Biochemical and Biophysical Research Communications
Interaction of serologically defined colon cancer antigen-3 with Arf6 and its predominant expression in the mouse testis
Introduction
ADP ribosylation factor 6 (Arf6) belongs to the Arf small GTPase family and localizes at the plasma membrane and endosomes where it regulates actin cytoskeleton remodeling and membrane trafficking by cycling between GDP-bound inactive and GTP-bound active states. Arf6 is implicated in a variety of cellular events related to morphological changes such as cytokinesis, migration, phagocytosis, endocytosis, exocytosis, and neurite extension [1], [2], [3]. Among these, cytokinesis is the division of the cytoplasm of a single cell to form two daughter cells at the final step of mitotic cell division, and represents an extreme example of cell phenomena accompanying drastic changes in cell shape. In addition to involvement of the actin cytoskeleton, membrane trafficking is known to play an essential role in cytokinesis through the addition of new membrane to compensate for the massive increase in surface area, and the local enrichment of protein and lipid components required for cleavage furrow ingression and abscission. Since an initial report showing the localization of a constitutively active Arf6 mutant at the cleavage furrow and midbody and the activation of Arf6 during cytokinesis [4], accumulating evidence has established Arf6 as an important regulator of cytokinesis. Several effectors of Arf6 during cytokinesis have been so far identified including Rab11 family-interacting proteins FIP3/4 [5], Sec10 subunit of the exocyst complex [5], [6], c-Jun N-terminal kinase-interacting proteins JIP3/4 [7], and mitotic kinesin-like protein MKLP1 [8], [9]. Through its interaction with these effectors, Arf6 is proposed to regulate various steps of cytokinesis, including endosomal transport along central spindle microtubules [7], tethering and fusion of transport vesicles carrying components required for cleavage furrow ingression and abscission [5], [10], and maintenance of structural integrity of the intercellular bridge and midbody [8], [9].
The serologically defined colon cancer antigen-3 (SDCCAG3) is an endosomal protein that was originally isolated by screening of cDNA expression libraries from human colon cancers with autologous patient antibodies [11]. Inhibition of SDCCAG3 activity by overexpression of the C-terminal region containing a coiled-coil domain was shown to decrease the presentation of tumor necrosis factor (TNF) receptor-1 on the cell surface and to protect cells from TNF-induced apoptosis, suggesting the involvement of SDCCAG3 in membrane trafficking [12]. Indeed, SDCCAG3 localizes at a subpopulation of early/recycling endosomes that partially overlap with early endosome antigen 1 (EEA1) and the transferrin receptor [13]. Recent proteomic analysis revealed that SDCCAG3 forms a protein complex with the retromer complex through an interaction with the Wiskott–Aldrich syndrome protein and scar homolog (WASH) complex [14] and regulates sorting nexin27-retromer-mediated endosomal trafficking of the glucose transporter GLUT1 [15]. During cytokinesis, SDCCAG3 undergoes dynamic subcellular changes and accumulates at the midbody [13]. SDCCAG3 was shown to regulate the completion of cytokinesis through its interaction with the protein tyrosine phosphatase PTPN13 and the Arf GTPase-activating protein GIT1 [13].
In this study, we identified SDCCAG3 as an Arf6-interacting protein by yeast two-hybrid screening with a GTP-locked Arf6 mutant as bait and characterized the tissue distribution of SDCCAG3 at the mRNA and protein levels. Furthermore, we provide evidence suggesting the importance of an interaction between SDCCAG3 and Arf6 in the midbody localization of SDCCAG3.
Section snippets
Animals
We purchased male C57BL/6 mice at postnatal week 10 from CLEA Japan (Tokyo, Japan) for western blot, northern blot and immunohistochemical analyses, and female Hartley guinea pigs weighing 250–300 g from Japan Laboratory Animals, Inc. (Tokyo, Japan) for antibody production. All animal experiments were approved by the Animal Experimentation and Ethics Committee of the Kitasato University School of Medicine and conducted in compliance with the Animal Use Guidelines of the Kitasato University
Interaction of SDCCAG3 with Arf6
In our previous screening of a mouse brain cDNA library with Arf6(Q67L) as bait [16], [18], we isolated five independent cDNA fragments for SDCCAG3, all of which contained a coiled-coil domain at the C-terminal region (Fig. 1A). To examine the specificity of the interaction of SDCCAG3 with Arf family members, yeast cells were cotransformed with cDNA clone #18 encoding amino acids 92–382 of SDCCAG3 and constitutively active or inactive Arf mutants (Fig. 1B). Only yeast cells coexpressing SDCCAG3
Discussion
Our yeast two-hybrid screening of a mouse brain cDNA library with Arf6(Q67L) showed SDCCAG3 to be a newly identified protein interacting with Arf6. Among the Arf family, SDCCAG3 specifically interacted with Arf6(Q67L) through its 101 C-terminal amino acids. Overexpression of the C-terminal region of SDCCAG3 containing an Arf6-binding site was previously shown to protect NIH3T3 and HeLa cells from TNF-induced apoptosis by suppressing the trafficking of TNF receptor-1 to the plasma membrane [13].
Acknowledgments
We thank Dr. Kazuhisa Nakayama (Kyoto University) for Arf cDNAs, and Mr. Osamu Katsumata and members of Research Center for Biological Imaging in Kitasato University School of Medicine for their technical support in electron microscopy. This work was supported by KAKENHI Grant No. 25640025 to H.S. from the Japan Society for the Promotion of Science.
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