ScFKBP12 bridges rapamycin and AtTOR in Arabidopsis

Abstract

FKBP12 encodes a prolyl isomerase and highly conserved in eukaryotic species. In yeasts and animals, FKBP12 can interact with rapamycin and FK506 to form rapamycin-FKBP12 and FK506-FKBP12 complex, respectively. In higher plants, FKBP12 protein lost its function to bind rapamycin and FK506. Early studies showed that yeast and human FKBP12 protein can restore the rapamycin sensitivity in Arabidopsis, but the used concentration is 100–1000 folds higher than that in yeast and animals. High concentration of drugs would increase the cost and cause the potential secondary effects on plant growth and development. Here we further discovered that BP12 plants generated in our previous study are hypersensitive to rapamycin at the concentration as low as that is effective in yeast and animals. It is surprising to observe that WT and BP12 plants are not sensitive to FK506 in normal growth condition. These findings advance the current understanding of rapamycin-TOR signaling in plants.

Keywords: :

• yeast FKBP12
• rapamycin
• FK506
• Arabidopsis growth

From the earliest times, people have been looking for the medicine to achieve immortality. Both eastern and western alchemists were obsessed with creating an elixir of life. Unfortunately almost all attempts have been miserable failures. 2009, rapamycin as the first drug to significantly extend lifespan in mammals is scientifically proven,¹ which is selected by Science as one of the Top 10 Scientific Achievements. This is of vital importance for the well-being of human, especially for the Gereatrics and Health Care because no drug agents is able to extend lifespan untill rapamycin was found so far.² Rapamycin and its targets, therefore, have been attracted more and more extensive attentions and becoming the focus of study in current Gereatrics and Preventive Medicine.²

Rapamycin is originally found for a macrolide antifungal drug in the soil Streptomyces hygroscopicus from Easter Island in 1970s.³ Shortly after this discovery, its potent immunosuppressive and antiproliferative properties were detected in 1970s.^4,5 Thereafter it has been widely used as an immunosuppressant drug to prevent rejection in organ transplantation, especially in kidney transplants.^6–8 After the 1980s, many studies showed that rapamycin has the anti-cancer activities.^9–11 Its deritivatives, temsirolimus (CCI-779), everolimus (RAD001), and ridaforolimus (AP-23573) have been approved in clinical trails for several cancers by FDA (Food and Drug Adiministration).¹² Additionally, rapamycin is a very useful agent for studying signal transduction in eukaryotic systems and able to block many signal transduction pathways from yeast to mammalians.¹³ It was found that rapamycin is able to inhibit cellular proliferation and cell cycle progression mimicking the nutrient or energy starvation from yeast to mammals.¹⁴

The evolutionary distance between yeast and mammalian is more than 1 billion years, but their lifespan can be effectively extended by feeding rapamycin.¹⁵ This suggests that there are evolutionarily conserved proteins targeted by rapamycin, existing in all eukaryotic species from yeast to mammalian. The targets of rapamycin didn’t change even going through 1 billion years evolution and were identified as TOR1 (Target Of Rapamycin1) and TOR2 (Target Of Rapamycin1) in yeast in 1990s.¹⁶ The TOR proteins are the phosphatidylinositol protein kinase and are highly conserved from yeast to mammalian.¹⁵ Most detected eukaryotic species contain a single TOR while yeast has TOR1 and TOR2 and three TOR genes were recently found in Leishmania major.^16–18 TOR is critical for organisms growth and development. Loss function of TOR leads to lethal in all examined organisms from yeast to mammals.¹⁴ The protein kinase activity of TOR displayed by TORC1 (TOR complex1) and TORC2 (TOR complex2). TORC1 and TORC2 are distinguished by RAPTOR and RICTOR, respectively, but LST8 is the constitutive member in both complexes.^14–16 TOR complexes are the hub of various signaling cascades through mediating upsteam signals including nutrition, energy, growth factor and stress signals.^14,15 TOR phosphorylate a series of downstream substrate proteins including S6K, 4E-BP, AKT to spatiotemporally regulate cell growth and division.^14,15

FKBP12 (FK506 binding protein 12) bridged the interaction between rapamycin and TOR complexes.¹⁹ FKBP12 encodes a peptidyl-prolyl cis-trans isomerase and highly conserved from yeast to human. In the presence of rapamycin, FKBP12 is able to bind FRB domain of TOR protein to form ternary complex.^19,20 Rapamycin-FKBP12 disrupts the association between RAPTOR and TOR and therefore inhibited TOR protein kinase activities and block TOR signaling cascades, leading to G1 cell cycle arrest.²¹ Human FKBP12 enables to complement yeast fkbp12 mutant strain, suggesting that FKBP12 is a highly conserved protein in eukaryotes.²⁰

In contrast to yeasts and mammals, studies with land plants consistently showed that plants are insensitive to rapamycin since plants FKBP12 proteins loss the function to bind rapamycin and failed to inhibit land plants growth.^22,23 Several independent studies showed that plant FKBP12 cann’t interact with rapamycin and TOR in vivo or in vitro by using either the yeast-two-hybrid method or direct binding assay.^22–26 The protein structure analysis showed that only plant FKBP12s has the unique structural features with a disulfide bond.²² This unique structural feature disrupted FKBP12 protein to bind rapamycin in higher plants. In the presence of yeast or human FKBP12, rapamycin is able to block TOR signaling in plant cells, but the used concentration is 100–1000 folds (10μM) higher than that in yeast and animals.^22–27 High concentration of drugs would increase the cost and cause the potential secondary effects on plant growth and development. It should be important that plant TOR kinase activity can be effectively regulated by rapamycin at a concentration as low as that in yeast and animals.

In our early study, we generated the Arabidopsis BP12 plants containing yeast FKBP12 and found that BP12 plants treated with rapamycin significantly reduces TOR kinase activity, slow down the growth rate, extend the lifespan and failed to response the nutrition and light signals through using genetics, transcriptomic, and metabolomic approaches.²⁷ We observed that the rapamycin still effectively inhibited the growth of BP12 plants at 500 nM concentration in our early study.²⁷ In this study, we further reduced the rapamycin concentration to the same dosage as that used in yeast and animal cells (10–100nM). As indicated in Figure 1, rapamycin is able to significantly inhibit the primary roots, lateral roots, and root hair growth of BP12 plants at 10–100nM concentration in contrast to WT (Fig. 1A, C–F), whereas the inhibition is undetectable when the concentration is reduced to 1nM. The effects we saw were specific to TOR inhibition since WT plants with or without rapamycin treatment and BP12 plants without rapamycin treatment were essentially indistinguishable from each other for their growth, development and differentiation. This rapamycin inducible TOR suppression BP12 plants, especially the BP12–2 containing a single copy of ScFKBP12, will contribute to the further dissection of TOR signaling network in plants.

Figure 1. Characterization of rapamycin and FK506 sensitivity in WT, BP12–2, and BP12–5 plants. (A) Growth performance of WT, BP12–2, and BP12–5 plants grown on 0.5MS+DMSO, 100nM, 10nM, and 1nM rapamycin media for 25 d. WT plants showed insensitivity to rapamycin on any concentration of rapamycin media whereas BP12–2 and BP12–5 plants were not obviously inhibited by rapamycin until the concentration was reduced to 1nM as indicated in Figure A4. (B) FK506 does not inhibit the growth of WT and BP12 plants containing the exogenous yeast FKBP12. (C–F) The primary root length (C), lateral root number (D), lateral root length, (E) and root hair length (F) of 25 DAG WT and BP12 plants grown on rapamycin (10nM) or FK506 (20μM) medium were measured, respectively, indicating that rapamycin can specifically inhibit BP12 plants, but FK506 is unable to suppress plant growth in the absence or presence of exogenous yeast FKBP12. Error bars indicate SD for triplicates (n = 20).

FKBP12 acts as a cellular target not only for rapamycin but also for FK506, a macrolide sharing structure similar to rapamycin.^10,20 FKBP12-FK506 complex further binds to calcineurin in vivo and inhibits its phosphatase activities.²⁰ FK506 therefore acts to block CaN-dependent signaling and inhibit the cell growth at 10nM concentration in yeast and animals.²⁰ It should be very interesting to know whether WT and BP12 Arabidopsis plants can also mediate the action of FK506 by AtFKBP12 or ScFKBP12 protein. We therefore examined the plants with the increasing FK506 concentration: 50, 100, 500, 1,000, 10,000 and 20,000 nM, respectively. Interestingly, the root number, lateral root, root hair, leaf number and size of WT and BP12 plants didn’t exhibit obviously growth defects at 20μM FK506 concentration in contrast to 0.5xMS media (Fig. 1B–F). These observations suggest that FK506 is unable to inhibit cell growth in Arabidopsis even in the presence of yeast FKBP12. In yeast and animals, FK506 can target a family of proteins that possess the activity of peptidyl-prolyl cis-trans isomerase (PPIase). FKBP12 belongs to the FK506 Binding Protein superfamily and therefore has dual function to bind rapamycin and FK506. In yeast, the absence of FKBP12 results in rapamycin resistant phenotype and human FKBP12 is able to restore the mutant strain to gain the rapamycin sensitivity, suggesting a functional equivalence between human and yeast.²⁰ However, rapamycin-resistant yeast strain remains sensitive to FK506, revealing that the other proteins involved in the FK506 signal transduction in yeast. These results indicated that ScFKBP12 is the specific target for rapamycin, whereas FKBP12 is not essential for FK506 signal transduction. The unique feature of FK506-resistant was observed in plants revealed the different molecular mechanism for FK506 signaling transduction exists in plants.

Although a TOR ortholog was identified in Arabidopsis several years ago,^24,28 the progress in the understanding of TOR signaling in plants has been very limited so far. This is partly due to the lack of a well-established rapamycin sensitive system in Arabidopsis. In this study, we have developed rapamycin hypersensitive BP12 plants in Arabidopsis by using yeast FKBP12 gene. Rapamycin can effectively inhibit the growth and development of BP12 plants at the concentration as low as that in yeast and animals. Thus the in vitro high efficiency rapamycin sensitive system for suppression of TOR activity has been well established with BP12 plants in our study. TOR signaling rhythmically controls cell growth and development in response to growth factor such as insulin in heterotroph eukaryotic model systems.^14,15 However, no equivalents of insulin have been found in plants, but auxin, brassinosteriold, cytokinin are the major growth factor to affect growth and cell proliferation in plants. Thus, it will be important to examine the crosstalk and interplays among TOR signaling pathway, nutrition, light, and hormones signals. Further analyses of the BP12 lines may facilitates our understanding of TOR signaling network and contribute to reveal some key biological process and components of TOR signaling in plants.

Abbreviations:
ScFKBP12	=	Saccharomyces cerevisiae FK506 binding protein 12Kd
BP12	=	P35S:ScFKBP12 transgenic Arabidopsis plants
TOR	=	target of rapamycin
TORC1	=	TOR complex1
TORC2	=	TOR complex2

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by grant number 2013AA102601 (Functional genomic research and application in cotton) from National 863 Projects.

10.4161/psb.26115