(1995) Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26 S proteasome. a transmembrane 1 domain, resulted in no degradation, significantly reducing the ability of invasion and migration of NPC cells. This study provides a novel molecular mechanism for the low expression of NGX6a in NPC cells and an important molecular event in the process of invasion and metastasis of nasopharyngeal carcinoma cells. (nasopharyngeal cancer-related gene 6) is a candidate tumor metastasis suppressor gene that is cloned from the high frequency loss-of-heterozygosis region of chromosome 9p21-22 in nasopharyngeal carcinoma (1). Our previous studies demonstrated that the gene encodes a product of two isoforms, NGX6a and -b, from three different transcripts (2). NGX6b encodes 338 amino acids, which contain the extracellular domain of an EGF-like domain and two transmembrane domains, whereas NGX6a contains the extracellular domain of an EGF-like domain and seven transmembrane domains (3,C5). NGX6b mRNA expression is reduced or absent in nasopharyngeal carcinoma and colon cancer and is associated with tumor metastasis (6,C9). NGX6b expression in NPC 5-8F cells reduces the invasion capacity, increasing the rate of cell adhesion and restoring intercellular gap junction communication (10, 11); the tumor formation and lung metastases of NPC 5-8F cells that were transplanted in SCID mice were significantly inhibited by NGX6b expression. NGX6b can bind to the cell membrane via an intracellular region with ezrin and inhibit the cell proliferation, cell invasion, and metastasis of nasopharyngeal carcinoma through the EGF receptor signaling pathway (12, 13). NGX6b can also inhibit the invasion of colon cancer cells by inhibiting the Wnt/-catenin signaling pathway (4, 5, 14). The isoform NGX6a was recently found to be expressed in various organs, mainly in epithelial cells and neuronal cells in the brain, nasopharynx, and lung, whereas NGX6b is expressed in the brain, heart, kidney, nasopharynx, and lung, and the expression levels of NGX6a are much higher than are those of NGX6b (3). However, the function of NGX6a is not well defined. Ezrin is an important member of the ezrin/radixin/moesin (ERM)3 family of eukaryotic membrane proteins-cytoskeleton bridge molecules (15, 16). Ezrin is involved in cell morphology, cell adhesion, movement, cytoskeleton remodeling, and signaling processes (10, 11, 17). The ezrin protein contains three main parts: a CM-4620 spherical highly conserved amino terminus CM-4620 (85% identical) that binds with the membrane protein; an extending helix Rabbit Polyclonal to ATP5A1 domain in the middle; and a positively charged carboxyl terminus, which binds to actin. When ezrin is present as a soluble monomer protein, the CM-4620 amino terminus binds with the carboxyl end but does not bind to actin protein when ezrin is in the inactivated state; when ezrin is activated, the binding sites are exposed, and it plays an important role as a bridge between membrane protein and cytoskeleton actin. Many studies have demonstrated that ezrin expression is abnormally regulated in tumors with or without metastasis and have indicated that ezrin plays a key role in tumor metastasis (18,C21). We aimed to examine what roles NGX6a plays in the invasion and metastasis of nasopharyngeal carcinoma cells and to determine the molecular link between NGX6a and ezrin. We found that NGX6a is degraded through the proteasome pathway mediated by ezrin in NPC cells but is not ubiquitinated. Seven transmembrane domains of NGX6a and the N-ERMAD domain of ezrin are required for the degradation of NGX6a. The knockdown of ezrin expression or the increase in NGX6a expression inhibits the invasion and CM-4620 metastasis of nasopharyngeal carcinoma cells. MATERIALS AND METHODS Antibodies, siRNAs, and Plasmids The monoclonal mouse antibody anti-FLAG(M2) and anti-ubiquitin (Sigma), mouse anti-c-Myc (Clontech, Mountain View, CA), anti-His (Novagen, Darmstadt, Germany), anti-GAPDH, mouse anti–actin, Protein G Plus-agarose (Santa Cruz Biotechnology, Inc.), rabbit anti-ezrin (Upstate), anti-ISG15, anti-NEDD8, CM-4620 anti-SUMO1 (Cell Signaling Technology, Danvers, MA), goat anti-mouse IgG(H+L) antibody (HRP), goat anti-rabbit IgG(H+L) antibody (HRP) (KPL), sheep anti-mouse IgG Cy3 conjugate antibody (Boster, Wuhan, China), and sheep anti-rabbit IgG FITC conjugate antibody (SABC, Beijing, China) were purchased from the companies indicated, and polymonoclonal anti-NGX6a antibody was prepared as described (3). pCMV-Myc-NGX6a and pIRES-neo3-NGX6a constructs were made by cloning the human NGX6a ORF coding region (accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001042589.2″,”term_id”:”390407646″,”term_text”:”NM_001042589.2″NM_001042589.2) into pCMV-Myc and pIRES-neo3 (Clontech). pcDNA3.1(+)-ezrin was built by cloning the human ezrin coding region (accession number.