Materials and Reagents

W303a wild type yeast cells
Tris base (C₄H₁₁NO₃) (Thermo Fisher Scientific, catalog number: 77-86-1 )
NaCl (Thermo Fisher Scientific, catalog number: 7647-14-5 )
Na₂EDTA•2H₂O (EDTA) (Sigma-Aldrich, catalog number: ED2SS )
Triton X-100 (Thermo Fisher Scientific, catalog number: 9002-93-1 )
Phenylmethanesulfonyl fluoride (PMSF) (C₇H₇FO₂S) (Sigma-Aldrich, catalog number: P7626 )
Complete protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693159001 )
PhosSTOP tablet (F. Hoffmann-La Roche, catalog number: 04906837001 )
Glycerol (Thermo Fisher Scientific, catalog number: 56-81-5 )
MgCl₂ (USB, catalog number: 18641 500 GM )
Dithiothreitol (DTT) (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 20290 )
Rapamycin (Santa Cruz Biotechnology, catalog number: sc-3504 )
Glass beads (Sigma-Aldrich, catalog number: G-8772 )
HA antibody (12CA5) (Abcam, catalog number: ab16918
ATP (Sigma-Aldrich, catalog number: A2383-1G )
[γ-³²P]-ATP (PerkinElmer, catalog number: BLU002250UC )
Coomassie Blue R250 (National Diagnostics, catalog number: HS-605 )
HCl
2.5x SDS loading dye
IP buffer (see Recipes)
Kinase buffer (see Recipes)
PBS buffer (see Recipes)

Equipment

Standard bench-top centrifuge
Shaker
1.5 ml Eppendorf tubes
Authoradiograph

Procedure

Inoculate W303a wild type or W303a cells containing vector, pRS315-SCH9-HA3 and its kinase dead form (K441A) and hyperactive form (2D3E) overnight in 10 ml SC-His- medium. Shaking vigorously (300 rpm) at 30 °C.
Subculture yeast cells in 2 flasks containing 100 ml YPD each with starting OD₆₀₀=0.2.
Shake vigorously at 30 °C to OD₆₀₀=0.5 (important to use YPD rather than SD medium).
Add to one flask 200 nM of rapamycin and another vehicle control. Shake vigorously at 30 °C for 30 min.
Spin down at room temperature (RT) at 3,000 x g to collect cells (It is important to avoid freezing).
Discard most of the supernatant, then suspend yeast cells in the remaining medium and split into 5x 1.5 ml Eppendorf tubes (20 ml cell culture/tube, more cells in one tube do not breakdown sufficiently).
Collect cell pellets, immediately add 200 µl ice-cold IP buffer and the same amount of glass beads.
Immediately breakdown cells by beads beater at 4 °C for 1 min. Note: Do not exceed 3 min, otherwise kinase activity will begin to decrease due to overheating. 15 sec x 5 beating with 45 sec in between also decreases Sch9 kinase activity.
Collect cell lysate by spinning down at 20,000 x g, 10 min at 4 °C. Combine lysate.
Immediately add 1 µg HA antibody (12CA5) to 1 mg/500 μl cell lysate, gently rotate at 4 °C for 1 h.
Wash protein A/G beads 3x with IP buffer. Add 50 μl (100 μl 50% slurry) to cell lysate and further rotate for another 1 h.
Spin down and collect beads. Wash 3x with 0.5 ml ice-cold IP buffer.
Add 0.5 ml 1x with ice-cold kinase buffer. Save 50 µl in another 1.5 ml Eppendorf tube for western blot with HA antibody.
Spin down and add to the immunocomplex 100 µM ATP, 0.5 µg bacterially-expressed GST-Maf1 in 50 µl kinase.
Add to the reaction system 50 µCi [γ-³²P]-ATP. Vortex and incubate at 30 °C for 15-30 min.
Kinase reaction was stopped by heating at 100 °C for 5 min in 2.5x SDS loading dye.
Half of the sample should be subjected to SDS-PAGE. Substrate will be detected by Coomassie blue staining.
Dry the gel using gel dryer at 80 °C for 2 h. Phosphorylation of substrate is revealed by authoradiograph.

Recipes

IP buffer
50 mM Tris-HCl (pH 7.5)
150 mM NaCl
0.5 mM EDTA
0.5% Triton X-100
Add 2 mM PMSF, Roche Complete protease inhibitor cocktail and phosSTOP tablet before use. Vary NaCl concentration or/and Triton X-100 level to obtain optimum condition for different kinase and different antibodies.
1x PBS (pH 7.4)
Kinase buffer
1x PBS (pH 7.4)
20% glycerol
4 mM MgCl₂
10 mM DTT
Protease inhibitor

Acknowledgments

This protocol was adapted from and used in Wei and Zheng (2009) and Wei et al. (2009).

References

Wei, Y. and Zheng, X. F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
Wei, Y., Tsang, C. K. and Zheng, X. F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.