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

  • Wang, Xiaojing (College of Life Sciences, Inner Mongolia University) ;
  • Wang, Yanfeng (College of Life Sciences, Inner Mongolia University) ;
  • Zheng, Xu (College of Life Sciences, Inner Mongolia University) ;
  • Hao, Xiyan (College of Life Sciences, Inner Mongolia University) ;
  • Liang, Yan (College of Life Sciences, Inner Mongolia University) ;
  • Wu, Manlin (College of Life Sciences, Inner Mongolia University) ;
  • Wang, Xiao (College of Life Sciences, Inner Mongolia University) ;
  • Wang, Zhigang (College of Life Sciences, Inner Mongolia University)
  • Received : 2014.02.26
  • Accepted : 2014.07.14
  • Published : 2014.12.01


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.


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


  1. Dunlop, E. A., K. M. Dodd, L. A. Seymour, and A. R. Tee. 2009. Mammalian target of rapamycin complex 1-mediated phosphorylation of eukaryotic initiation factor 4E-binding protein 1 requires multiple protein-protein interactions for substrate recognition. Cell. Signal. 21:1073-1084.
  2. Bai, X., D. Ma, A. Liu, X. Shen, Q. J. Wang, Y. Liu, and Y. Jiang. 2007. Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science 318:977-980.
  3. Banasavadi-Siddegowda, Y. K., J. Mai, Y. Fan, S. Bhattacharya, D. R. Giovannucci, E. R. Sanchez, G. Fischer, and X. Wang. 2011. FKBP38 peptidylprolyl isomerase promotes the folding of cystic fibrosis transmembrane conductance regulator in the endoplasmic reticulum. J. Biol. Chem. 286:43071-43080.
  4. Basso, A. D., A. Mirza, G. Liu, B. J. Long, W. R. Bishop, and P. Kirschmeier. 2005. The farnesyl transferase inhibitor (FTI) SCH66336 (lonafarnib) inhibits Rheb farnesylation and mTOR signaling. Role in FTI enhancement of taxane and tamoxifen anti-tumor activity. J. Biol. Chem. 280:31101-31108.
  5. Bonneau, A. and N. Parmar. 2012. Effects of RhebL1 silencing on the mTOR pathway. Mol. Biol. Rep. 39:2129-2137
  6. Brown, H. L. D., K. R. Kaun, and B. A. Edgar. 2012. The small GTPase Rheb affects central brain neuronal morphology and memory formation in Drosophila. PLoS One 7(9):e44888 DOI: 10.1371/journal.pone.0044888
  7. Duran, R. V. and M. N. Hall. 2012. Regulation of TOR by small GTPases. EMBO Rep. 13:121-128
  8. Goorden, S. M. I., M. Hoogeveen-Westerveld, C. Cheng, G. M. van Woerden, M. Mozaffari, L. Post, H. J. Duckers, M. Nellist, and Y. Elgersma. 2011. Rheb is essential for murine development. Mol. Cell. Biol. 31:1672-1678.
  9. Hanker, A. B., N. Mitin, R. S. Wilder, E. P. Henske, F. Tamanoi, A. D. Cox, and C. J. Der. 2010. Differential requirement of CAAX-mediated posttranslational processing for Rheb localization and signaling. Oncogene 29:380-391
  10. Lam, E., M. Martin, and G. Wiederrecht. 1995. Isolation of a cDNA encoding a novel human FK506-binding protein homolog containing leucine zipper and tetratricopeptide repeat motifs. Gene 160:297-302.
  11. Haupt, K., G. Jahreis, M. Linnert, M. Maestre-Martinez, M. Malesevic, A. Pechstein, F. Edlich, and C. Lucke. 2012. The FKBP38 catalytic domain binds to Bcl-2 via a charge-sensitive loop. J. Biol. Chem. 287:19665-19673.
  12. Karassek, S., C. Berghaus, M. Schwarten, C. G. Goemans, N. Ohse, G. Kock, K. Jockers, S. Neumann, S. Gottfried, C. Herrmann, R. Heumann, and R. Stoll. 2010. Ras homolog enriched in brain (Rheb) enhances apoptotic signaling. J. Biol. Chem. 285:33979-33991
  13. Kim, H. W., S. H. Ha, M. N. Lee, E. Huston, D. H. Kim, S. K. Jang, P. G. Suh, M. D. Houslay, and S. H. Ryu. 2010. Cyclic AMP controls mTOR through regulation of the dynamic interaction between Rheb and phosphodiesterase 4D. Mol. Cell. Biol. 30:5406-5420.
  14. Lee, M. N., A. Koh, D. Park, J. H. Jang, D. Kwak, H. Jeon, J. Kim, E. J. Choi, H. Jeong, P. G. Suh, and S. H. Ryu. 2013. Deacetylated alphabeta-tubulin acts as a positive regulator of Rheb GTPase through increasing its GTP-loading. Cell Signal 25:539-551.
  15. Liu, J. O. 2003. Endogenous protein inhibitors of calcineurin. Biochem. Biophys. Res. Commun. 311:1103-1109.
  16. Ma, D., X. Bai, H. Zou, Y. Lai, and Y. Jiang. 2010. Rheb GTPase controls apoptosis by regulating interaction of FKBP38 with Bcl-2 and Bcl-XL. J. Biol. Chem. 285:8621-8627.
  17. Ma, D., X. Bai, S. Guo, and Y. Jiang. 2008. The switch I region of Rheb is critical for its interaction with FKBP38. J. Biol. Chem. 283:25963-25970.
  18. Maehama, T., M. Tanaka, H. Nishina, M. Murakami, Y. Kanaho, and K. Hanada. 2008. RalA functions as an indispensable signal mediator for the nutrient-sensing system. J. Biol. Chem. 283:35053-35059.
  19. Sato, T., A. Nakashima, L. Guo, and F. Tamanoi. 2009. Specific activation of mTORC1 by Rheb G-protein in vitro involves enhanced recruitment of its substrate protein. J. Biol. Chem. 284:12783-12791.
  20. Maestre-Martinez, M., K. Haupt, F. Edlich, P. Neumann, C. Parthier, M. T. Stubbs, G. Fischer, and C. Lucke. 2011. A charge-sensitive loop in the FKBP38 catalytic domain modulates Bcl-2 binding. J. Mol. Recognit. 24:23-34.
  21. Parkhitko, C. A., C. O. Favorova, and E. P. Henske. 2011. Rabin8 protein interacts with GTPase Rheb and inhibits phosphorylation of Ser235/Ser236 in small ribosomal subunit protein S6. Acta Naturae 3:71-76.
  22. Patel, P. H., N. Thapar, L. Guo, M. Martinez, J. Maris, C. L. Gau, J. A. Lengyel, and F. Tamanoi. 2003. Drosophila Rheb GTPase is required for cell cycle progression and cell growth. J. Cell Sci. 116:3601-3610.
  23. Sciarretta, S., P. Zhai, D. Shao, Y. Maejima, J. Robbins, M. Volpe, G. Condorelli, and J. Sadoshima. 2012. Rheb is a critical regulator of autophagy during myocardial ischemia: pathophysiological implications in obesity and metabolic syndrome. Circulation 125:1134-1146.
  24. Shirane, M., M. Ogawa, J. Motoyama, and K. I. Nakayama. 2008. Regulation of apoptosis and neurite extension by FKBP38 is required for neural tube formation in the mouse. Genes Cells 13:635-651.
  25. Shirane, M. and K. I. Nakayama. 2003. Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis. Nat. Cell Biol. 5:28-37.
  26. Sucher, N. J., E. Yu, S. F. Chan, M. Miri, B. J. Lee, B. Xiao, P. F. Worley, and F. E. Jensen. 2010. Association of the small GTPase Rheb with the NMDA receptor subunit NR3A. Neurosignals 18:203-209.
  27. Uhlenbrock, K., M. Weiwad, R. Wetzker, G. Fischer, A. Wittinghofer, and I. Rubio. 2009. Reassessment of the role of FKBP38 in the Rheb/mTORC1 pathway. FEBS Lett. 583:965-970.
  28. Wang, X., B. D. Fonseca, H. Tang, R. Liu, A. Elia, M. J. Clemens, U. A. Bommer, and C. G. Proud. 2008. Re-evaluating the roles of proposed modulators of mammalian target of rapamycin complex 1 (mTORC1) signaling. J. Biol. Chem. 283:30482-30492.
  29. Weiwad, M., F. Edlich, F. Erdmann, F. Jarczowski, S. Kilka, M. Dorn, A. Pechstein, and G. Fischer. 2005. A reassessment of the inhibitory capacity of human FKBP38 on calcineurin. FEBS Lett. 579:1591-1596.
  30. Zheng, X., J. F. Yang, X. J. Wang, Y. Liang, M. L. Wu, J. J. Shi, T. Zhang, Y. Qin, S. Y. Li, X. Y. Hao, Z. G. Wang, and D. J. Liu. 2011. Molecular characterization and expresiion pattern of Rheb gene in Inner Mogolia cashmere goat (Capra hircus). Agric. Sci. China 10:1452-1458.
  31. Zheng, M., Y. H. Wang, X. N. Wu, S. Q. Wu, B. J. Lu, M. Q. Dong, H. Zhang, P. Sun, S. C. Lin, K. L. Guan, and J. Han. 2011. Inactivation of Rheb by PRAK-mediated phosphorylation is essential for energy-depletion-induced suppression of mTORC1. Nat. Cell Biol. 13:263-272.
  32. Zheng, X., X. Y. Hao, Y. H. Chen, X. Zhang, J. F. Yang, Z. G. Wang, and D. J. Liu. 2012. Molecular characterization and tissue-specific expression of a novel FKBP38 gene in the cashmere goat (Capra hircus). Asian Australas. J. Anim. Sci. 25:758-763.
  33. Yadav, R. B., P. Burgos, A. W. Parker, V. Iadevaia, C. G. Proud, R. A. Allen, J. P. O'Connell, A. Jeshtadi, C. D. Stubbs, and S. W. Botchway. 2013. mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging. BMC Cell Biol. 14:3. doi: 10.1186/1471-2121-14-3.

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