Journal of Molecular Biology
A Divergent Substrate-Binding Loop within the Pro-oncogenic Protein Anterior Gradient-2 Forms a Docking Site for Reptin
Introduction
Anterior gradient-2 (AGR2) is a protein whose function is proving to play an increasingly critical role in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. AGR2 was identified initially as a secretory factor expressed in the anterior region of the dorsal ectoderm in Xenopus laevis embryos, where it was postulated to mediate the specification of dorsoanterior ectodermal fate, particularly in the formation of the cement gland.1, 2 AGR2 was subsequently cloned as a gene whose expression is induced by the estrogen receptor α,3 and subsequent studies in primary breast carcinomas have also shown significant associations between AGR2 expression and estrogen receptor-α positivity or tamoxifen resistance.4, 5 Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung.4, 6, 7, 8, 9 More biological studies in cell lines have shown a significant role for AGR2 in tumor-associated pathways, including tumor growth, cellular transformation, cell migration, limb regeneration, and metastasis.8, 10, 11, 12
AGR2 protein was also identified as part of a clinical proteomics screen aimed at discovering novel inhibitors of the tumor suppressor p53, and it was subsequently validated as a potent inhibitor of p53 activity and of the p53-dependent response to DNA damage.11 The latter data provide a specific oncogenic pathway into which AGR2 integrates; however, the signaling mechanisms that drive AGR2 to inhibit p53 are not defined. Although there are no well-validated binding proteins in human cells that can explain how AGR2 can act as a pro-oncogenic protein, peptide aptamer screens have identified a specific peptide-binding activity for the AGR2 protein for peptides containing an (S/T)xIΦΦ consensus motif, suggesting that the AGR2 protein might prove to have a peptide groove able to interact with cellular proteins containing such a consensus motif.13 Furthermore, penetratin peptides linked to this AGR2-binding (S/T)xIΦΦ motif or EGFP fusions to the (S/T)xIΦΦ motif can stabilize AGR2 in cells and stimulate p53 activity, indicating that the AGR2 protein can interact with this peptide consensus motif in vivo.14 A yeast two-hybrid screen has also been used to previously identify the prometastatic proteins C4.4 and DYS1 as interactors of AGR215; however, there was no biological validation of C4.4 and DYS1 as a bona fide protein–protein interaction in human cells. However, potential extracellular receptor functions for AGR2 in human cells remain possible, because an interaction between the newt extracellular receptor PROD1 and newt AGR2 was identified using a yeast two-hybrid screen and validated to demonstrate a direct signaling role for AGR2 in amphibian limb regeneration.10
Although the general biochemical functions of AGR2 in human cells remain undefined, AGR2 is part of the protein disulfide isomerase (PDI) superfamily that contains core thioredoxin folds (CxxC or CxxS motif), which have the potential to act as molecular chaperones that regulate protein folding via regulation of disulfide-bond formation.16 There are five protein members of this family: TRX1 (thought to be predominantly the nuclear thioredoxin), TRX2 (thought to be predominantly the mitochondrial thioredoxin), endoplasmic reticulum (ER) protein 18 (ERP18; the ancestral protein in the AGR2/AGR3 group that has potent reducing potential),17 AGR2, and the AGR2 ortholog AGR3. AGR2 and AGR3 are confined to vertebrates, and both have the CxxS core motif instead of the CxxC motif of TRX1, TRX2, and ERP18.18 The majority of PDIs/ERPs harbor a typical H/KDEL ER retrieval signal. A putative ER retention sequence that has been shown to regulate the intracellular localization of AGR2 in human cells has been identified at the C-terminus of AGR2.14 It is therefore possible that at least one function of the AGR2 is to act as a PDI and hence as a protein molecular chaperone. A recent study has confirmed that AGR2 is essential for the production of the intestinal mucin MUC2, a cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. The cysteine residue within the AGR2 thioredoxin-like domain was shown to form a mixed disulfide bond with a cysteine in the N-terminus or C-terminus of MUC2 as it is being processed.19 However, there are currently no established biochemical mechanisms to explain the function or the regulation of AGR2 protein.
In this report, a yeast two-hybrid screen was used to identify a potentially novel interacting protein for the AGR2 protein from a human breast cancer library. A protein named Reptin was identified by the yeast two-hybrid screen and validated as an interacting protein of AGR2 in human cells. Reptin is a highly conserved member of the AAA+ family that can be found in numerous multiprotein complexes linked to transcription, DNA damage response, and nonsense-mediated RNA decay.20, 21, 22, 23, 24, 25 This protein is a member of the highly conserved RuvBl1/2 superfamily containing ATP binding motifs and DNA binding and helicase functions and an ability to form biologically relevant protein–protein interactions with proteins implicated in cancer, including Myc, Tip60, APPL1, Pontin, and telomerase holoenzyme complexes.20, 23, 26, 27, 28, 29 The validation of Reptin as an AGR2 binding protein gives rise to a potentially novel signaling complex involved in prometastatic cancer development. Our mapping of the determinants that mediate a specific Reptin–AGR2 protein–protein complex in vitro provides biochemical insights for understanding how the Reptin–AGR2 complex can be regulated in cells. These data also provide ideas for development of in vitro enzyme assays for the screening of small molecules that might be used to disrupt the AGR2–Reptin complex in cancer cells as potential therapeutic leads.
Section snippets
Reptin is overexpressed in primary human cancers and forms a stable protein–protein complex with AGR2 protein in cancer cells
In our search for proteins interacting with human AGR2 protein, we used a yeast two-hybrid assay with LexA fused to AGR2 as bait screened against a cDNA library derived from breast cancer cells (Fig. 1a). These hits appear relatively specific for the AGR2 bait because a parallel yeast two-hybrid screen performed on the AGR2 ortholog AGR3 (sharing approximately 75% homology) yielded a completely distinct set of interacting proteins (data not shown).
Two extracellular prometastatic receptors
Defining regulatory motifs in Reptin that regulate binding to AGR2
AGR2 is a prometastatic and p53 inhibitory protein involved in a range of oncogenic pathways, such as tamoxifen resistance and cell migration, as well as additional biological functions in limb regeneration and inflammatory responses.5, 10, 12, 19 Despite the wealth of data accumulating on AGR2, there are no validated interacting proteins for the AGR2 protein in human cells, with only the newt receptor PROD1 identified as an AGR2-interacting protein in yeast two-hybrid that functions in newt
Reagents
Fetal bovine serum was from Autogen Bioclear. Dulbecco's modified Eagle's medium and RPMI were provided by Gibco. Trypsin/EDTA solution and penicillin–streptomycin were supplied by Invitrogen. Attractene was from Qiagen. Hybond-C nylon membrane for immunoblotting was supplied by Amersham Pharmacia Biotech. ATP-γS [adenosine 5′-O-(3-thiotriphosphate)] and ATP (adenosine 5′ thiotriphosphate) were from Calbiochem. The following antibodies were used: anti-HA tag monoclonal antibody and anti-Myc tag
Acknowledgements
B.V. was supported by the European Regional Development Fund through IGA MZCR NS/9812-4 and RECAMO CZ.1.05/2.1.00/03.0101. R.H. was supported with GACR P301/10/1615. This work was funded by Cancer Research UK Program Grant C483/A6354 (T.R.H.) and a Cancer Research UK PhD Studentship to M.M.M. (C483/A8033). We acknowledge our use of the Edinburgh Biophysical Characterization Facility (supported by the Scottish University Life Sciences Alliance and the Biotechnology and Biological Sciences
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