Ding, Z. et al. A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proc. Natl. Acad. Sci. USA 103, 15014-15019

Department of Biochemistry & Molecular Biology, Baylor College of Medicine, Houston, Texas, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2006; 103(41):15014-9. DOI: 10.1073/pnas.0606917103
Source: PubMed


We developed a retrovirus-based protein-fragment complementation assay (RePCA) screen to identify protein-protein interactions in mammalian cells. In RePCA, bait protein is fused to one fragment of a rationally dissected fluorescent protein, such as GFP, intensely fluorescent protein, or red fluorescent protein. The second, complementary fragment of the fluorescent protein is fused to an endogenous protein by in-frame exon traps in the enhanced retroviral mutagen vector. An interaction between bait and host protein (prey) places the two parts of the fluorescent molecule in proximity, resulting in reconstitution of fluorescence. By using RePCA, we identified a series of 24 potential interaction partners or substrates of the serine/threonine protein kinase AKT1. We confirm that alpha-actinin 4 (ACTN4) interacts physically and functionally with AKT1. siRNA-mediated ACTN4 silencing down-regulates AKT phosphorylation, blocks AKT translocation to the membrane, increases p27(Kip1) levels, and inhibits cell proliferation. Thus, ACTN4 is a critical regulator of AKT1 localization and function.

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Available from: Shiaw-Yih Lin, May 03, 2015
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    • "Plant scientists have frequently taken advantage of this technique to identify novel proteins that interact with a known bait protein (Ballas and Citovsky, 1997; Bakó et al., 2003; Hwang and Gelvin, 2004; Tao et al., 2004). Although peptide complementation (two-hybrid) assays have been developed in animal systems (Fearon et al., 1992; Shioda et al., 2000; Remy and Michnick, 2004; Ding et al., 2006), screening in yeast remains the favored route for identifying proteins that interact with a given bait protein. "
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    ABSTRACT: Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein- or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ~2 × 10(5) cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.
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    • "Finally, the variety of protein interactions visualized through BiFC indicates that the methodology could be used more globally [53] for screening for novel interacting partners in a developing organism. This could readily be achieved combining BiFC with the strength of Drosophila genetics. "
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    ABSTRACT: Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.
    Full-text · Article · Jan 2011 · BMC Biology
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    • "For example Actn4, an actinin-bundling protein, has been implicated in breast cancer, and progressed stages of esophageal squamous cell carcinoma and lung cancer (Honda et al., 1998, 2005; Fu et al., 2007). Moreover, recently Actn4 was found to be one of the binding partners of Akt1, thereby having a crucial function in activation and translocation of Akt1 to the cell membrane (Ding et al., 2006). Interestingly, we find Actn4, Agrn, Dbs, FlnC, FosB, Hrs and PdgfB capable of cooperating with c-Myc alone independent from p19Arf. "
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    ABSTRACT: c-Myc drives uncontrolled cell proliferation in various human cancers. However, in mouse embryo fibroblasts (MEFs), c-Myc also induces apoptosis by activating the p19Arf tumor suppressor pathway. Tbx2, a transcriptional repressor of p19Arf, can collaborate with c-Myc by suppressing apoptosis. MEFs overexpressing c-Myc and Tbx2 are immortal but not transformed. We have performed an unbiased genetic screen, which identified 12 oncogenes that collaborate with c-Myc and Tbx2 to transform MEFs in vitro. One of them encodes the LPA2 receptor for the lipid growth factor lysophosphatidic acid (LPA). We find that LPA1 and LPA4, but not LPA3, can reproduce the transforming effect of LPA2. Using pharmacological inhibitors, we show that the in vitro cell transformation induced by LPA receptors is dependent on the Gi-linked ERK and PI3K signaling pathways. The transforming ability of LPA1, LPA2 and LPA4 was confirmed by tumor formation assays in vivo and correlated with prolonged ERK1/2 activation in response to LPA. Our results reveal a direct role for LPA receptor signaling in cell transformation and tumorigenesis in conjunction with c-Myc and reduced p19Arf expression.
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