Research ArticleA cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95
Introduction
The RING finger motif, found in many functionally distinct proteins, was first identified in the early 1990′s in the protein encoded by the Really Interesting New Gene 1 [1]. The RING finger domain contains eight metal-binding residues that coordinate two zinc atoms in an interleaved pattern for folding and biological actions [2]. RING finger proteins act by mediating diverse protein–protein interactions and one of their main biological functions is catalyzing ubiquitylation as a ubiquitin-protein isopeptide ligase. Ubiquitylation is a regulated process through which ubiquitin, a highly conserved 76-amino acid polypeptide, is covalently attached to other proteins [3], [4]. This modification is realized by the action of three classes of enzymes; ubiquitin-activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin-protein isopeptide ligase (E3). E1 forms a thiol-ester linkage between its active site cysteine and the carboxyl-terminal glycine of ubiquitin. Ubiquitin is then transferred to a ubiquitin conjugating enzyme E2 through a thiol-ester linkage. E3 eventually interacts with E2 and a substrate, facilitating formation of an isopeptide linkage between the carboxyl-terminal glycine of ubiquitin and lysines either on a substrate protein or the last ubiquitin of a protein-bound multi-ubiquitin chain. E3 is primarily responsible for determination of specificity for ubiquitin conjugation [5]. Poly-ubiquitylation targets proteins for degradation by a multisubunit, ATP-dependent protease termed the proteasome [6]. It is worth noting that a representative RING finger E3 ligase, MDM2, is implicated in tumorigenesis by degrading a tumor suppressor protein, p53 [7].
Colorectal cancer is one of the most common causes of cancer-related deaths in the Western world [8]. Effective intervention strategy requires targeted therapy with earlier detection of colorectal cancer. Hence, in search for a new molecular target or an invaluable biomarker, we explored genes differentially expressed in colon adenocarcinoma using the commercially available BioExpress database [9], [10], [11]. Through the data mining, we identified RNF43, a gene encoding a RING finger protein, upregulated in colon adenocarcinoma. RNF43 has already been reported to be one of the genes overexpressed in colorectal tumors [12], [13]. Strikingly, overexpression of RNF43 promotes cell growth and, conversely, knockdown of its expression by specific short interference RNAs exerts a growth-suppressing effect [13]. These findings suggest that RNF43 plays a crucial role in the colon cancer pathogenesis.
Although the presence of the RING finger domain implies the relationship of RNF43 with the ubiquitylation pathway, no biochemical characterization of RNF43 with this respect has yet been described and, importantly, the mechanism of growth stimulation by RNF43 remains elusive. In this study, therefore, we conducted biochemical and intracellular characterizations of RNF43 to clarify the underlying mechanism of its action. Especially, since RING finger proteins exist throughout the subcellular compartments to accomplish diverse functions, unlike the zinc fingers that reside exclusively in the nucleus for binding DNA sequences [14], we made an attempt to localize RNF43 subcellularly as a clue to unraveling its biological role. Our results revealed that this RING finger protein is localized in the ER and, at least partially, in the inner nuclear membrane and displays ubiquitin ligase activity.
Section snippets
Identification of RNF43 upregulated in colon cancers
Expression profiles of RNF43 in various normal and diseased tissues were analyzed by virtue of the BioExpress database (Gene Logic Inc., Gaithersburg, MA) as described previously [10], [11]. Cell culture and RT-PCR were conducted by the previous method [10]. Primers employed for RNF43-specific PCR were 5′-AGCCACCTTCTCCTGATCAGCAA-3′ (forward) and 5′ -TCACACAGCTCCTCGAGTTCCT-3′ (reverse).
Plasmids construction
Amino acid numbering is based on GenBank accession no. NM_017763 and nucleotide residue one corresponds to
Identification of RNF43 as a gene upregulated in colon adenocarcinoma as well as in colon adenoma
Through the search for genes differentially expressed in colon cancer using the BioExpress expression profile database, we found the probe set 218704_at upregulated in colon adenocarcinoma (Fig. 1A). This probe set corresponds to RNF43, a recently discovered RING finger protein gene, whose expression is elevated in colon cancers [12], [13]. The coincidence of our RNF43 expression results with the published ones underscores the reliability of the BioExpress database. From this database we also
RNF43 as a putative inner nuclear membrane protein
In eukaryotic cells the nucleoplasm is separated from the cytoplasm by the nuclear envelope that comprises the outer and inner nuclear membranes. The outer nuclear membrane is continuous with the rough ER whereas the inner nuclear membrane faces the nucleoplasm and is covered by the nuclear lamina [22]. The two membranes are separated by a narrow lumen and joined periodically at the pore membrane [23]. Several proteins are identified as inner nuclear membrane proteins in mammalian cells such as
Acknowledgments
We thank Y. Nagano and Y. Noguchi for technical assistance.
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