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. 2014 Dec;27(12):1671-7.
doi: 10.5713/ajas.2014.14145.

Direct Interaction between Ras Homolog Enriched in Brain and FK506 Binding Protein 38 in Cashmere Goat Fetal Fibroblast Cells

Xiaojing Wang  1 , Yanfeng Wang  1 , Xu Zheng  1 , Xiyan Hao  1 , Yan Liang  1 , Manlin Wu  1 , Xiao Wang  1 , Zhigang Wang  1
Affiliations

Direct Interaction between Ras Homolog Enriched in Brain and FK506 Binding Protein 38 in Cashmere Goat Fetal Fibroblast Cells

Xiaojing Wang et al. Asian-Australas J Anim Sci. 2014 Dec.

Abstract

Ras homolog enriched in brain (Rheb) and FK506 binding protein 38 (FKBP38) are two important regulatory proteins in the mammalian target of rapamycin (mTOR) pathway. There are contradictory data on the interaction between Rheb and FKBP38 in human cells, but this association has not been examined in cashmere goat cells. To investigate the interaction between Rheb and FKBP38, we overexpressed goat Rheb and FKBP38 in goat fetal fibroblasts, extracted whole proteins, and performed coimmunoprecipitation to detect them by western blot. We found Rheb binds directly to FKBP38. Then, we constructed bait vectors (pGBKT7-Rheb/FKBP38) and prey vectors (pGADT7-Rheb/FKBP38), and examined their interaction by yeast two-hybrid assay. Their direct interaction was observed, regardless of which plasmid served as the prey or bait vector. These results indicate that the 2 proteins interact directly in vivo. Novel evidence is presented on the mTOR signal pathway in Cashmere goat cells.

Keywords: FK506 Binding Protein 38 [FKBP38]; Interaction; Ras homolog enriched in brain [Rheb]; mammalian Target of Rapamycin [mTOR].

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Figures

Figure 1
Figure 1
Western blot of total protein and co-IP with anti-Rheb. (A) Western blot analysis of total cell protein. Overexpressed cells had an enhanced expression of Rheb and FKBP38 versus normal control GFb cells; (B) co-IP analysis of eluates. Rheb and FKBP38 could be detected in anti-Rheb immunoprecipitates; β-actin is the internal control. Rheb, Ras homolog enriched in brain; co-IP, coimmunoprecipitation.
Figure 2
Figure 2
Toxicity and self-activation of the bait proteins analyzed in Yeast cells (4×). (A) Phenotype for yeast transformation, 1, Y2HpGBKT7; 2, Y2HpGBKT7-Rheb; 3, Y2HpGBKT7-FKBP38; 4, Y187pGADT7; 5, Y187pGADT7-Rheb; 6, Y187 pGADT7-FKBP38. (B) Self-activation assay of Rheb and FKBP38. 1′ is Y2HGold with pGBKT7-Rheb and 2′ is Y2HGold with pGBKT7-FKBP38. Rheb, Ras homolog enriched in brain.
Figure 3
Figure 3
PCR amplification of Rheb and FKBP38 using yeast colonies as templates; electrophoresis of 3 PCR products of (a) Rheb in yeast Y2H; (b) FKBP38 in yeast Y2H; (c) Rheb in yeast Y187; and (d) FKBP38 in yeast Y187; lane M: DL2000 marker. PCR, polymerase chain reaction; Rheb, Ras homolog enriched in brain.
Figure 4
Figure 4
Growth of yeast in two-hybrid screen on SD/-Leu-Trp medium (4×). α1, pGBKT7-Rheb+pGADT7-FKBP38; β1, pGBKT7-FKBP38+pGADT7-Rheb; γ1, pGBKT7+pGADT7-FKBP38; δ1, pGBKT7+pGADT7-Rheb. SD, standard dropout; Rheb, Ras homolog enriched in brain.
Figure 5
Figure 5
Colony screening using yeast two-hybrid system (4×). 1, α1 mating yielded blue colonies were selected on SD medium (SD/-Leu/-Trp/Aba/X-α-Gal [DAX]). 2, β1 mating yielded blue colonies on SD medium (SD/-Leu/-Trp/Aba/X-α-Gal [DAX]). 3, Blue yeast colonies of α1 mating yeast strain were selected on SD medium (SD/-Leu/-Trp/-His/X-α-Gal/Aba [HAX]). 4, Blue yeast colonies of β1 mating yeast strain were selected on SD medium (SD/-Leu/-Trp/-His/X-α-Gal/Aba [HAX]). 5, α1 grew white colonies on SD medium (SD/-Leu/-Trp/-His/-Ade/Aba/X-α-Gal [QAX]). 6, Blue yeast colonies of β1 mating yeast strain were selected in the fourth round on SD medium (SD/-Leu/-Trp/-His/-Ade/Aba/X-α-Gal [QAX]). SD, standard dropout.
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