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Y. Zhang, R.L. Converse, C.–Y. Liu, L.–K. Yeh, H. Liu, W.W. Y. Kao; Identification of Talin and 14–3–3 as SARG Interacting Proteins in Corneal Epithelium by Two–Hybrid System . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1599.
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© ARVO (1962-2015); The Authors (2016-present)
SARG (specifically androgen regulated gene) exhibits expression in the basal cells of the stratified corneal epithelium. Little is known about the role of SARG in vivo. Based on our immunohistochemical results we propose that SARG likely functions through specific interactions with other protein binding partners. This study is designed to identify these candidates and provide insight toward understanding SARG function.
Two cDNA (1 kb) fragments encoding mouse N–terminal SARG (aa 1∼312) and C–terminal SARG (aa 301∼606) were used independently as bait to screen a mouse lung cDNA library using a bacterial two–hybrid system (Stratagene). Putative clones grown on selective media (3–AT) were patched and further selected on dual selective media (3–AT/strep). Candidate clones were characterized by PCR, sequencing, and sequence homology comparisons via BLAST. These interactions of SARG with candidate clones were further characterized in vivo and in vitro.
An initial screening of our mouse lung cDNA library with an N–terminal SARG bait plasmid yielded 278 clones on 3–AT selective media, with a 42% reduction on 3–AT strep media. Likewise, an initial screening of this library with a C–terminal SARG bait plasmid yielded 182 clones on 3–AT selective media, with a 36% reduction on 3–AT strep media. From this population, clones were grouped according to sequence homology via BLAST. A group of 13 unique positive clones encoding known proteins or EST’s were chosen due to their potential role in cell polarity and cell signalling in the corneal epithelium. Among these, 14–3–3 and talin were isolated and verified by co–transformation with the original bait and the full–length SARG cDNA. We found that talin interacts with N–terminal SARG but not C–terminal SARG, in contrast, 14–3–3 interacts with C–terminal SARG but not N–terminal SARG. Both of these clones were also shown to interact with a full–length SARG bait plasmid. These interactions were further confirmed by GST pull–down experiments and IP–western blotting analysis. Moreover, immunocytochemistry showed that some but not all SARG did co–localize with 14–3–3 and talin in corneal epithelial cells.
These data indicate that SARG may have a role in intracellular scaffolding–associated epithelial cell polarity, migration, and signal transduction.
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