Characterization of the Phosphoproteins and Protein Kinase Activity in mTOR Immunoprecipitates

Nishiuma, Teruaki; Hara, Kenta; Tsujishita, Yosuke; Kaneko, K.; Shii, Kozui; Yonezawa, Kazuyoshi

doi:10.1006/bbrc.1998.9671

Cited by 27 publications

(27 citation statements)

References 26 publications

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“…Although mTORcatalyzed p70␣ phosphorylation was detectable with all washing conditions, the mTOR autophosphorylation and kinase activity toward p70␣ is substantially enhanced when the immunoprecipitate is washed with the high salt wash buffer containing 1% Nonidet P-40 or the RIPA buffer, compared with that prepared after washing with the high salt wash buffer without detergent. The result was unexpected, as we have previously shown that washing of mTOR immunoprecipitates with 1% Nonidet P-40 reduces greatly the ability of mTOR to catalyze eIF-4E BP1 phosphorylation (20). These differences in mTOR-catalyzed p70␣ phosphorylation are not because of differences in the recovery of mTOR polypeptide, as demonstrated by the immunoblot with the anti-mTOR antibody (Fig.…”

Section: Resultsmentioning

confidence: 85%

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Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro

Isotani

Hara

Tokunaga

et al. 1999

Journal of Biological Chemistry

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313

246

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p70 S6 kinase ␣ (p70␣) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70␣ activation in a manner that can be overcome by coexpression of p70␣ with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70␣ polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70␣ phosphorylation in vitro is accompanied by a substantial restoration in p70␣ kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70␣ by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70␣ activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined. p70 S6 kinase ␣ (p70␣), 1 whose major substrate is the 40 S ribosomal protein S6, plays a critical role in the translation of a subclass of mRNAs that contain a short oligopyrimidine sequence immediately following the transcriptional start site (1). p70␣ is activated in response to insulin/mitogens in vivo through a multisite phosphorylation of serine and threonine residues (2). Several sets of independently regulated p70␣ phosphorylation sites have been identified (3-6); one set consists of Ser/Thr-Pro motifs, five of which are clustered in a psuedosubstrate autoinhibitory domain in the noncatalytic carboxyl-terminal tail (Ser-434, Ser-441, Ser-447, Ser-452, and Thr-444 in p70␣), and two others, Thr-390 and Ser-394, are located in a 65-amino acid segment immediately carboxyl-terminal to the kinase catalytic domain. A second set of regulated phosphorylation sites, Thr-412 and Ser-427, exhibit the sequence motif Phe-Ser/Thr-Phe/Tyr. Thr-252, located on the activation loop in catalytic subdomain VIII, is the site at which 3-phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates p70␣ (7,8). Among these, the phosphorylation of Thr-252, Ser-394, and Thr-412 is necessary for the activation of p70␣ kinase catalytic function; the attainment of physiologic levels of p70␣ activity results from a strongly synergistic, positive site-site interaction between the phosphorylated Thr-252 and Thr-412 residues (7).In addition to its regulation by insulin and mitogens through PI-3 kinase-dependent pathways, p70␣ can also be activated by increasing concentrations of extracellular amino acids in the absence of serum or mitogens to the level attained by maximal mitogen stimulation (9-11). Moreover, a thr...

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Section: Resultsmentioning

confidence: 85%

“…Antibodies and cDNAs-The anti-mTOR antibody was described previously (20). The anti-phosphopeptide antibodies against Thr-412, Ser-434, and Thr-444/Ser-447 of p70␣ (the anti-412-P Ab, the anti-434-P Ab, and the anti-444/447-P Ab, respectively), the anti-FLAG antibody, and the anti-HA antibody were described previously (21).…”

Section: Methodsmentioning

confidence: 99%

Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro

Isotani

Hara

Tokunaga

et al. 1999

Journal of Biological Chemistry

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313

246

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“…We examined the possibility that the region might be required for post-translational modi®cations such as lipid modi®cation, because the C-terminal amino acid sequence of mTOR was expected to play a similar role to the CAAX motif, that is known to be essential for the function of the ras family proteins by targeting them to the membrane, although Pro and Phe in the CPFW motif are not aliphatic. In support of this possibility, it has been shown that mTOR is identi®ed in the membrane fraction in HEK293 cells (Withers et al 1997;Nishiuma et al 1998) and that yeast TOR2 protein is localized on the surface of the yeast vacuole and this localization is important for TOR2 function in vivo (Cardenas & Heitman 1995). However, substitution of Cys2546 with Ala did not affect the mTOR function in vitro and in vivo.…”

Section: Discussionmentioning

confidence: 96%

“…The anti-mTOR antibody (Ab) was produced as previously described (Nishiuma et al 1998). The anti-phosphopeptide Ab against Thr412 of p70a (the anti-412-P Ab) was purchased from New England Biolabs.…”

Section: Antibodies and Cdnasmentioning

confidence: 99%

Carboxyl‐terminal region conserved among phosphoinositide‐kinase‐related kinases is indispensable for mTOR function in vivo and in vitro

et al. 2000

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“…The Tel2 fragment tagged with N-terminal glutathione S-transferase was expressed in Escherichia coli strain BL21(DE3)pLysS (Novagen) and then used to immunize rabbits. Anti-mTOR mouse monoclonal antibody (N5D11) was previously described (21) and used for immunoprecipitation. Anti-LC3 (microtubule-associated protein 1 light chain 3) rabbit polyclonal antibody was previously described (22).…”

Section: Methodsmentioning

confidence: 99%

Tti1 and Tel2 Are Critical Factors in Mammalian Target of Rapamycin Complex Assembly

Kaizuka

Hara

Oshiro

et al. 2010

Journal of Biological Chemistry

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226

193

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Mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family and is a major regulator of translation, cell growth, and autophagy. mTOR exists in two distinct complexes, mTORC1 and mTORC2, that differ in their subunit composition. In this study, we identified KIAA0406 as a novel mTOR-interacting protein. Because it has sequence homology with Schizosaccharomyces pombe Tti1, we named it mammalian Tti1. Tti1 constitutively interacts with mTOR in both mTORC1 and mTORC2. Knockdown of Tti1 suppresses phosphorylation of both mTORC1 substrates (S6K1 and 4E-BP1) and an mTORC2 substrate (Akt) and also induces autophagy. S. pombe Tti1 binds to Tel2, a protein whose mammalian homolog was recently reported to regulate the stability of PIKKs. We confirmed that Tti1 binds to Tel2 also in mammalian cells, and Tti1 interacts with and stabilizes all six members of the PIKK family of proteins (mTOR, ATM, ATR, DNA-PKcs, SMG-1, and TRRAP). Furthermore, using immunoprecipitation and size-exclusion chromatography analyses, we found that knockdown of either Tti1 or Tel2 causes disassembly of mTORC1 and mTORC2. These results indicate that Tti1 and Tel2 are important not only for mTOR stability but also for assembly of the mTOR complexes to maintain their activities. Mammalian target of rapamycin (mTOR)4 is a member of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, which also includes ataxia telangiectasia mutated (ATM), ATM-and Rad3-related (ATR), DNA-PKcs, suppressor with morphological effect on genitalia 1 (SMG-1), and the catalytically inactive transformation/transcription domainassociated protein (TRRAP) (1-3). mTOR forms two distinct complexes that differ in their subunit composition, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 includes mTOR, mLST8 (also known as G␤L), and Raptor, whereas mTORC2 includes mTOR, mLST8, mSin1, and Rictor (4 -7). mTORC1 is activated by several factors, including amino acids. It promotes protein translation through phosphorylation of S6K1 and 4E-BP1 (7), and inhibits autophagy through phosphorylation of ULK1 and Atg13 (8-10). On the other hand, mTORC2 is activated by growth factors and phosphorylates Akt at Ser-473, and plays key roles in cell survival, metabolism, proliferation, and organization of the cytoskeleton (7).Although growing numbers of mTOR-interacting proteins have been identified, little is known about the assembly of these mTOR complexes. Deletion of the mTORC2 components such as Rictor, mSin1, and mLST8 induces disassembly of mTORC2, impairing Akt phosphorylation (at Ser-473) (11-13), whereas mLST8 is not required for mTOR-Raptor interaction and S6K1 activation (11). Prolonged treatment with rapamycin, which is an mTORC1 inhibitor, inhibits mTORC2 assembly (14). Recently, it was proposed that phosphatidic acid is required for the assembly of mTOR complexes, because depletion of phosphatidic acid caused by treatment with 1-butanol and expression of dominant negative mutant phospholipase D results...

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Characterization of the Phosphoproteins and Protein Kinase Activity in mTOR Immunoprecipitates

Cited by 27 publications

References 26 publications

Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro

Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro

Carboxyl‐terminal region conserved among phosphoinositide‐kinase‐related kinases is indispensable for mTOR function in vivo and in vitro

Tti1 and Tel2 Are Critical Factors in Mammalian Target of Rapamycin Complex Assembly

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