Anti-c-(monoclonal, C-8), anti-cyclin D1 (monoclonal, HD11), anti-human and -mouse MET (rabbit, C-28 and SP260), antiubiquitin (monoclonal, P1A6), and anti-JNK1 (rabbit, FL) antibodies were from Santa Cruz. The amount of uncomplexed cytoplasmic -catenin is usually tightly ACVRLK7 regulated by a multiprotein complex made up of axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3 (GSK3). Physical conversation between these proteins promotes -catenin phosphorylation on serine residues by GSK3, an event leading to -catenin ubiquitination and proteasomal degradation (8). Increased cellular -catenin due to mutations in APC tumor suppressor or -catenin genes occurs in many human cancers, including those of colon and skin (14, 57, 68, 92). Mutations in axin leading to -catenin accumulation have been found in hepatocellular carcinomas (16, 96). All these mutations result in reduced degradation of -catenin, which is usually believed to promote tumor formation by constitutive activation of -catenin targets (68, 84). Another pathway leading to -catenin stabilization is usually activation of the Wnt signaling, the vertebrate homologue of (75, 82, 102). Wnt genes are tumorigenic in mice (75) and may also be implicated in human malignancy (84). The MET receptor tyrosine kinase (RTK) family contains three users: MET (17, 34, 81), RON (89), and c-Sea (42) (Sea may be a chicken RON orthologue). MET is the receptor for hepatocyte growth factor (HGF) (10). MET is usually expressed in a number of cell types, including epithelial cells (23, 105), endothelial cells (13, 35), myoblasts (2), spinal motor neurons (27), and hematopoietic cells (30, 74). Conversation of HGF with MET activates multiple intracellular signaling pathways involved in Akebiasaponin PE muscle and liver formation (32, 62, 97), cell proliferation (64, 73, 91), morphogenesis (66), and motility (105, 106). In addition to regulation of normal cell functions, MET is usually implicated in development and progression of a number of tumors. Increased MET expression has been found in papillary carcinomas of the thyroid gland; carcinomas of colon, pancreas, and ovary; and osteogenic sarcomas (23C25, 28, 33, 50). Point mutations in the MET kinase domain name have been recognized in hereditary Akebiasaponin PE and sporadic papillary renal carcinomas (98, 99). RON, the receptor for macrophage-stimulating protein (MSP, also known as HGF-like protein) (115), is usually another member of the MET RTK family sharing many common features with MET (89). RON is usually expressed in different cell types, including macrophages (47), epithelial cells (113, 114), osteoclasts (59), and hematopoietic cells, such as erythroid and myeloid progenitor cells (48) and bone marrow megakaryocytes (3). Ligand-stimulated RON activates the pathways regulating cell adhesion and motility, growth, and survival (20, 21). Recent investigations have shown that activated RON is expressed in human Akebiasaponin PE main breast carcinomas (61) and in a number of malignancy cell lines that are removed from the host tissue environment (15, 31). Truncated RON confers susceptibility to Friend virus-induced erythroleukemia in mice (83), and the avian oncogene v-and cyclin D1 oncogenes. Biochemical data suggest that the initiating step may be -catenin tyrosine phosphorylation by mutated RON and MET. Inhibition of Akebiasaponin PE the -catenin pathway resulted in reduction of RON and MET mutant transforming ability. In light of numerous data reporting increased cellular -catenin in malignancy (7, 68, 84), Akebiasaponin PE this pathway may contribute to cell transformation by mutated growth factor receptors of the MET family. MATERIALS AND METHODS Antibodies. RON receptor was immunoprecipitated from cell lysates by using mouse monoclonal anti-RON antibodies (clone ID2; a gift from F. Montero-Julian) (65). Rabbit anti-RON antibodies (C-20; Santa Cruz, Santa Cruz, Calif.) were used for Western blotting. Protein tyrosine phosphorylation was detected.
Anti-c-(monoclonal, C-8), anti-cyclin D1 (monoclonal, HD11), anti-human and -mouse MET (rabbit, C-28 and SP260), antiubiquitin (monoclonal, P1A6), and anti-JNK1 (rabbit, FL) antibodies were from Santa Cruz