Met, the Hepatocyte Growth Factor Receptor, Localizes to the Nucleus in Cells at Low Density Sharon Pozner-Moulis, Derek J. Pappas, and David L. Rimm Department of Pathology, Yale University School of Medicine, New Haven, Connecticut Abstract Some breast cancer cases in our previous immunohistochem- ical studies show Met expression in the nucleus. Given nuclear localizationofotherreceptortyrosinekinases,weproceededto investigate Met. Nuclear Met is seen in numerous cell lines and in germinal regions of many tissues using four unique antibodies.Cellfractionationrevealsa60-kDabandrecognized by COOH-terminal Met antibodies that is present independent of hepatocyte growth factor treatment. Green fluorescent protein (GFP) fusion proteins of the cytoplasmic domain of Met transfected into HEK293 cells are found in the nucleus whereas the full-length Met-GFP fusion is membranous. Further deletions of the Met-GFP fusions identify a region of the juxtamembrane domain required for nuclear trans- location. In a CaCo2 cell line model for epithelial maturation, we find that Met is initially nuclear, and then becomes membranous, after confluence. This work suggests processing oftheMetreceptor,analogoustoErbB4,resultingintherelease of the cytoplasmic domain and its translocation to the nucleus in cells at low density. (Cancer Res 2006; 66(16): 7976-82) Introduction Met (the c-Met gene product) is a receptor tyrosine kinase (RTK) for the hepatocyte growth factor (HGF) ligand that is expressed on the surface of epithelial and endothelial cells. The primary 150-kDa transcript is partially glycosylated to form the 170-kDa Met precursor that is then cleaved forming a heterodimer consisting of two subunits (45 and 150 kDa) joined by disulfide bonds. The smaller a subunit is entirely extracellular, whereas the larger h subunit traverses the plasma membrane and includes a juxtamem- brane region responsible for negatively regulating Met, a tyrosine kinase domain with intrinsic kinase activity, and a COOH-terminal region that provides binding sites for target substrates (for review, see ref. 1). Binding of HGF to Met induces dimerization, which in turn triggers autophosphorylation of tyrosine residues in the activation loop of the kinase domain. Autophosphorylation then activates the intrinsic kinase activity of Met via the subsequent phosphorylation of two tyrosine residues in the COOH terminus, forming a multisubstrate docking site. Chimeric receptors with these residues can induce mitogenic, morphogenic, and motogenic responses similar to wild-type Met (reviewed in ref. 2). Activation of the docking site triggers signaling cascades, such as Gab1, Grb2, and phosphatidylinositol 3-kinase, leading to proliferation, scatter- ing, increased motility, invasion, and branching morphogenesis (reviewed in ref. 3). Met has also been shown to be activated in the absence of HGF ligand. Constitutive activation of Met can occur via mutations in the cytoplasmic domain and are associated with the genesis and progression of some human tumors (4). A growing list of membrane proteins has been found to translocate to the nucleus. Members of the epidermal growth factor (EGF) and fibroblast growth factor family of RTKs have been shown to translocate to the nucleus as either intact receptors, or for ErbB4, as a truncated fragment, activating transcription of target genes (5, 6). ErbB4, a member of the EGF receptor family, plays an important role in mammary gland development and controls cell proliferation and differentiation. Ligand binding to ErbB4, such as Met, stimulates dimerization and autophosphor- ylation followed by substrate phosphorylation. ErbB4 undergoes cleavage of its intracellular domain via presenilin-dependent g-secretase-like proteolysis, which then localizes to the nucleus and plays a role in regulating gene transcription (7, 8). Other membrane proteins that undergo similar cleavage events and subsequent nuclear localization include Notch, APP, CSF-1, E- cadherin, and CD44 (9–11). g-Secretase cleavage of these proteins occurs in the transmembrane domain and is usually preceded by ectodomain shedding by a matrix metalloproteinase. Our previous immunohistochemical study on f600 cases of breast cancer showed that expression of the Met cytoplasmic domain and not the extracellular domain was correlated with poor patient outcome in lymph node–negative breast carcinomas (12). This difference is not easily explained, but one possibility is a processing event where a COOH-terminal fragment is present in the absence of the NH 2 terminus. Other RTKs are cleaved, and the resultant cleavage product translocates to the nucleus as for ErbB4 (7). Although we cannot yet prove cleavage, here we show a 60-kDa fragmentofMetthatlocalizestothenucleusinaligand-independent manner and is associated with lower density cell colonies. Materials and Methods Cell lines and treatments. HEK293, A431, NIH3T3, and HT-29 cell lines wereobtainedfromAmericanTypeCultureCollection(ATCC,Manassas,VA) and cultured in DMEM with 10% fetal bovine serum (FBS). Mel1241 and Mel1335 cells were cultured in RPMI 1640 with 10% FBS. HMEC cells were obtained from ATCC and cultured in MEGM with 10% FBS. MCF-10A cells wereobtainedfromATCCandculturedinDMEM/F12with10%horseserum. CaCo2 cells were generously donated by the laboratory of Dr. Jon Morrow (Yale University School of Medicine, New Haven, CT). A431 cells were serum starved in DMEM containing 0.5% FBS for 18 hours and treated with 250 Amol/L ALLN (Calbiochem, La Jolla, CA) or 20 ng/mL HGF (R&D Systems, Minneapolis, MN). Treatment with ALLN and HGF combined was for the indicated time with a 30-minute pretreatment of ALLN. Primary antibodiestotheCOOHterminusofMetweremonoclonalantibody3D4and polyclonal antibody CVD13 (Zymed Laboratories, Inc., San Francisco, CA) andpolyclonalantibodiesC12andC28(SantaCruzBiotechnology,Inc.,Santa Cruz, CA). Other antibodies include lamin, E-cadherin, and extracellular signal-regulated kinase (ERK) 1 (BD Transduction Laboratories, San Diego, CA), a-tubulin(ZymedLaboratories),andpERK1/2(Upstate,LakePlacid,NY). Requests for reprints: David L. Rimm, Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510. Phone: 203-737-4204; Fax: 203-737-5089; E-mail: david.rimm@yale.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-4335 Cancer Res 2006; 66: (16). August 15, 2006 7976 www.aacrjournals.org Research Article Research. on January 4, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from