scholarly journals Identification of Protor as a novel Rictor-binding component of mTOR complex-2

2007 ◽  
Vol 405 (3) ◽  
pp. 513-522 ◽  
Author(s):  
Laura R. Pearce ◽  
Xu Huang ◽  
Jérôme Boudeau ◽  
Rafał Pawłowski ◽  
Stephan Wullschleger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Mammalian Target Of Rapamycin ◽  
Specific Component ◽  
S6 Kinase ◽  
Important Regulator ◽  
Hydrophobic Motif ◽  
Triton X ◽  
Binding Subunit ◽  
Binding Component

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR–Raptor (regulatory associated protein of mTOR)–mLST8 (termed mTORC1), and complex 2, comprising mTOR–Rictor (rapamycininsensitive companion of mTOR)–mLST8–Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.

scholarly journals Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)

2009 ◽  
Vol 421 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Juan M. García-Martínez ◽  
Jennifer Moran ◽  
Rosemary G. Clarke ◽  
Alex Gray ◽  
Sabina C. Cosulich ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Mammalian Target Of Rapamycin ◽  
Specific Inhibitor ◽  
Initiation Factor ◽  
Target Of Rapamycin ◽  
S6 Kinase ◽  
Class 1 ◽  
Hydrophobic Motif ◽  
Ribosomal S6 Kinase

mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of ∼10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.

Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney

2011 ◽  
Vol 436 (1) ◽  
pp. 169-179 ◽  
Author(s):  
Laura R. Pearce ◽  
Eeva M. Sommer ◽  
Kei Sakamoto ◽  
Stephan Wullschleger ◽  
Dario R. Alessi
Keyword(s):  
Protein Kinase ◽  
Knockout Mice ◽  
Mammalian Target Of Rapamycin ◽  
The Other ◽  
Downregulated Gene ◽  
S6 Kinase ◽  
Interacting Protein ◽  
Active Complex ◽  
Protein Kinase Cα ◽  
Important Regulator

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1), mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K (S6 kinase), whereas mTORC2 regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1). To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1- and Protor-2-knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex. Moreover, Protor knockout mice display no defects in the phosphorylation of Akt and PKCα at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1 knockout mice displayed markedly reduced hydrophobic motif phosphorylation of SGK1 and its physiological substrate NDRG1 (N-Myc downregulated gene 1) in the kidney. Taken together, these results suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney.

Lupeol Inhibits Proliferation and Promotes Apoptosis of Ovarian Cancer Cells by Inactivating Phosphoinositide-3-Kinase/Protein Kinase B/ Mammalian Target of Rapamycin Pathway

2020 ◽  
Vol 19 (2) ◽  
pp. 206-210
Author(s):  
Feng Chen ◽  
Bei Zhang
Keyword(s):  
Cell Cycle ◽  
Ovarian Cancer ◽  
Protein Kinase ◽  
Cell Viability ◽  
Cancer Cells ◽  
Protein Kinase B ◽  
Mammalian Target Of Rapamycin ◽  
Target Of Rapamycin ◽  
Ovarian Cancer Cells ◽  
Phosphoinositide 3 Kinase

Lupeol exhibits multiple pharmacological activities including, anticancerous, anti-inflammatory, and antioxidant. The aim of this study was to explore the anticancerous activity of lupeol on ovarian cancer cells and examine its mechanism of action. To this end, increasing concentrations of lupeol on cell viability, cell cycle, and apoptosis in Caov-3 cells were evaluated. Lupeol inhibited cell viability, induced G1 phase arrest in cell cycle, increased cell apoptosis, and inhibited the ratio of phospho-Akt/protein kinase B and phospho-mammalian target of rapamycin/mammalian target of rapamycin. In conclusion, these data suggest that lupeol may play a therapeutic role in ovarian cancer.

scholarly journals Akt/Protein Kinase B Promotes Organ Growth in Transgenic Mice

2002 ◽  
Vol 22 (8) ◽  
pp. 2799-2809 ◽  
Author(s):  
Tetsuo Shioi ◽  
Julie R. McMullen ◽  
Peter M. Kang ◽  
Pamela S. Douglas ◽  
Toshiyuki Obata ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transgenic Mice ◽  
Cell Size ◽  
Protein Kinase B ◽  
Mammalian Target Of Rapamycin ◽  
Pi3k Pathway ◽  
Mutant Protein ◽  
Heart Weight ◽  
Heart Size ◽  
Constitutively Active

ABSTRACT One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth.

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