Akt Kinase Phosphorylation Assay by Immunoprecipitation

The protein kinases belong to the great family of kinases and are involved in the mechanisms of phosphorylation. They are activated by phosphorylation which in turn activates a cascade of events leading to the phosphorylation of different amino acids. The protein kinases are responsible for cellular signal transduction that play an important role in modulating the localization of transcription factors. They are found to be overactive, malfunctional or overexpressed in a variety of diseases, mostly tumors.

Immunoprecipitation of the kinase phosphorylation is an invaluable tool for assessing the activation status of intracellular signaling cascades within a specific cellular state and confirming the enzymatic activity of a specific kinase towards the putative substrate.

Here we describe a non-radioactive method to assess Akt kinase activity, however the basic protocol may be applied to most kinases and putative substrates of interest.

Figure 1. The general workflow for immunoprecipitation.

Materials and Reagents

• Recombinantly produced substrate of interest (full-length murine Oct4 was cloned into a mammalian expression vector containing a T3 promoter sequence and a carboxy-terminal 6X His-TEV-3X FLAG epitope tag)
• TnT coupled reticulocyte lysate systems
• Expression vector (with Sp6, T3, or T7 promoter sequence) containing protein of interest (test substrate) wild-type, putative mutant and empty vector control
• Transcend tRNA
• RNaseOUT
• SDS polyacrylamide gel
• Polyvinyl difluoride membrane (PVDF)
• Primary antibody
• Kinase agonist to augment kinase activity (Ro-31-8220)
• Non-radioactive Akt kinase assay kit (Immobilized phospho-Akt (Ser473) rabbit mAb (bead conjugate), Phospho-GSK-3 (Ser21/9) rabbit mAb, GSK-3 fusion protein, 10 mM ATP)
• Bead conjugated primary antibody or primary antibody and protein A/G agarose
• Phenyl methlsulfonyl fluoride (PMSF)
• 1X Cell lysis buffer (25 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM Na₄P₂O₇, 1 mM β-Glycerophosphate, 1 mM Na₃VO₄, 1 µg/µL Leupeptin, stored at 4°C for 1-2 weeks only)
• 1X Kinase buffer (25 mM Tris (pH 7.5), 5 mM β-Glycerophosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂, stored at -20°C.)
• 3X SDS sample buffer (187.5 mM Tris (pH 6.8), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue, aliquot and stored at -20°C, add DTT fresh before each use)

Equipment

• Cell scraper
• 1.5 mL microfuge tube
• Refrigerated microcentrifuge
• Microtiter plate reader
• End-over-end rotator

Assay Procedure

• In vitro transcription
  1) Prepare the recombinantly produced substrate of interest using the TnT coupled reticulocyte lysate system. Wild-type, kinase mutant, and empty vector control will be required.
  2) Following the manufacturer’s recommended protocol proceed to set up the following reaction mixes for each sample. All reagents should be kept on ice.

Component	Volume (μL)
TnT lysate	25
TnT reaction buffer	2
T3 RNA polymerase	1
Amino acids-Met	0.5
Amino-acids-Leu	0.5
RNaseOUT	1
Transcend tRNA	1
H2O	14
DNA (0.2 µg/µL for 1µg total)	5

Note: This assay requires that the protein of interest be cloned in an expression vector containing a Sp6, T3, or T7 promoter sequence. The appropriate polymerase must be selected based on vector utilized.
3) Incubate the reaction mixes for 90 min at 30°C.
4) Typical yields from coupled transcription are 50-500 ng/µL.
5) Analyze the product by western blot. Combine 5 µL of the reaction with 20 µL of 1X SDS loading buffer. Denature at 95°C for 3 min and load 10 µL onto an SDS polyacrylamide gel. Transfer gel to PVDF membrane. To confirm the expression by using a primary antibody raised against the protein of interest and/or epitope tag contained in the selected vector.
6) Store the remaining in vitro transcription reaction at -20°C.

• Cell lysate preparation
  1) Culture actively growing fibroblasts to 75% confluence.
  2) Incubate the cells in the presence of 10 µM Ro-31-8220 at 37°C for one hour to increase Akt activation.
  3) Aspirate media and quickly rinse the cells twice with ice-cold PBS.
  4) Lyse cells with complete 1X cell lysis buffer supplemented with 1 mM PMSF. Incubate on ice for 5 min.
  5) Remove cells from plate with cell scraper. Place lysate in 1.5 mL microcentrifuge tube on ice for 30 min. Vortex two times for 10 sec each at 10 and 20 min.
  6) Centrifuge the lysate at 10,000 x g for 10 min at 4°C. Transfer the supernatant to a fresh tube. Store lysate at -80°C until use.
• Immunoprecipitation
  1) Each experiment will require eight immunoprecipitations; four with cell lysate and four mock immunoprecipitations with 1X cell lysis buffer. For each set of four immunoprecipitations, one will be a positive control for kinase activity, employing a previously confirmed (in the literature) substrate. The remaining three will be used for the recombinantly produced test substrate (wild-type, putative kinase mutant, and empty vector). The mock immunoprecipitations are negative controls used to ensure that the kinase activity emanates from the cell lysate and no other reagents used during this protocol.
  2) Add 20 µL of immobilized antibody-bead slurry to 200 µL of lysate (or 1X cell lysis buffer) in a 1.5 mL microcentrifuge tube. Incubate overnight at 4°C with end-over-end rotation. Immobilized phospho-Akt (Ser473) is included in the non-radioactive Akt-kinase assay kit.
  3) Alternatively, add primary antibody to 200 µL of lysate. The exact amount may need to be titrated. Approximately 1 µg of an affinity-purified antibody is generally sufficient.
  4) Incubate overnight at 4°C with end-over-end rotation.
  5) Add prepared protein A/G agarose beads (25 µL of 50% slurry). Incubate at 4°C with end-over-end rotation for 2 h.
  6) Centrifuge the immunoprecipitate at 10,000 x g for 30 sec at 4°C. Aspirate off supernatant and wash the pellet (on ice) two times for 3 min each with 500 µL 1X cell lysis buffer.
  7) Wash the pellet (on ice) two times for 3 min with 500 µL 1X kinase buffer.
• Non-radioactive kinase assay
  1) Resuspend the final pellet in 50 µL of 1X kinase buffer.
  2) Add 1 µL of 10 mM ATP (from the non-radioactive Akt kinase assay kit) and 1 or 2 µL of kinase substrate generated in step in vitro transcription 6).
  3) Incubate for 30 min at 30°C.
  4) To terminate the reaction by adding 25 µL of 3X SDS sample buffer. Vortex gently, and then microcentrifuge at 10,000 x g.
  5) Store at -80°C or proceed directly to analyze by western blot.

References

1. Fatima Ardito, et al.; The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy. Int J Mol Med, 2017, 40(2): 271-280.
2. Peck S C. Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates. Plant J, 2006, 45(4): 512-522.

For research use only. Not for any other purpose.