Elsevier

Experimental Cell Research

Volume 314, Issue 7, 15 April 2008, Pages 1519-1528
Experimental Cell Research

Research Article
A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95

https://doi.org/10.1016/j.yexcr.2008.01.013Get rights and content

Abstract

RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with 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.

References (46)

  • N. Stuurman et al.

    Nuclear lamins: their structure, assembly, and interactions

    J. Struct. Biol.

    (1998)
  • R. Foisner et al.

    Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation

    Cell

    (1993)
  • F. Lin et al.

    MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin

    J. Biol. Chem.

    (2000)
  • D.M. Hodzic et al.

    Sun2 is a novel mammalian inner nuclear membrane protein

    J. Biol. Chem.

    (2004)
  • R. Ashery-Padan et al.

    Distinct regions specify the targeting of otefin to the nucleoplasmic side of the nuclear envelope

    J. Biol. Chem.

    (1997)
  • S. Fang et al.

    Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53

    J. Biol. Chem.

    (2000)
  • L.A. Passmore et al.

    Getting into position: the catalytic mechanisms of protein ubiquitylation

    Biochem. J.

    (2004)
  • A.M. Weissman

    Themes and variations on ubiquitylation

    Nat. Rev., Mol. Cell Biol.

    (2001)
  • P. Chene

    Inhibiting the p53–MDM2 interaction: an important target for cancer therapy

    Nat. Rev., Cancer

    (2003)
  • N.P. Crowford et al.

    Tumor markers and colorectal cancer utility in management

    J. Surg. Oncol.

    (2003)
  • G.L. Shen-Ong et al.

    Expression profiling identifies a novel alpha-methylacyl-CoA racemase exon with fumarate hydratase homology

    Cancer Res.

    (2003)
  • T. Sugiura et al.

    DDX39, upregulated in lung squamous cell cancer, displays RNA helicase activities and promotes cancer cell growth

    Cancer Biol. Ther.

    (2007)
  • R. Yagyu et al.

    A novel oncoprotein RNF43 functions in an autocrine manner in colorectal cancer

    Int. J. Oncol.

    (2004)
  • Cited by (44)

    • The Clinical Utility of Biomarkers in the Management of Pancreatic Adenocarcinoma

      2014, Seminars in Radiation Oncology
      Citation Excerpt :

      These relatively frequent pancreatic cystic neoplasms have the potential to become invasive and metastatic after progression to PDAC.132 RNF43 catalyzes the ubiquitin-dependent degradation of frizzled and LRP5/6; 2 important Wnt-activated membrane proteins that mediate inhibition of the adenomatosis polyposis coli destruction complex and subsequent survival of β-catenin, a prominent transcription factor involved in cell adhesion and growth.134-136 Interestingly, pancreatic adenocarcinoma cell lines with inactivating mutations in RNF43 were all sensitive to LGK974, a porcupine inhibitor specific for inhibiting the Wnt pathway, whereas the wild-type cells were insensitive to LGK974.137

    • RNF43 mutations are recurrent in Chinese patients with mucinous ovarian carcinoma but absent in other subtypes of ovarian cancer

      2013, Gene
      Citation Excerpt :

      Nevertheless, larger sample size for several subtypes of ovarian carcinomas should be recruited to test this conclusion, such as ovarian sex cord–stromal tumor, Krukenberg tumor and the remaining rare subtypes. As RNF43 is a critical enzyme in the process of ubiquitin-dependent protein degradation (Hao et al., 2012; Koo et al., 2012; Sugiura et al., 2008), it is quite natural to speculate that RNF43 mutations might affect RNF43 enzyme activity, and then intracellular protein degradation, ultimately contribute to the development of human neoplasms. Functional assays are performing in our laboratory to determine whether the RNF43 mutations could promote ovarian cancer progression.

    • New insights in ubiquitin-dependent Wnt receptor regulation in tumorigenesis

      2024, In Vitro Cellular and Developmental Biology - Animal
    View all citing articles on Scopus
    View full text