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ABSTRACT: BACKGROUND: mTOR is a genetically conserved serine/threonine protein kinase, which controls cell growth, proliferation, and survival. A multifunctional protein CAD, catalyzing the initial three steps in de novo pyrimidine synthesis, is regulated by the phosphorylation reaction with different protein kinases, but the relationship with mTOR protein kinase has not been known. RESULTS: CAD was recovered as a binding protein with mLST8, a component of the mTOR complexes, from HEK293 cells transfected with the FLAG-mLST8 vector. Association of these two proteins was confirmed by the co-immuoprecipitaiton followed by immunoblot analysis of transfected myc-CAD and FLAG-mLST8 as well as that of the endogenous proteins in the cells. Analysis using mutant constructs suggested that CAD has more than one region for the binding with mLST8, and that mLST8 recognizes CAD and mTOR in distinct ways. The CAD enzymatic activity decreased in the cells depleted of amino acids and serum, in which the mTOR activity is suppressed. CONCLUSION: The results obtained indicate that mLST8 bridges between CAD and mTOR, and plays a role in the signaling mechanism where CAD is regulated in the mTOR pathway through the association with mLST8.
Journal of Biomedical Science 04/2013; 20(1):24. · 2.01 Impact Factor
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ABSTRACT: Glutamine : fructose-6-phosphate amidotransferase 1 (GFAT1) was identified as a protein phosphorylated in glucose-deprived cells by immunoprecipitation using the anti-phospho Akt substrates (PAS) antibody, which recognizes the phosphorylation motif site by AMP-activated protein kinase (AMPK), followed by mass fingerprinting analysis. Glucose depletion-induced phosphorylation of endogenous GFAT was potentiated by 2-deoxyglucose (2-DG), an AMPK activator, and the 2-DG-stimulated phosphorylation of FLAG-tagged GFAT1 in transfected cells was suppressed by Compound C, an AMPK inhibitor. The 2-DG induced phosphorylation of GFAT1 was attenuated by the introduction of the kinase-negative mutant of AMPK, and the phosphorylation was observed in the cells expressing the constitutively active mutant of AMPK even in the absence of 2-DG. Subsequent analysis revealed that the PAS antibody recognized GFAT1 phosphorylated at Ser243, which is conserved among different species. The assay of the GFAT enzymatic activity in the cell lysates indicated that the 2-DG-treatment inhibited the enzymatic activity, and Compound C-preincubation partially prevented the 2-DG-induced decrease of the activity. Furthermore, the mutant replacing Ser243 by alanine partially prevented the decrease of GFAT activity by 2-DG treatment. These results indicate that the phosphorylation of GFAT1 at Ser243 by AMPK has an important role in the regulation of the GFAT1 enzymatic activity.
Genes to Cells 03/2009; 14(2):179-89. · 2.68 Impact Factor
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ABSTRACT: Sufficiency and depletion of nutrients regulate the cellular activities through the protein phosphorylation reaction; however, many protein substrates remain to be clarified. GBF1 (Golgi-specific brefeldin A resistance factor 1), a guanine nucleotide exchange factor for the ADP-ribosylation factor family associated with the Golgi apparatus, was isolated as a phosphoprotein from the glucose-depleted cells by using the phospho-Akt-substrate antibody, which recognizes the substrate proteins of several protein kinases. The phosphorylation of GBF1 was induced by 2-deoxyglucose (2-DG), which blocks glucose utilization and increases the intracellular AMP concentration, and by AICAR, an AMP-activated protein kinase (AMPK) activator. This phosphorylation was observed in the cells expressing the constitutively active AMPK. The 2-DG-induced phosphorylation of GBF1 was suppressed by Compound C, an AMPK inhibitor, and by the overexpression of the kinase-negative AMPK. Analysis using the deletion and point mutants identified Thr(1337) as the 2-DG-induced phosphorylation site in GBF1, which is phosphorylated by AMPK in vitro. ATP depletion is known to provoke the Golgi apparatus disassembly. Immunofluorescent microscopic analysis with the Golgi markers indicated that GBF1 associates with the fragmented Golgi apparatus in the cells treated with 2-DG and AICAR. The expression of the kinase-negative AMPK and the GBF1 mutant replacing Thr(1337) by Ala prevented the 2-DG-induced Golgi disassembly. These results indicate that GBF1 is a novel AMPK substrate and that the AMPK-mediated phosphorylation of GBF1 at Thr(1337) has a critical role, presumably by attenuating the function of GBF1, in the disassembly of the Golgi apparatus induced under stress conditions that lower the intracellular ATP concentration.
Journal of Biological Chemistry 03/2008; 283(7):4430-8. · 4.77 Impact Factor
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ABSTRACT: TBC7, a TBC (Tre-2/Bub2/Cdc16) 1 domain protein, was identified as a novel binding protein to the TSC1-TSC2 tumor suppressor complex by peptide mass fingerprinting analysis of the proteins immunoprecipitated with FLAG-epitope tagged TSC1 and TSC2 from the transfected mammalian cells. The in vivo and in vitro association of TBC7 and the TSC1-TSC2 complex was confirmed by the co-immunoprecipitation and pull-down analysis, respectively, and TBC7 was revealed to bind to the C-terminal half region of TSC1, which is distinct from the binding site with TSC2. The immunofluorescence microscopy and subcellular fractionation showed that TBC7 co-localizes with the tumor suppressor complex in the endomembrane. Overexpression of TBC7 enhanced ubiquitination of TSC1 and increased phosphorylation of S6 protein by S6 kinase, that is located in the mTOR-signaling pathway. These results indicate TBC7 could take a part in the negative regulation of the tumor suppressor complex through facilitating the downregulation of TSC1.
Biochemical and Biophysical Research Communications 10/2007; 361(1):218-23. · 2.48 Impact Factor
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Noriko Oshiro,
Rinako Takahashi,
Ken-ichi Yoshino,
Keiko Tanimura,
Akio Nakashima, Satoshi Eguchi,
Takafumi Miyamoto,
Kenta Hara,
Kenji Takehana,
Joseph Avruch,
Ushio Kikkawa,
Kazuyoshi Yonezawa
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ABSTRACT: The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser(183)). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe(129) to Ala). Immediately carboxyl-terminal to Phe(129) are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser(183), to Asp. PRAS40 (Ser(183)) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser(183)) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser(183)) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.
Journal of Biological Chemistry 08/2007; 282(28):20329-39. · 4.77 Impact Factor
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Noriko Oshiro,
Rinako Takahashi,
Ken-ichi Yoshino,
Keiko Tanimura,
Akio Nakashima, Satoshi Eguchi,
Takafumi Miyamoto,
Kenta Hara,
Kenji Takehana,
Joseph Avruch,
Ushio Kikkawa,
Kazuyoshi Yonezawa
Journal of Biological Chemistry. 05/2007;
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ABSTRACT: The translational regulator protein 4E-BP1, that binds to eukaryotic initiation factor-4E (eIF4E) to prevent the formation of the active translation complex, dissociates from eIF4E by phosphorylation through the mammalian target of rapamycin (mTOR) in the cells stimulated by amino acids. 4E-BP1 has been shown to associate with the scaffold protein raptor through its TOS and RAIP motifs to be recognized by mTOR. We revealed that the TOS motif mutant was phosphorylated by mTOR only at the priming sites of Thr37/46 but the RAIP motif mutant was phosphorylated not only at the priming sites but also at the subsequent site of Thr70 in vitro and in response to amino acid treatment in HEK293 cells. Analysis using the phosphorylation site mutants indicated that phosphorylation of the priming and subsequent sites of 4E-BP1 was required for dissociation from raptor as well as for the release of eIF4E. The expression of the 4E-BP1 mutants replacing the TOS motif and phosphorylation sites, that are poor substrates for mTOR and have no or little dissociation ability from raptor and eIF4E, respectively, significantly reduced the size of K562 cells. These results indicate that the the TOS motif has an essential function whereas the RAIP motif has an accessory role in the association with raptor and mTOR-mediated phosphorylation of 4E-BP1 to dissociate it from raptor and release eIF4E in response to amino acid stimulation leading to the control of cell size.
Genes to Cells 08/2006; 11(7):757-66. · 2.68 Impact Factor
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ABSTRACT: The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that plays a crucial role in a nutrient-sensitive signalling pathway that regulates cell growth. TOR signalling is potently inhibited by rapamycin, through the direct binding of a FK506-binding protein 12 (FKBP12)/rapamycin complex to the TOR FRB domain, a segment amino terminal to the kinase catalytic domain. The molecular basis for the inhibitory action of FKBP12/rapamycin remains uncertain. Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that is essential for mTOR signalling in vivo, and whose binding to mTOR is critical for mTOR-catalysed substrate phosphorylation in vitro. Here we investigated the stability of endogenous mTOR/raptor complex in response to rapamycin in vivo, and to the direct addition of a FKBP12/rapamycin complex in vitro. Rapamycin diminished the recovery of endogenous raptor with endogenous or recombinant mTOR in vivo; this inhibition required the ability of mTOR to bind the FKBP12/rapamycin complex, but was independent of mTOR kinase activity. Rapamycin, in the presence of FKBP12, inhibited the association of raptor with mTOR directly in vitro, and concomitantly reduced the mTOR-catalysed phosphorylation of raptor-dependent, but not raptor-independent substrates; mTOR autophosphorylation was unaltered. These observations indicate that rapamycin inhibits mTOR function, at least in part, by inhibiting the interaction of raptor with mTOR; this action uncouples mTOR from its substrates, and inhibits mTOR signalling without altering mTOR's intrinsic catalytic activity.
Genes to Cells 05/2004; 9(4):359-66. · 2.68 Impact Factor
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ABSTRACT: The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Herein we demonstrate that raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs to be required for amino acid- and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring their sensitivity to rapamycin and amino acid sufficiency.
Journal of Biological Chemistry 06/2003; 278(18):15461-4. · 4.77 Impact Factor
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ABSTRACT: TBC7, a TBC (Tre-2/Bub2/Cdc16) 1 domain protein, was identified as a novel binding protein to the TSC1–TSC2 tumor suppressor complex by peptide mass fingerprinting analysis of the proteins immunoprecipitated with FLAG-epitope tagged TSC1 and TSC2 from the transfected mammalian cells. The in vivo and in vitro association of TBC7 and the TSC1–TSC2 complex was confirmed by the co-immunoprecipitation and pull-down analysis, respectively, and TBC7 was revealed to bind to the C-terminal half region of TSC1, which is distinct from the binding site with TSC2. The immunofluorescence microscopy and subcellular fractionation showed that TBC7 co-localizes with the tumor suppressor complex in the endomembrane. Overexpression of TBC7 enhanced ubiquitination of TSC1 and increased phosphorylation of S6 protein by S6 kinase, that is located in the mTOR-signaling pathway. These results indicate TBC7 could take a part in the negative regulation of the tumor suppressor complex through facilitating the downregulation of TSC1.
Biochemical and Biophysical Research Communications.
