c-Kit-kinase induces a cascade of protein tyrosine phosphorylation in normal human melanocytes in response to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas.

Abstract

The proto-oncogene c-Kit, a transmembrane receptor tyrosine kinase, is an important regulator of cell growth whose constitutively active oncogenic counterpart, v-kit, induces sarcomas in cats. Mutations in murine c-kit that reduce the receptor tyrosine kinase activity cause deficiencies in the migration and proliferation of melanoblasts, hematopoietic stem cells, and primordial germ cells. We therefore investigated whether c-Kit regulates normal human melanocyte proliferation and plays a role in melanomas. We show that normal human melanocytes respond to mast cell growth factor (MGF), the Kit-ligand that stimulates phosphorylation of tyrosyl residues in c-Kit and induces sequential phosphorylation of tyrosyl residues in several other proteins. One of the phosphorylated intermediates in the signal transduction pathway was identified as an early response kinase (mitogen-activated protein [MAP] kinase). Dephosphorylation of a prominent 180-kDa protein suggests that MGF also activates a phosphotyrosine phosphatase. In contrast, MGF did not induce proliferation, the cascade of protein phosphorylations, or MAP kinase activation in the majority of cells cultured from primary nodular and metastatic melanomas that grow independently of exogenous factors. In the five out of eight human melanoma lines expressing c-kit mRNAs, c-Kit was not constitutively activated. Therefore, although c-Kit-kinase is a potent growth regulator of normal human melanocytes, its activity is not positively associated with malignant transformation.

Suppression of autocrine cell proliferation and tumorigenesis of human melanoma cells and fibroblast growth factor transformed fibroblasts by a kinase-deficient FGF receptor 1: evidence for the involvement of Src-family kinases

“…However, although bFGF is a potent mitogen for normal human melanocytes that acts in synergy with dbcAMP or ET-1, the levels of induced tyrosyl phosphorylation and activation in response to this ligand are extremely low (as compared with HGF/SF or the Kit ligand SC/MGF), including that of ERK2 (BoÈ hm et al, 1995). Although the autonomous growing melanoma cells described here expessed several tyrosyl phosphorylated proteins not detected in unstimulated normal melanocytes [as shown before (Funasaka et al, 1992;Zakut et al, 1993)], their intensity was not decreased by the truncated, mutant receptor suggesting that they were not originally induced by the bFGF autocrine loop.…”

“…Three lines are MET + and KIT + (501 mel, 888 mel and 697 mel) and the other two are MET + and KIT 7 (586 mel and YU SIT1) (see Funasaka et al, 1992;Zakut et al, 1993). The human metastatic melanoma cell lines were maintained in Ham's F10 medium supplemented with 200 U/ml penicillin, 100 mg/ml streptomycin (Gibco/ BRL Laboratories, Grand Island, NY) plus fetal calf and calf serum as described (Zakut et al, 1993).…”

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

“…The Ras/MAP kinase pathway, that has been demonstrated to be of utmost importance for mitogenic signaling in a number of growth factor systems, is activated by the Kit/SCFR following ligand-stimulation (Blume-Jensen et al, 1995;Funasaka et al, 1992). In order to investigate the role of Tyr568 and Tyr570 in Kit/SCFR mediated Ras/MAP kinase signaling, PAE cells expressing either wild-type, Y568F mutant, Y570F mutant or the Y568F/Y570F double mutant Kit/SCFR were challenged with SCF and the time course of Erk2 activation was measured by an in vitro kinase assay using myelin basic protein as a substrate.…”

v-Myb can transform and regulate the differentiation of melanocyte precursors

“…Likewise, vitronectin receptor (integrin avb3), ligand binding to which has been observed to enhance invasiveness of melanoma cells (Seftor et al, 1992), was frequently increased upon Myb inactivation. Untransformed melanocytes are dependent for growth in culture on stem cell factor (Kit-ligand or SCF) signalling through c-Kit (Lahav et al, 1994) whereas melanoma cells, in particular those lines which are aggressively metastatic, are factorindependent with corresponding down-regulation of cKit (Funasaka et al, 1992). We were unable to detect c-Kit expression at the cell surface by immunocytochemistry, correlating with a lack of dependence of growth or di erentiation on exogenous SCF (not shown).…”