In vitro mTORC1 Kinase Assay for Mammalian Cells Protocol

Kayleigh Margaret Dodd; Andrew Robert Tee

doi:10.21769/BioProtoc.1827

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In vitro mTORC1 Kinase Assay for Mammalian Cells Protocol

Kayleigh Margaret Dodd email

AT Andrew Robert Tee

Published: Vol 6, Iss 11, Jun 5, 2016 DOI: 10.21769/BioProtoc.1827 Views: 13043

Edited by: HongLok Lung Reviewed by: Shravani Mukherjee

Original research article

The authors used this protocol in:

http://www.ncbi.nlm.nih.gov/pubmed/24931163

Apr 2015

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Abstract

Historically, mechanistic target of rapamycin (mTOR) was purified from mammalian cells using mild nonionic detergents such as NP-40 and or Triton-X100 that resulted in dissociation of core regulatory components essential for its native kinase activity. Consequently, these older kinase assays required MnCl₂ to artificially enhance the weak phosphotransfer activity observed (Bai et al., 2007; Kim et al., 2002). With the use of the zwitterionic detergent 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), the mTOR complex 1 (mTORC1) containing Regulatory-associated protein of mTOR (Raptor) and Lst8 (also known as GbetaL) can be successfully purified as a complex. This in vitro kinase assay allows for purification of mTORC1 that resembles its physiological state and retains kinase activity under physiological MgCl₂ concentrations (Sancak et al., 2007). The activity of mTORC1 can be further enhanced through the use of hyperactive mutations within the kinase domain of mTOR or inclusion of GTP-bound RAS enriched in brain (Rheb) that is supplemented into the in vitro kinase assays. Rheb is a small-G-protein that binds to and activates mTORC1 to phosphorylate downstream substrates, such as eukaryotic initiation factor 4E-BP1 (4E-BP1) (Burnett et al., 1998), ribosomal protein S6 kinase 1 (S6K1) (Kim et al., 2002), Signal transducer and activator of transcription 3 (STAT3) (Dodd et al., 2015), and proline-rich Akt substrate of 40 kDa (PRAS40) (Dunlop et al., 2009).

Materials and Reagents

HEK293E cells
Plasmids and vectors
1. HA-Raptor (Addgene, catalog number: 8513 )
2. myc-mTOR (Addgene, catalog number: 1861 )
3. Rheb (National Center for Biotechnology Information, Gene, catalog number: 6009 ) cloned into pDEST27 using the gateway cloning system in accordance with manufacturer protocol (Life Technologies, catalog number: 11812-013 )
  Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 11812-013 ”.
4. GST-4E-BP1/pGEX vectors generated as previously described (Dunlop et al., 2009)
Antibodies
1. Clone 9E10 anti-Myc antibodies (Sigma-Aldrich, catalog number: M5546 )
2. Clone 9B11 anti-Myc antibodies (Cell Signaling Technology, catalog number: 2276 )
3. Anti-HA (Roche Diagnostics, catalog number: 11867431001 )
4. Anti-GST (Merck Millipore Corporation, catalog number: 05-782 )
Cell culture and transfection
1. Dulbecco’s modified eagle’s medium (DMEM)
2. 10% foetal bovine serum (FBS), EU Approved (South American) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270-106 )
3. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15070-063 )
  Note: HEK293E cells were cultured in DMEM supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin.
Insulin (Sigma-Aldrich, catalog number: I9278 )
Rapamycin (EMD Millipore Corporation, catalog number: 553210 )
Chemicals of analytical grade
1. HEPES (Sigma-Aldrich, catalog number: H3375 )
2. EDTA (Sigma-Aldrich, catalog number: 431788 )
3. β-glycerophosphate (disodium salt, pentahydrate) (Sigma-Aldrich, catalog number: 50020 )
4. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
5. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
6. Adenine triphosphate (ATP) (Sigma-Aldrich, catalog number: A26209 )
7. Leupeptin (Sigma-Aldrich, catalog number: L5793 )
8. Antipain (Sigma-Aldrich, catalog number: 10791 )
9. Benzamidine (Sigma-Aldrich, catalog number: 12072 )
10. Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
11. Sodium vanadate (Sigma-Aldrich, catalog number: 289361 )
12. Dithiothreitol (Sigma-Aldrich, catalog number: 43815 )
13. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: 78830 )
14. Wortmannin (Sigma-Aldrich, catalog number: W1628 )
15. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) (Sigma-Aldrich, catalog number: 226947 )
mTOR lysis buffer (see Recipes)
Low salt mTOR wash buffer (see Recipes)
High salt mTOR wash buffer (see Recipes)
mTOR wash buffer (see Recipes)
3x mTOR kinase assay buffer (see Recipes)
Rheb lysis buffer (see Recipes)
Rheb storage buffer (see Recipes)
Phosphate buffered saline (see Recipes)
mTOR assay start buffer (see Recipes)
Protease inhibitors (see Recipes)

Equipment

Thermomixer heating block (Eppendorf AG, model: Eppendorf thermomixer^® compact )
Refrigerated mini centrifuge (Thermo Fisher Scientific, Thermo Scientific™, model: Heraeus and Fresco 17 centrifuge )
Stuart^TM SB2 fixed speed rotator (Bibby Scientific Limited, Stuart Scientific)

Procedure