Hepatocyte growth factor induces activation of Nck and phospholipase C-gamma in lung carcinoma cells.

Hepatocyte growth factor (HGF), a mesenchyme derived growth factor, promotes cell growth, cell motility, and morphogenesis in a variety of epithelial cells. The diverse responses are transduced across the cell membrane by the met/HGF receptor, a product of c-met protooncogene. The met/HGF receptor recruits a variety of second messenger molecules which relay the diverse intracellular responses of HGF. In this study, we show that HGF autophosphorylates and activates met/HGF receptor. The activated met/HGF receptor then physically associates with and activates phospholipase C-gamma (PLC-gamma). Furthermore, upon ligand stimulation, tyrosine-autophosphorylated met/HGF receptor also activates Nck oncogene product. Taken together, our results suggest that the receptor activation leads to formation of a complex in which PLC-gamma and Nck oncogene product co-exist with the activated met/HGF receptor, and that the Nck oncogene product is an important component of HGF signaling in Calu-1 and A549 cells.

Evidence for autocrine basis of transformation in NIH-3T3 cells transfected with met/HGF receptor gene.

NIH-3T3 cells transformed with met/HGF receptor gene proliferate in the absence of serum and growth factors. Immunocytochemical staining with anti-HGF antibody revealed intense HGF staining in the transfected cells. Additionally, these cells secrete bioactive HGF as evidenced by the ability of the conditioned media to stimulate met/HGF receptor phosphorylation in epithelial cells, and to promote migration of bovine adrenal capillary endothelial cells in a modified Boyden chamber assay. The migration of endothelial cells could be specifically inhibited by anti-HGF antibody but not by an irrelevant antibody. Suramin, a drug known to disrupt ligand-receptor interactions, inhibits the serum and growth-factor free proliferation, and the endogenous phosphorylation of met/HGF receptor in the transformed cells. Taken together, our data suggests an autocrine mode of transformation in NIH-3T3 cells transfected with met/HGF receptor gene.

PDGF-independent activation of PDGF-beta receptors in NIH-3T3 cells transformed by c-met protooncogene.

We have previously reported that c-met protooncogene, a member of a new class of receptor tyrosine-kinase gene family, is transforming when overexpressed in NIH-3T3 cells. In this paper, we report that the c-met protooncogene-transformed cells proliferate in a serum- and growth factor-free medium and exhibit constitutive tyrosine phosphorylation of several cellular proteins including the met protooncogene-encoded p145 and p185. Further investigations revealed platelet-derived growth factor (PDGF)-independent phosphorylation of PDGF-beta receptors in the transformed cells. Phosphoamino acid analysis revealed phosphorylation of PDGF receptors at tyrosine and serine residues. The PDGF receptor phosphorylation is unlikely to occur via autocrine production of PDGF since we could not detect PDGF activity both at the RNA level and at a functional protein level. Additionally, phospholipase C-gamma (PLC-gamma) a substrate of activated PDGF receptors, was found to be physically associated with PDGF receptors in the absence of PDGF stimulation in transformed cells. Furthermore, PDGF receptors coimmunoprecipitated along with PLC-gamma. Taken together, our results demonstrate a PDGF-independent phosphorylation and activation of PDGF-beta receptor in NIH-3T3 cells transformed by c-met protooncogene.