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Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway.

Ahn CS, Ahn HK, Pai HS - J. Exp. Bot. (2014)

Bottom Line: DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation.Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels.These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Yonsei University, Seoul 120-749, Korea.

No MeSH data available.


Related in: MedlinePlus

Determination of the relationship between Tap46 and the TOR signalling pathway. (A) Phosphorylation of S6K1 and its mutant in DOE-11 seedlings upon DEX treatment. Flag-tagged WT S6K1 or mutant S6K1 [S6K1(m); T449A, non-phosphorylated form] were transiently expressed in protoplasts. Western blot analyses were performed with anti-Flag antibodies to detect total S6K1 protein and with anti-S6K1-P(T449) antibodies to detect S6K1 phosphorylated at T449. (B) Phosphorylation of S6K1 and its mutant in DEX-inducible Tap46 RNAi-16 seedlings upon DEX treatment. (C) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves that expressed HA:Tap46 with S6K1:Myc or S6K2:Myc fusion proteins. After immunoprecipitation (IP) with anti-Myc antibodies, co-immunoprecipitated HA:Tap46 was detected by immunoblotting with anti-HA antibodies. To check IP efficiency, the precipitated fractions were also immunoblotted with anti-Myc antibodies (input). (D) Co-immunoprecipitation of HA:Tap46 with S6K1:Myc, S6K1(A):Myc, or S6K1(D):Myc fusion proteins. S6K1(T449A) is a non-phosphorylated form and S6K1(T449D) is a phospho-mimetic form. (E) Visualization of interactions of Tap46 with S6K1 and S6K2 using bimolecular fluorescence complementation (BiFC). YFPN:Tap46 was expressed together with YFPC:S6K1 or YFPC:S6K2 fusion proteins in N. benthamiana leaves by agroinfiltration for confocal laser scanning microscopy. (F) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in TOR RNAi lines. Thirty micrograms of total protein isolated from Arabidopsis estradiol-inducible TOR RNAi seedlings after 3 and 7 days of ethanol (–EST) or 10 µM estradiol (+EST) treatment were subjected to immunoblotting with anti-Tap46 and anti-PP2Ac antibodies. Coomassie-stained Rubisco large subunit (rbcL) is shown as a loading control. (G) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in WT and Arabidopsis FKP12 overexpression lines upon rapamycin treatment. Seedlings were treated with 1 or 10 µM rapamycin for 24 hours prior to immunoblotting. This figure is available in colour at JXB online.
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Figure 6: Determination of the relationship between Tap46 and the TOR signalling pathway. (A) Phosphorylation of S6K1 and its mutant in DOE-11 seedlings upon DEX treatment. Flag-tagged WT S6K1 or mutant S6K1 [S6K1(m); T449A, non-phosphorylated form] were transiently expressed in protoplasts. Western blot analyses were performed with anti-Flag antibodies to detect total S6K1 protein and with anti-S6K1-P(T449) antibodies to detect S6K1 phosphorylated at T449. (B) Phosphorylation of S6K1 and its mutant in DEX-inducible Tap46 RNAi-16 seedlings upon DEX treatment. (C) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves that expressed HA:Tap46 with S6K1:Myc or S6K2:Myc fusion proteins. After immunoprecipitation (IP) with anti-Myc antibodies, co-immunoprecipitated HA:Tap46 was detected by immunoblotting with anti-HA antibodies. To check IP efficiency, the precipitated fractions were also immunoblotted with anti-Myc antibodies (input). (D) Co-immunoprecipitation of HA:Tap46 with S6K1:Myc, S6K1(A):Myc, or S6K1(D):Myc fusion proteins. S6K1(T449A) is a non-phosphorylated form and S6K1(T449D) is a phospho-mimetic form. (E) Visualization of interactions of Tap46 with S6K1 and S6K2 using bimolecular fluorescence complementation (BiFC). YFPN:Tap46 was expressed together with YFPC:S6K1 or YFPC:S6K2 fusion proteins in N. benthamiana leaves by agroinfiltration for confocal laser scanning microscopy. (F) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in TOR RNAi lines. Thirty micrograms of total protein isolated from Arabidopsis estradiol-inducible TOR RNAi seedlings after 3 and 7 days of ethanol (–EST) or 10 µM estradiol (+EST) treatment were subjected to immunoblotting with anti-Tap46 and anti-PP2Ac antibodies. Coomassie-stained Rubisco large subunit (rbcL) is shown as a loading control. (G) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in WT and Arabidopsis FKP12 overexpression lines upon rapamycin treatment. Seedlings were treated with 1 or 10 µM rapamycin for 24 hours prior to immunoblotting. This figure is available in colour at JXB online.

Mentions: Mammalian TOR (mTOR) regulates cap-dependent translation machinery through phosphorylation of its downstream substrate S6K and 4E-BP in response to nutrients, energy, and environmental stresses (Ma and Blenis, 2009). Inactivation of mTOR kinase by rapamycin leads to dephosphorylation of S6K, and PP2A was reported to interact with and dephosphorylate S6K in mammalian cells (Peterson et al., 1999; Westphal et al., 1999). In Arabidopsis, TOR activation triggers phosphorylation of S6K at specific sites (S6K1 at T449 and S6K2 at T455), suggesting that S6K is a true substrate of TOR kinase in plants (Schepetilnikov et al., 2011; Xiong and Sheen, 2012). Since Tap46, a regulatory subunit of PP2A, functions as a downstream effector of plant TOR (Ahn et al., 2011), we investigated whether Tap46 overexpression or Tap46 RNAi affects S6K phosphorylation (Fig. 6A, B). DEX-inducible Tap46 overexpression (DOE-11) and Tap46 RNAi (RNAi-16) lines were grown for 2 weeks in MS medium and transferred to new MS medium containing ethanol [(–)DEX] or 10 µM DEX [(+)DEX] for 3 days. Flag-tagged WT S6K1 or mutant S6K1 [S6K1(m); T449A] were transiently expressed in protoplasts isolated from DOE-11 and RNAi-16 seedlings as previously described (Xiong and Sheen, 2012). Immunoblotting of the protein extracts with anti-Flag and anti-phospho-S6K antibodies, followed by quantification of the immunoblot band intensities using ImageJ (http://imagej.nih.gov/ij/), demonstrated that the levels of phosphorylated S6K1 at T449 increased and decreased in the DOE-11 and RNAi-16 plants, respectively, as compared with total S6K1 protein levels (Fig. 6A, B; Supplementary Figure S3A). The T449A mutation in S6K1 completely abrogated the phosphorylation (Figure 6A, B). Therefore, Tap46 expression positively correlated with S6K phosphorylation, supporting its function as a positive effector of the TOR signalling pathway.


Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway.

Ahn CS, Ahn HK, Pai HS - J. Exp. Bot. (2014)

Determination of the relationship between Tap46 and the TOR signalling pathway. (A) Phosphorylation of S6K1 and its mutant in DOE-11 seedlings upon DEX treatment. Flag-tagged WT S6K1 or mutant S6K1 [S6K1(m); T449A, non-phosphorylated form] were transiently expressed in protoplasts. Western blot analyses were performed with anti-Flag antibodies to detect total S6K1 protein and with anti-S6K1-P(T449) antibodies to detect S6K1 phosphorylated at T449. (B) Phosphorylation of S6K1 and its mutant in DEX-inducible Tap46 RNAi-16 seedlings upon DEX treatment. (C) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves that expressed HA:Tap46 with S6K1:Myc or S6K2:Myc fusion proteins. After immunoprecipitation (IP) with anti-Myc antibodies, co-immunoprecipitated HA:Tap46 was detected by immunoblotting with anti-HA antibodies. To check IP efficiency, the precipitated fractions were also immunoblotted with anti-Myc antibodies (input). (D) Co-immunoprecipitation of HA:Tap46 with S6K1:Myc, S6K1(A):Myc, or S6K1(D):Myc fusion proteins. S6K1(T449A) is a non-phosphorylated form and S6K1(T449D) is a phospho-mimetic form. (E) Visualization of interactions of Tap46 with S6K1 and S6K2 using bimolecular fluorescence complementation (BiFC). YFPN:Tap46 was expressed together with YFPC:S6K1 or YFPC:S6K2 fusion proteins in N. benthamiana leaves by agroinfiltration for confocal laser scanning microscopy. (F) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in TOR RNAi lines. Thirty micrograms of total protein isolated from Arabidopsis estradiol-inducible TOR RNAi seedlings after 3 and 7 days of ethanol (–EST) or 10 µM estradiol (+EST) treatment were subjected to immunoblotting with anti-Tap46 and anti-PP2Ac antibodies. Coomassie-stained Rubisco large subunit (rbcL) is shown as a loading control. (G) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in WT and Arabidopsis FKP12 overexpression lines upon rapamycin treatment. Seedlings were treated with 1 or 10 µM rapamycin for 24 hours prior to immunoblotting. This figure is available in colour at JXB online.
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Figure 6: Determination of the relationship between Tap46 and the TOR signalling pathway. (A) Phosphorylation of S6K1 and its mutant in DOE-11 seedlings upon DEX treatment. Flag-tagged WT S6K1 or mutant S6K1 [S6K1(m); T449A, non-phosphorylated form] were transiently expressed in protoplasts. Western blot analyses were performed with anti-Flag antibodies to detect total S6K1 protein and with anti-S6K1-P(T449) antibodies to detect S6K1 phosphorylated at T449. (B) Phosphorylation of S6K1 and its mutant in DEX-inducible Tap46 RNAi-16 seedlings upon DEX treatment. (C) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves that expressed HA:Tap46 with S6K1:Myc or S6K2:Myc fusion proteins. After immunoprecipitation (IP) with anti-Myc antibodies, co-immunoprecipitated HA:Tap46 was detected by immunoblotting with anti-HA antibodies. To check IP efficiency, the precipitated fractions were also immunoblotted with anti-Myc antibodies (input). (D) Co-immunoprecipitation of HA:Tap46 with S6K1:Myc, S6K1(A):Myc, or S6K1(D):Myc fusion proteins. S6K1(T449A) is a non-phosphorylated form and S6K1(T449D) is a phospho-mimetic form. (E) Visualization of interactions of Tap46 with S6K1 and S6K2 using bimolecular fluorescence complementation (BiFC). YFPN:Tap46 was expressed together with YFPC:S6K1 or YFPC:S6K2 fusion proteins in N. benthamiana leaves by agroinfiltration for confocal laser scanning microscopy. (F) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in TOR RNAi lines. Thirty micrograms of total protein isolated from Arabidopsis estradiol-inducible TOR RNAi seedlings after 3 and 7 days of ethanol (–EST) or 10 µM estradiol (+EST) treatment were subjected to immunoblotting with anti-Tap46 and anti-PP2Ac antibodies. Coomassie-stained Rubisco large subunit (rbcL) is shown as a loading control. (G) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in WT and Arabidopsis FKP12 overexpression lines upon rapamycin treatment. Seedlings were treated with 1 or 10 µM rapamycin for 24 hours prior to immunoblotting. This figure is available in colour at JXB online.
Mentions: Mammalian TOR (mTOR) regulates cap-dependent translation machinery through phosphorylation of its downstream substrate S6K and 4E-BP in response to nutrients, energy, and environmental stresses (Ma and Blenis, 2009). Inactivation of mTOR kinase by rapamycin leads to dephosphorylation of S6K, and PP2A was reported to interact with and dephosphorylate S6K in mammalian cells (Peterson et al., 1999; Westphal et al., 1999). In Arabidopsis, TOR activation triggers phosphorylation of S6K at specific sites (S6K1 at T449 and S6K2 at T455), suggesting that S6K is a true substrate of TOR kinase in plants (Schepetilnikov et al., 2011; Xiong and Sheen, 2012). Since Tap46, a regulatory subunit of PP2A, functions as a downstream effector of plant TOR (Ahn et al., 2011), we investigated whether Tap46 overexpression or Tap46 RNAi affects S6K phosphorylation (Fig. 6A, B). DEX-inducible Tap46 overexpression (DOE-11) and Tap46 RNAi (RNAi-16) lines were grown for 2 weeks in MS medium and transferred to new MS medium containing ethanol [(–)DEX] or 10 µM DEX [(+)DEX] for 3 days. Flag-tagged WT S6K1 or mutant S6K1 [S6K1(m); T449A] were transiently expressed in protoplasts isolated from DOE-11 and RNAi-16 seedlings as previously described (Xiong and Sheen, 2012). Immunoblotting of the protein extracts with anti-Flag and anti-phospho-S6K antibodies, followed by quantification of the immunoblot band intensities using ImageJ (http://imagej.nih.gov/ij/), demonstrated that the levels of phosphorylated S6K1 at T449 increased and decreased in the DOE-11 and RNAi-16 plants, respectively, as compared with total S6K1 protein levels (Fig. 6A, B; Supplementary Figure S3A). The T449A mutation in S6K1 completely abrogated the phosphorylation (Figure 6A, B). Therefore, Tap46 expression positively correlated with S6K phosphorylation, supporting its function as a positive effector of the TOR signalling pathway.

Bottom Line: DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation.Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels.These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Yonsei University, Seoul 120-749, Korea.

No MeSH data available.


Related in: MedlinePlus