Expression of AIE-75 PDZ-domain protein induces G2/M cell cycle arrest in human colorectal adenocarcinoma SW480 cells
Introduction
AIE-75/harmonin is a protein of 75-kDa identified as an autoantigen specific to X-linked autoimmune enteropathy (AIE) [1], and as a colon cancer-related antigen [2]. AIE-75/harmonin germline mutation has been found to be a cause of Usher syndrome type 1C, an autosomal recessive disorder characterized by congenital sensorineural deafness and retinitis pigmentosa [3], [4]. Alternatively spliced transcripts of AIE-75/harmonin [4] predict at least 10 protein isoforms which can be grouped into three classes: harmonin a, b, and c [5]. All the three isoform classes of AIE-75/harmonin have three PSD-95/Dlg/ZO-1 (PDZ) domains [1], [5], which have been known to interact with the specific carboxyl terminus sequence motif X-S/T/F/Y-X-Φ(Φ: hydrophobic amino acid residue) of target proteins [6], [7]. Several PDZ domain-containing proteins have been known to interact with tumor suppressor proteins, for example, binding of discs large-1 (dlg) tumor suppressor (hDLG) to APC [8], [9], membrane-associated guanylate kinases (MAGI) to PTEN [10], [11], and Na+/H+ exchanger regulatory factor-1 to merlin [12]. We have recently identified an interaction of AIE-75 with a novel protein MCC2 which has high homology to the tumor suppressor MCC (mutated in colon cancer) [13]. AIE-75/harmonin binds to cadherin 23 and myosin VIIa, via its PDZ domains [5], [14]. In addition, the longest isoform b of AIE-75/harmonin is also known to interact with F-actin through the second coiled-coil domain [5].
AIE-75 is physiologically expressed in epithelial cells with microvilli such as those of small intestine, colon, kidney, pancreas, and inner ear [1], [4], [13], [15]. It is present at the apical membranes of ileal villous cells and renal proximal tubular cells, while it is present diffusely in the cytoplasm of colonic mucosa cells. In colon cancer cells, loss of AIE-75 expression is a rather rare phenomenon, whereas it is frequent in renal cancer cells [13]. Although the above pieces of evidence suggest a unique functional role of AIE-75 in colonic mucosa cells and colon cancer cells, it remains totally unknown. In order to find out the potential function of AIE-75, we transfected AIE-75 gene into SW480 human colon adenocarcinoma cells. Here, we report that expression of AIE-75 induces G2/M cell cycle arrest. We also identified a serine/threonine phosphatase PP2A as a candidate mediator of the checkpoint induction.
Section snippets
Cells
Human colon cancer cell line SW480 [16] was provided by Institute of Development, Aging and Cancer, Tohoku University. They were maintained in the mixture of DMEM and Ham's F12 (NISSUI, Tokyo) with 10% fetal bovine serum (FBS; Bio Whittaker, Walkersville, MD) at 37 °C in a humidified 5% CO2 atmosphere.
AIE-75 transfection and cell cloning
AIE-75 expression vector pcDNA3.1/HisC-AIE-75 was made by subcloning an AIE-75 (harmonin a; GenBank AB006955) coding region into a vector, pcDNA3.1/HisC-Neo (Invitrogen, Carlsbad, CA). The
Expression of AIE-75
Expression of AIE-75 mRNA in each transfectant clone was assessed by a semi-quantitative RT-PCR assay. Expression level of AIE-75 was high in Clone 1-2 and Clone 1-4, and relatively low in Clone 1-5 and Clone 1-6. In contrast, neither parent cells nor the cells transfected with vector only (Neo1, Neo2) showed expression of AIE-75(Fig. 1).
Cell growth and morphological changes
Using AIE-75 transfectants, we evaluated the effect of AIE-75 expression on cell growth (Fig. 2). Compared to Neo1 and Neo2 control vector transfectants, all
Discussion
AIE-75 is a protein of 75 kDa recently identified as the autoantigen that reacts to the autoantibody in the sera of patients with AIE [1]. This protein was also identified by another group as the antigen reactive to autoantibody in the sera of colon cancer patients [2], [15]. We have recently demonstrated that AIE-75 can bind to a novel candidate tumor suppressor MCC2 that is homologous to the tumor suppressor MCC (mutated in colon cancer) [13]. In this context, we were interested in the
Acknowledgements
We thank Ms Narumi Furuuchi for technical help and Ms Masako Yanome for help in preparing manuscript.
References (29)
- et al.
Identification of an autoimmune enteropathy-related 75-kilodalton antigen
Gastroenterology
(1999) - et al.
Crystal structures of a complexed and peptide-free membrane protein- binding domain: molecular basis of peptide recognition by PDZ
Cell
(1996) - et al.
Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase
J. Biol. Chem.
(2000) - et al.
Interaction of MCC2, a novel homologue of MCC tumor suppressor, with PDZ-domain Protein AIE-75
Gene
(2001) - et al.
Isoforms of the human PDZ-73 protein exhibit differential tissue expression
Biochim. Biophys. Acta
(1999) - et al.
Localization of the novel serine/threonine protein phosphatase 6 gene (PPP6C) to human chromosome Xq22.3
Genomics
(1997) - et al.
Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in xenopus laevis oocytes
Fed. Eur. Biochem. Soc. Lett.
(1989) - et al.
Characterization of human colon cancer antigens recognized by autologous antibodies
Int. J. Cancer
(1998) - et al.
A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene
Nat. Genet.
(2000) - et al.
A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C
Nat. Genet.
(2000)
Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle
Eur. Mol. Biol. Org. J.
Recognition of unique carboxyl-terminal motifs by distinct PDZ domains
Science
Binding of APC to the human homolog of the Drosophila discs large tumor suppressor protein
Science
The APC-hDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase
Oncogene
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