ANKRD26 and Its Interacting Partners TRIO, GPS2, HMMR and DIPA Regulate Adipogenesis in 3T3-L1 Cells

HMMR and DIPA Regulate Adipogenesis in 3T3- L1 Cells. PLoS ONE 7(5): e38130. doi:10.1371/journal.pone.0038130 ANKRD26 and Its Interacting Partners TRIO, GPS2, HMMR and DIPA Regulate Adipogenesis in 3T3-L1 Cells Xiu-Fen Liu. 0 1 Tapan K. Bera. 0 1 Charissa Kahue 0 1 Thelma Escobar 0 1 Zhaoliang Fei 0 1 Gregory A. Raciti 0 1 Ira Pastan 0 1 Rebecca Berdeaux, University of Texas Health Science Center at Houston, United States of America 0 Current address: Moffitt Cancer Center , Tampa, Florida , United States of America 1 Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland , United States of America Partial inactivation of the Ankyrin repeat domain 26 (Ankrd26) gene causes obesity and diabetes in mice and increases spontaneous and induced adipogenesis in mouse embryonic fibroblasts. However, it is not yet known how the Ankrd26 protein carries out its biological functions. We identified by yeast two-hybrid and immunoprecipitation assays the triple functional domain protein (TRIO), the G protein pathway suppressor 2 (GPS2), the delta-interacting protein A (DIPA) and the hyaluronan-mediated motility receptor (HMMR) as ANKRD26 interacting partners. Adipogenesis of 3T3-L1 cells was increased by selective down-regulation of Ankrd26, Trio, Gps2, Hmmr and Dipa. Furthermore, GPS2 and DIPA, which are normally located in the nucleus, were translocated to the cytoplasm, when the C-terminus of ANKRD26 was introduced into these cells. These findings provide biochemical evidence that ANKRD26, TRIO, GPS2 and HMMR are novel and important regulators of adipogenisis and identify new targets for the modulation of adipogenesis. - Funding: This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work and should both be considered first authors. The current epidemic of obesity and diabetes has stimulated research to improve the understanding of these diseases and to identify and characterize new genes involved in their pathogenesis [15]. We recently demonstrated that the Ankyrin repeat domain 26 (Ankrd26) gene plays an important role in the development of these pathologies. Mice with partial inactivation of the Ankrd26 gene develop marked hyperphagia, severe obesity and an unusual form of diabetes in which white adipose tissue preserves its sensitivity to insulin [6,7]. Moreover, when young Ankrd26 deficient mice were placed on a pair-feeding diet with normal mice, they maintained normal body weight, showed a better glucose tolerance, and increased insulin sensitivity in the white adipose tissue, indicating a dual role of the Ankrd26 gene in the control of appetite and of adipose tissue insulin sensitivity [7]. In addition, mouse embryonic fibroblasts (MEFs) from these mice have a higher rate of spontaneous adipogenesis compared to normal MEFs and their differentiation to adipocytes is greatly increased when they are exposed to a mixture of adipogenic inducers [8], indicating a prominent role of the Ankrd26 gene in fat cells. The Ankrd26 protein is highly expressed in the hypothalamus and other regions of the brain, as well as in many tissues and organs, including white adipose tissue. The protein is located in the cytosol close to the inner aspect of the cell membrane in HeLa and 293T cells expressing ANKRD26-EGFP, and it contains both ankyrin repeats and spectrin helices, through which it is potentially able to interact with other proteins [6]. However, it is not yet known what these proteins are and how the Ankrd26 protein carries out its biological functions. In the present work, we used yeast two-hybrid and immunoprecipitation assays to identify interacting partners for the Ankrd26 protein, and performed knock-down experiments to establish whether any of these interacting proteins were functionally relevant in adipogenesis and whether this process was carried out by their interaction with Ankrd26 protein. Identification of ANKRD26 protein interaction partners To identify potential interacting partners of the ANKRD26 protein, we performed yeast two-hybrid screening using 18 baits, encoding overlapping fragments from the full-length ANKRD26 protein sequence (Figure 1). The baits were screened with three different cDNA expression libraries generated from human adult brain, human testis and mouse 11.5-day embryo, tissues known to express ANKRD26 [6]. Thirteen independent interacting proteins were identified by the screening, and 12 of them bound to the coiled-coil domain in the C-terminus, suggesting a prominent role of this domain for Ankrd26 protein-protein interactions (Table 1). Validation of ANKRD26 protein interaction partners by IP Among the 13 interacting partners, we selected five to validate their interactions with ANKRD26 in mammalian cells. These are the triple functional domain protein (Trio), G-protein pathway Figure 1. ANKRD26 yeast two-hybrid strategy. The position of ANKRD26s ankyrin and spectrin coiled-coil repeats are depicted on a schematic diagram of the full-length, 1710 amino acid, protein. The line below indicates the relative locations of ANKRD26 bait clones used in the GAL4-based yeast two-hybrid cDNA library screens. DIPA interacts with baits 7, 8, 9 and 17; GPS2 interacts with bait 17; HMMR interacts with baits 9 and 17; and TRIO interacts with bait 10. doi:10.1371/journal.pone.0038130.g001 suppressor 2 (Gps2), delta interacting protein A (DIPA; also called coiled-coil domain-containing protein 85B, Ccdc85b), hyaluronan-mediated motility receptor (HMMR), and Ras association (RalGDS/AF-6) domain family (N-terminal) member 7 (Rassf7; also referred to as LOC66985). Because all five partners interact with the C-terminus region of ANKRD26, we generated an expression plasmid containing the last 498 amino acids of human ANKRD26 (12121710 fragment) fused to a FLAG epitope tag located at the N-terminus of the protein (Flag-ANKRD26-C). Then a series of IP assays were performed in 293/T cells transiently co-transfected with Flag-ANKRD26-C and with different vectors, each expressing one of the selected proteins. IP analysis confirmed positive interactions for TRIO, GPS2, DIPA, ANKRD26/TRIO interaction by IP Among the 13 interacting candidates, TRIO was pulled out once during the two-hybrid screening from the mouse embryo cDNA library (Table 1). TRIO is a triple functional domain protein that promotes Ras homolog gene family (Rho) and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation through the exchange of GDP by GTP [9]. Also with the protein tyrosine phosphatase (LAR) it plays a role in coordinating cell-matrix and cytosk (...truncated)