Merck: Calbiochem: Product Spotlight/K-LISA mTOR Activity Kit

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K-LISA™ mTOR Activity Kit

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mTOR (mammalian Target of Rapamycin) was originally identified in Saccharomyces cerevisiae where mutations of the protein kinase, TOR, confer rapamycin resistance. mTOR is a large (>250 kDa) class IV PI-3 kinase family member with protein kinase activity, but lacks any lipid kinase activity. mTOR forms a complex with the 12 kDa cytosolic protein, FKBP-12 and rapamycin that functions to arrest the cell cycle in the G₁ phase. Cell cycle arrest by the mTOR complex requires the presence of the intact kinase domain of mTOR and, in particular, a conserved serine within this domain, Ser²⁴⁴⁸, which has been identified as an Akt/PKB-mediated phosphorylation site. Biomarkers indicate that the mTOR pathway is hyperactive in certain types of cancers, suggesting that mTOR could be an attractive target for cancer therapy. Activated mTOR may provide tumor cells with a growth advantage by promoting protein synthesis, which is the best-described physiological function of mTOR signaling. mTOR regulates Akt activity, a crucial downstream effector in the PI-3K–PTEN pathway, which controls cell proliferation and survival. Targeting this function of mTOR may also have therapeutic potential.

The K-LISA™ mTOR Activity Kit (Cat. No. CBA055) is suitable for measuring the kinase activity of purified or partially purified preparations of mTOR or mTOR that have been immunoprecipitated from crude cell lysates. This assay is also useful for in vitro mTOR inhibitor screening and for assessing the regulation of mTOR in cell signaling.

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The K-LISA™ mTOR Activity Kit is:
•	Versatile: can detect kinase activity or be used for inhibitor screening
•	Useful: mTOR Standard, Substrate, and Inhibitors are provided
•	Convenient: 96-well ELISA-based format, non-radioactive detection
•	Detection: Colorimetric, using HRP-Antibody Conjugate and TMB Substrate

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Figure 1. Activity of mTOR Standard

The mTOR Standard supplied in the kit is an enriched rat brain fraction isolated using proprietary methods. The mTOR standard phosphorylates p70S6K specifically on Thr³⁸⁹, and is inhibited by FKBP12/Rapamycin [(20 mM; Cat. No. 553210 or 553211)], a specific inhibitor of the mTOR/Raptor complex, as well as wortmannin (10 mM; Cat. No. 681675; also included with the kit), a more general PI-3K inhibitor. Addition of Wortmannin serves as a positive control for inhibitor analysis. Inhibition profiles can be generated based on mTOR activity in the presence and absence of test inhibitor(s).

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Rapamycin + FKBP12 Inhibition of mTOR Kinase Activity

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Antibodies

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Assay Kits

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Inhibitors

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