Protocol for checking ³²P incorperation into proteins after cAMP stimulation

Janet Smith | Boston Biomedical Research Institute

This protocol was developed for looking at changes in phosphorylation of myosin II (heavy and light chains) and myosin light chain kinase (MLCK) after cAMP stimulation, but it can also be used to look at phosphorylation of other proteins. The cAMP pulsing protocol was gotten from Tom Egelhoff's lab, and is similar to protocols used by the Devreotes lab.

Materials

Buffers

Equipment

Cell Starving and Pulsing

1. Resuspend two almost confluent 15 cm (25 ml) plates of Dictyostelium cells. Pool into a 50 ml disposible conical tube. Remove an aliquot, and count it during the spin.
2. Spin the cells in a clinical centrifuge, preferably at RT but 4°C is okay too. 1500 rpm (??? x g) for 3 min.
3. Aspirate media from the cell pellet. Resuspend the cells in 10 ml of MES-SB and spin as before.
4. Wash again in 10 ml MES-SB, then resuspend in MES-SB so that the cell density is 2 x 10⁷.
5. Transfer 5 ml to a 50 ml plastic beaker with the rim cut off, cover with aluminum foil, and shake at 200 rpm at RT.
6. After 2 hrs, start a cAMP drip. Use a syringe pump outfitted with a 5 ml syringe with a BioRad luer-lock attachment and chromatography tubing bent at an angle so that it will drip down into the beaker. Fill the syringe with a cAMP stock solution so that the final concentration in the beaker will be 60 nM, assuming that the volume of the drop is 25 μl. As long as the volume in the beaker is 5 ml, this comes to 12 μM. Set the syringe pump to deliver one drop every six minutes. On our pump, at setting of "23" delivers one drop every 5'30". This the closest to 6 min we can get with this set-up, and seems to be fine. Allow the drip to go for 4 hrs.
7. After 4 hrs, remove the drip and add caffeine to 5 mM. We assume that the volume added by the drip is offset by evaporation, and use the starting cell volume. If the starting volume was 5 ml, add 250 μl of a 100 mM stock. The caffeine stock needs to be thawed to RT before use so that it is fully dissolved, and this might also require some warming.

³²P-Labeling

1. 30 min later, remove cells so that 1.7 ml remains. Add 850 μCi ³²P inorganic phosphate. Shake 30 min more.
2. Have tubes labeled, and a boiling water bath with a rack nearby. Need screw-cap tubes labeled -15", 10", 20", 30", 60", 90", 180", 300". Each tube should contain 10 μl of 20% SDS. Also, prepare a "slug" of cAMP/DTT. This "slug" will raise the cAMP concentration to 10 μM, and the DTT concentration to 10 mM. Mix 60 μl 1M DTT and 3 μl 20 mM cAMP; you will add 17.85 μl of this.
3. Take a 200 μl aliquot at -15". Immediately place the tube in the boiling water bath and turn the shaker back on. At zero time, add 15.75 μl of the slug solution, turn the shaker off, and take the remaining time points. Turn the shaker back on after the 30" and 60" time points, but not between the earlier time points - there isn't time. It is helpful to slightly tilt the beaker in the clamp so there is no problem getting enough volume for the last time points. When you get a spare moment (after the 90" time point is a good time), add 1.25 μl slug to the boiling -15" sample, so that it has the same amount of cAMP/DTT as the other ones.
4. After the last time point, allow all the samples to boil for 5 min, then place in ice.

Immunoprecipitations

1. Add 40 μl pansorbin suspension, and spin 5' at top speed at RT in a microfuge.
2. Remove 200 μl supe to fresh tubes containing
   • 20 μl protein A/G sepharose
   • 10 μl anti-RLC
   • 10 μl anti-MLCK
   • 2 μl anti-LMM (myosin)
   • 1 ml IP buffer without SDS.
   Prepare this as a mix, but vortex frequently when aliquoting so that each tube gets the same amount of beads.
3. Rotate O/N in cold-room - well shielded and labeled.
4. Next day- Spin tubes at 2,000 rpm in a microfuge for 1 minute. Remove supes to labeled screw-cap tubes and save, labeled (³²P, date, number of counts) and shielded, in the freezer.
5. Wash each sample with 1 ml IP Buffer with SDS. Spin as above, and remove supes to a 50 ml orange cap tube. Label (³²P, date, number of counts (guess 1/20th of total) and store shielded in freezer. Dispose in drain when counts have decayed to 10 μlCi. After this step, radioactivity has decreased to a relatively safe level. I do the next wash behind a screen, and then do the rest unshielded.
6. Perform additional washes
   • 1 ml IP Buffer w/SDS
   • 1 ml Urea Buffer
   • 1 ml High Salt Buffer
   • 1 ml Tris NaCl Buffer
   For each wash, spin as above, and then remove supe by aspiration, using a bent syringe needle. Put the bevel of the needle against the side of the tube so that you don't suck up the beads. After the last wash is removed, give the tubes a quick spin, and aspirate the pellets dry.
7. Add 25 μl 2X SDS sample buffer, boil for 5 min, and load 20 μl on a 10-well minigel.
8. Stain and dry gel. Expose to Kodak BioMax film with 1 screen for about 10 hrs to get a high quality image, and then expose to a phosphorimager screen for a set amount of time(10 hrs) for quanitation.