Alexandra Newton Lab

Protocols

Protein Kinase C Activity Assay

The following is a modification of Kikkawa et al., (1982) J. Biol. Chem. 257, 13341. This assay measures phosphorylation (32P) of a protein kinase C-peptide substrate, which is subsequently separated by binding to phosphocellulose paper.

Materials:

Protein kinase C: This assay system can be used to measure activity of the purified kinase, or the activity in crude cellular extracts. Protein kinase C sample can be diluted in 20 mM HEPES/ 2 mM DTT mmediately prior to assay.

Peptide substrate: The peptide Ac-FKKSFKL-NH2, derived from the MARCKS protein, is used as a substrate. The Km for this peptide is ~50 µM. A 10 mg/ml stock in ddH2O (10 mM) is convenient. Other basic, protein kinase C-selective peptides can also be used, at a concentration of at least 2 -3 x their Km.

Cofactors: Conventional protein kinase C's are activated by Ca2+ and lipids (phosphatidylserine and diacylglycerol), which must be present in the assay for maximal activity. EGTA replaces cofactors for "non-activating conditions".

1) Lipid (phosphatidylserine/diacylglycerol membranes): A 10x stock of activating membranes, comprising 1.4 mM phosphatidylserine / 38 µM diacylglycerol (97.5 mol % and 2.5 mol %, respectively), is prepared as follows: Brain phosphatidylserine (2800 nmol) and diacylglycerol (76 nmol) (in CHCl3 stocks) are mixed in a glass tube, and the solvent evaporated under a stream of nitrogen. The lipids are then resuspended in 2 ml 20 mM HEPES, pH 7.4 by vigorous vortexing, and dispersed by brief (30 s) sonication in a bath sonicator. The resulting turbid solution can be stored for 1 month at 4oC.

2) Ca2+: CaCl2 (1 mM) provides a 10x stock for activating protein kinase C. For "non-activating" conditions, a 10x stock of the chelator EGTA (5 mM, pH 7.4) is substituted.

ATP: A concentrated stock of 10 mM ATP is convenient. The exact concentration of the ATP stock may be determined by measuring the absorbance at 260 nM. (Extinction coefficient = 15.4 x 103 (absorbance units M-1). Protein kinase C has a Km of ~ 50 µM for ATP.

5x "GO" solution: A mixture of Mg/ATP is prepared at 5X concentration. 
This mixture contains:

for 2 ml:
20 mM HEPES, pH 7.4 (40 µL of 1 M stock)
500 µM ATP (100 µl of 10 mM stock)
25 mM MgCl (50 µl of 1 M stock)
500 µg/ml peptide substrate (100 µl of 10 mg/ml stock =10 mM)
gamma-32P-ATP, 100-150 µCi
bring to 2 ml with ddH2O

"STOP" solution: to terminate the assay reaction:

0.1 M ATP, 0.1 M EDTA, pH approx. 8.

 
Assay:

Typical reaction: 20 mM HEPES, pH 7.4, 1-2 mM DTT, 5 mM MgCl2, 100 µM ATP, ~1 µCi gamma-32P-ATP, 100 µg/ml peptide substrate (~100 µM), 140 µM / 3.8 µM phosphatidylserine/diacylglycerol membranes, 100 µM Ca2+ (or 500 µM EGTA).

Total reaction volume is 80 µl.

1) mix, in 12 x 75 mm polystyrene tubes:

Activating conditions
Non-activating conditions
8 µl 10 x lipid
OR
20 mM HEPES
8 µl 1 mM CaCl2
OR
5 mM EGTA

 

 



and 48 µl enzyme sample, diluted in HEPES/DTT (volume = 64 µl).

2) To initiate the reaction, add 16 µl "5x GO solution", for an end volume of 80 µl. Vortex after each addition.

3) The reaction should proceed at 30oC for 5 to 10 min (initial rate).

4) After the desired time, terminate the reaction by adding 25 µL "STOP solution". Vortex after each addition. The resulting volume is now 105 µl.

5) Spot 85 µl of each reaction onto a 1 1/2 by 3/4 inch rectangle of Whatman P81 cellulose phosphate filter.  (See Photo 1 below)

6) Wash the P81 filters four times with 500 ml 0.4% phosphoric acid (5 - 10 min. per wash). Unreacted [32P] ATP is removed during these washes, and thus the liquid must be disposed of as radioactive waste. Blank filters can be washed alongside to monitor background. (See Photo 2 below)

7) Wash filters once with 500 ml 95% EtOH, for 2-5 min.

8) Put filters into scintillation vials, and measure the 32P-incorporation by scintillation counting. The specific activity of the ATP (cpm per nmol) can be determined by spotting a small sample of the "5X GO solution" onto a P81 paper, and counting directly (i.e. no washes).

9) Units of protein kinase C activity, defined as nmol phosphate transferred per min, are calculated as follows:

The activity, in UNITS of nmol/min is:

= (cpm on paper) x (105 µl total /85 µl spotted) 
(assay time, min) (specific activity of GO, cpm/nmol)
 

Photo 1: Whatman papers pinned onto styrofoam for ease of spotting assay samples.

 

Photo 2: Wire cage for washing spotted papers, with stir bar beneath.