constitutive activity was higher in malignant than in benign melanomas, indicating that STAT5 activation is correlated with the malignancy of these tumors.

Activation of STAT5 triggers proliferation and contributes to anti-apoptotic signalling mediated by the oncogenic Xmrk kinase

“…Earlier we have shown that activation of the Xmrk kinase in a chimeric Xmrk-receptor (HER-mrk), when expressed in BaF/3 cells (BaF Hm cells) leads to induction of DNA synthesis (Wellbrock et al, 1998). In contrast, Ba/F3 cells expressing the closely related EGFR (BaF HER cells) showed no signi®cant induction of DNA synthesis (Figure 1a), which is in agreement with reported data (Shibuya et al, 1992).…”

“…Accordingly, constitutive STAT5 activation is found in a variety of transformed hematopoietic cells as well as in melanoma cells of Xiphophorus (Wellbrock et al, 1998). However, STAT proteins such as STAT1 and 2 on the other hand are playing an important role in the anti-proliferative function of various cytokine receptors acting also on melanoma cells (Lazar-Molnar et al, 2000).…”

“…In Xiphophorus, overexpression of Xmrk is the initiator of melanoma formation (Wittbrodt et al, 1989), but cell-type speci®c manifestation of the transformed state is due to a Xmrk speci®c intracellular signalling (Winkler et al, 1994). This signalling involves recruitment and activation of the adaptor proteins Shc and GRB2 (Wellbrock and Schartl, 1999), the src kinase p59 fyn and Phosphatidylinositol-3 kinase (PI3K) (Wellbrock and Schartl, 2000) as well as STAT5 (Wellbrock et al, 1998). However, although it was shown that PI3K, fyn and activation of the MAP kinase pathway through GRB2 contribute to mitogenic signalling in ®sh melanoma cells, nothing is known about the role of STAT5 in Xmrk triggered cell transformation.…”

“…Stable transfectants were generated by electroporation as described (Wellbrock et al, 1998). A whole cell ELISA assay (Lischke et al, 1996) using a monoclonal antibody (mAb 108.1) against the extracellular receptor part was employed to identify clones expressing similar amounts of the respective receptor versions.…”

“…We have shown recently that the melanoma inducing receptor tyrosine kinase Xmrk (Xiphophorus melanoma receptor kinase) speci®cally activates STAT5 but not STAT1 and STAT3 in melanoma cells (Baudler et al, 1999) and that constitutive STAT5 signalling correlates with the malignancy of melanomas of the ®sh Xiphophorus (Wellbrock et al, 1998). Xmrk belongs to the EGFR family and represents an additional member to the four widely expressed receptors EGFR, HER2, 3 and 4.…”