Constitutive Activation of c-Met in Liver Metastatic B16 Melanoma Cells Depends on Both Substrate Adhesion and Cell Density and Is Regulated by a Cytosolic Tyrosine Phosphatase Activity

Abstract

Serial selection in vivo for liver colonization of B16 murine melanoma cells consistently resulted in cell lines expressing elevated amounts of the hepatocyte growth factor/scatter factor receptor (c-Met), which is constitutively activated in the absence of its cognate ligand. In this paper we present evidence suggesting that c-Met constitutive activation in liver-specific B16 melanoma cells depends on both receptor concentration on the cell surface and a cytosolic tyrosine phosphatase activity. In fact, c-Met constitutive activation is suddenly lost upon detachment of the cells from the substrate and is dramatically decreased in adherent cells plated at low density. The loss of tyrosine phosphorylation of c-Met in suspension appears to depend, at least partly, on an increased cytosolic tyrosine phosphatase activity. Instead, lower activation of c-Met at low density mostly results from a decrease in receptor concentration on the membrane. Moreover, we show that c-Met activation does not occur homogeneously on the surface of adherent cells. In fact, receptor concentration and activation appear to be higher on the ventral surface (adherent to the substrate) than on the apical surface. Upon detachment, compartmentalization is lost, leading to a decrease in average receptor density on the plasma membrane and hence to a lower activation.

Non-autocrine, constitutive activation of Met in human anaplastic thyroid carcinoma cells in culture

“…Constitutive activation of Met, as observed in the present study, has previously been reported from a gastric carcinoma cell line (GTL-16) , and also in murine melanoma cells (B16-LS9) (Rusciano et al, 1996). GTL-16 cells overexpress Met as a result of MET gene amplification .…”

“…Thus, it is possible that an overexpression leads to constitutive activation of the receptor proteins. Rusciano et al (1996) suggested that the ligand-independent activation observed in the B16-LS9 cells may be caused by the high number of receptors present on the cell surface, leading to spontaneous dimerization and phosphorylation. However, Ferracini et al (1995) reported very high expression of Met in the osteosarcoma cell line, U-2 OS, without any constitutive phosphorylation of the receptor.…”

Presence of autocrine hepatocyte growth factor-Met signaling and its role in proliferation and migration of SNU-484 gastric cancer cell line

“…Similarly, we observed that abundantly expressed Met in SNU-5 and SNU-638 was constitutively autophosphorylated without exogenous HGF treatment or autocrine HGF stimulation. Rusciano et al suggested that the ligandindependent activation observed in the B16-LS9 cells was caused by the high number of receptors present on the cell surface, leading to spontaneous dimerization and phosphorylation (Rusciano et al, 1996). Consistent with these findings, the ligand-independent activation of Met was observed only in cell lines where Met was overexpressed (Ponzetto et al, 1991).…”

“…As expected, constitutive activation of Met was observed in SNU-484 cells (Figure 3, middle panel). Interestingly, the Met expressed in SNU-5 and SNU-638 was also highly phosphorylated, even though the HGF produced by these cells was negligible according to ELISA assay (data not shown) possibly due to the ligand-independent activation of Met via spontaneous dimerization and phosphorylation when overexpressed (Rusciano et al, 1996). To further demonstrate autocrine HGF-Met signaling in SNU-484 cells, Met in SNU-484 cells was immunoprecipitated and the degree of tyrosine phosphorylation was assessed.…”

A src-Related Kinase in the Brush Border Membranes of Gastrointestinal Cells Is Regulated by c-met

“…In contrast to this finding, other reports have revealed that c-met is predominantly expressed in the basolateral membrane domains of human gut cells and a variety of other polarized epithelial cells [10,11,24]. Immunofluorescent staining of mouse intestinal tissue was performed using D1 monoclonal antibodies that selectively bind c-met [19].…”