mTOR complexes kinase assays

HEK-293 cells were freshly lysed in Hepes lysis buffer. Lysate (1 ~ 4 mg) was pre-cleared by incubating with 5 ~ 20 μL of Protein G-Sepharose conjugated to pre-immune IgG. The lysate extracts were then incubated with 5 ~ 20 μL of Protein G-Sepharose conjugated to 5 ~ 20 μg of either anti-Rictor or anti-Raptor antibody, or pre-immune IgG. All antibodies were covalently conjugated to Protein G-Sepharose. Immunoprecipitations were carried out for 1 hr at 4 ℃ on a vibrating platform. The immunoprecipitates were washed 4 times with Hepes lysis buffer, followed by two washes with Hepes kinase buffer. For Raptor immunoprecipitates used for phosphorylating S6K1, for the initial two wash steps the buffer included 0.5 M NaCl to ensure optimal kinase activity. GST-Akt1 was isolated from serum-deprived HEK-293 cells incubated with PI-103 (1 μM for 1 hr). GST-S6K1 was purified from serum-deprived HEK-293 cells incubated with Rapamycin (0.1 μM for 1 hr). mTOR reactions were initiated by adding 0.1 mM ATP and 10 mM MgCl2 in the presence or absence of KU-0063794 and GST-Akt1 (0.5 μg) or GST-S6K1 (0.5 μg). Reaction were carried out for 30 mins at 30 ℃ on a vibrating platform and stopped by addition of SDS sample buffer. Reaction mixtures were then filtered through a 0.22-μm-poresize Spin-X filter and samples were subjected to electrophoresis and immunoblot analysis.

Cell lines

Wild-type and mLST8-deficient MEFs

Preparation method

The solubility of this compound in DMSO is limited. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 ℃ for several months.

Reaction Conditions

~ 3 μM; 24, 48 and 72 hrs

Applications

In wild-type and mLST8-deficient MEFs, KU-0063794 inhibited cell growth.

Ku-0063794 is a specific mTOR inhibitor, which inhibits both mTORC1 and mTORC2 with IC50 values of ~ 10nM.[1]
The mTOR (mammalian target of rapamycin) protein kinase lies at the nexus of signaling pathways that regulate cell growth and proliferation. Various cancer-driving mutations in genes such as receptor tyrosine kinases, Ras, PI3K (phosphoinositide 3-kinase) and PTEN (phosphatase and tensin homologue deleted on chromosome 10) stimulate proliferation, growth and survival by the activation of mTOR kinase. MTOR 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 phosphorylates the hydrophobic motif of S6K, while mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. Ku-0063794 inhibits both mTORC1 and mTORC2 in vitro and in vivo and can be used to dissect cellular functions of the mTOR pathway.[1]
In HEK-293 cells, Ku-0063794 inhibited the activity of endogenous immunoprecipitated mTORC1 and mTORC2, employing S6K1 and Akt as substrates, with IC50 values of ~10nM. The ability of Ku-0063794 to suppress S6K1 activity and phosphorylation was rapid,Concentrations
of 100–300 nM Ku-0063794 were required to fully suppress amino-acid-induced phosphorylation of S6K1 and S6 protein. Ku-0063794 inhibits phosphorylation of Akt at Thr 308. Ku-0063794 was also assessed for other targets. However, it does not significantly inhibit 76 other protein kinases tested as well as seven lipid kinases, including class 1a PI3K α and PI3K β.[1,2]
The antileukemic effects of Ku-0063794 of the PI3K signaling pathway were evaluated by methylcellulose colony assays with primary murine CML cells obtained from mice that underwent bone marrow reconstitution with hematopoietic cells transduced with a retrovirus vector-expressing BCR/ABL. It showed that Ku-0063794 fully suppressed colony formation of primary murine CML at 1000nM. Moreover, KU-0063794 strikingly reduces cell proliferation in BA/F3 cells expressing BCR/ABL independently of mutation status, which indicates Ku-0063794 is a desirable therapeutic strategy to affect oncogenes signaling downstream of BCR/ABL.[2]
References:
1.Garcia-Martinez J, Moran J, Clarke R, et al. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)[J]. Biochem. J, 2009, 421: 29-42.
2.Schuster K, Zheng J, Arbini A A, et al. Selective targeting of the mTORC1/2 protein kinase complexes leads to antileukemic effects in vitro and in vivo[J]. Blood cancer journal, 2011, 1(9): e34.