Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme

Materials and Reagents

Note: Unless specified, all reagents are stored at room temperature (RT, 15-25 °C).

1. P81 Whatman cellulose chromatography paper (Sigma-Aldrich, catalog number: Z753645 ), in 2 x 2 cm pieces
   Note: There should be as many squares as conditions, plus 9 extra squares for controls, all in triplicates. All pieces should be labeled with a pencil to differentiate them after washing.
2. 7 ml Copolymer Plastic Vial, Unlined White Poly Screw Cap (RPI, catalog number: 125509 )
3. Eppendorf tube (1.5 ml and 2 ml)
4. 0.45 µm filter
5. Male mouse (8-20 weeks old)
6. 70% ethanol
7. Tris(hydroxymethyl)aminomethane (Tris base) (Cicarelli, catalog number: 1131214 )
8. Triton X-100 (Neo Lab, catalog number: 0 1685 )
9. NaCl (Cicarelli, catalog number: 750 )
10. MgCl₂ (Sigma-Aldrich, catalog number: M8266 )
11. Adenosine 5′-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A7699 ), store at -20 °C
12. Sodium orthovanadate (Na₃VO₄) (Sigma-Aldrich, catalog number: 450243 ), store at -20 °C
13. β-Glycerophosphate disodium salt hydrate (G3P) (Sigma-Aldrich, catalog number: G6251 ), store at -20 °C
14. Para-Nitrophenyl Phosphate (NPP) (Cayman chemicals, catalog number: 400090 )
15. cOmplete^TM, EDTA-free Protease Inhibitor Cocktail (Protease Cocktail) (Roche, catalog number: 4693132001 ), store at 4 °C
16. Bovine Serum Albumin fatty-acid free (BSA) (Sigma-Aldrich, catalog number: A7906 ), store at 4 °C
17. Adenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (cAMP) (Sigma-Aldrich, catalog number: 3A6885 ), alternatively, 8-BrcAMP or db-cAMP can be used, store at -20 °C
18. 3-Isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 ), store at -20 °C
19. Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) (Sigma-Aldrich, catalog number: K1127 ), store at -20 °C
20. HEPES (Sigma-Aldrich, catalog number: H3375 )
21. Trichloroacetic acid (TCA) (BioChemica, catalog number: PAA1431 )
22. ortho-Phosphoric acid 85% (Merck, catalog number: 100573 )
23. ATP (ATP-³²Pγ) 3000 Ci/mmol; 10 mCi/ml (Perkin Elmer, catalog number: BLU502A )
24. Specific drugs to be tested in the experiment (inhibitors, agonists, etc.)
25. KCl (Sigma-Aldrich, catalog number: S-7653 )
26. Na-Pyruvate (Sigma-Aldrich, catalog number: P-4562 )
27. CaCl₂·2H₂O (Sigma-Aldrich, catalog number: C-7902 )
28. KH₂PO₄ (Sigma-Aldrich, catalog number: P-5655 )
29. MgSO₄·7H₂O (Sigma-Aldrich, catalog number: 63138 )
30. Glucose (Sigma-Aldrich, catalog number: G-6152 )
31. DMSO (Sigma-Aldrich, catalog number: D2650 )

Solutions
For detailed instructions on how to prepare the following solutions, refer to the Recipes heading:

Equipment

1. Scintillation Counter (LKB Wallac Rackbeta 1209, catalog number: 0 90161 )
2. Neubauer chamber
3. Surgery scissors (WPI, catalog number: 14393 )
4. Tweezers
5. Orbital shake
6. Incubator
7. Centrifuge
   

Software

1. Microsoft^® Office Excel (Microsoft Office 365), or similar

Procedure

1. Prepare Lysis buffer (Recipe 2) on the day of use. Leave the protease inhibitor cocktail to be added just before usage. Keep buffer on ice.
   
   
2. Calculate the volume of Kinase buffer that will be needed, considering 10 µl per reaction tube, and taking into account that each condition (and controls) will be performed in triplicates. While prepare Kinase buffer (Recipe 4) adding all components but Kemptide, protease inhibitors, and ATP-³²Pγ, which will be added before use.
   
   
3. Isolate the insoluble PKA fraction from mouse sperm extracts
   Note: This section describes how to obtain PKA from the Triton-insoluble fraction of mouse sperm extracts, to be used as source of PKA in the activity assay.
   1. Euthanize 1 male mouse (8-20 weeks old) and perform surgery to extract the cauda regions from both epididymides (Figure 1). To do this:
      1. Lay the euthanized mouse dorsally on a dissection board.
      2. Spray the ventral area with 70% ethanol to prevent dry hair from detaching
      3. Using scissors, incise the skin and muscle tissue from the abdominal wall, exposing the lower abdominal viscera (Figure 1A).
      4. Using forceps, remove the fat pads that cover the testis to get the epididymis exposed (Figure 1A).
      5. Identify the epididymis attached to the testis, excise it, and localize the cauda region (Figure 1B, red area). Remove it from the caput and corpus regions (Figure 1B grey area), and adipose tissue (Figure 1B blue area).
      6. While holding the cauda with forceps, perform 3 to 4 incisions, using surgery scissors (Figure 1C).
   2. Place both epididymides in a round-bottom 2 ml Eppendorf containing 500 µl of H-TYH medium (Figure 1D) and incubate for 15 min at 37 °C.
      
      
      Figure 1. Sperm preparation. A. Ventral incision to excise the epididymis. Note the tweezers holding the cauda epididymis, attached to the testicle (red circled). B. Whole epididymis excised. Red, cauda; grey, caput and corpus; blue, adipose tissue. The cauda region is used for sperm swim out. C. Three to four incisions should be performed in each cauda, before allowing the sperm to swim out. D. Place both epididymides in a round-bottom Eppendorf tube containing 500 µl of H-TYH medium.
      
      
   3. Transfer the sperm suspension (500 µl) to a new round-bottom 2 ml Eppendorf tube, leaving the epididymides behind.
   4. Check sperm concentration by counting in a Neubauer chamber. Adjust concentration to 25 x 10⁶ sperm/ml in H-TYH medium.
   5. Transfer the swim-out to a 1.5 ml Eppendorf tube.
   6. Centrifuge the sperm suspension at 10,000 x g for 3 min at RT
   7. Discard supernatant. If the amount of swim out was of 25 x 10⁶ sperm in 1 ml, resuspend the pellet in 330 µl of ice-cooled Lysis buffer supplemented with protease inhibitor cocktail before use. If a different amount of sperm is used, modify buffer addition accordingly.
   8. Incubate cells in Lysis buffer on ice for 30 min
   9. Centrifuge cells at 10,000 x g for 10 min at 4 °C.
   10. Discard supernatant and resuspend the pellet in 330 µl of Lysis buffer (or same amount used in Step C7), with freshly added protease inhibitor cocktail. This fraction contains PKA. Each condition with 10 µl of fraction will have the soluble PKA fraction extracted from 7.5 x 10⁵ sperm. Consider that a minimum of 1.5 x 10⁵ sperm are needed for each condition, which should be tested in triplicate.
   11. Keep on ice for PKA activity assays. From now on this fraction is called PKA fraction.
       
       
       
4. Kinase reaction assay
   1. Consider as many tubes as needed, keeping in mind that each condition should be performed in experimental triplicates, plus the following controls:
      
      1. Negative control: it contains all the reaction reagents except for PKA fraction, which is replaced by Lysis buffer. It is used to determine the amount of Kemptide that is marked by ³²P in the absence of PKA.
      2. Positive control: it is a control of maximum PKA activity, i.e., in the presence of 1 mM cAMP and 100 µM IBMX.
   2. Add 10 µl of Lysis buffer containing any drug to be analyzed. Consider that it will be diluted 3x at the time of the reaction. Whatever drug to be tested must be considered within the 10 µl corresponding to the addition of this 10 µl of Lysis buffer.
   3. Add to each tube 10 µl of PKA fraction, except for the Negative control, in which PKA fraction should be replaced by 10 µl of Lysis buffer.
   4. Keep all tubes on ice until used.
   
   From Step D5 onwards the procedures must be performed in the radioisotope room. Please note that security measures must be taken when working with radioactive material. Proper shielding for the personnel is required, as well as adequate waste disposal according to institutional regulations.
   5. Complete preparation of the Kinase buffer by adding the proper amount of ATP-³²Pγ (see Table 1), taking into consideration of the decay time and manufacture date, then add 1 µCi/assay.
      
      Table 1. Stock solutions and concentration for the preparation of Kinase buffer
      
      Notes:
      
      1. All reagents are dissolved in distilled water, unless specified otherwise.
      2. The protease inhibitor cocktail is added as to prepare a 2x concentrated buffer, since the PKA fraction already contains cocktail.
      3. Addition of BSA is done to help precipitate all proteins after TCA addition, but keeping the Kemptide soluble.
         
         
   6. Sequentially add 10 µl of Kinase buffer to all tubes and place them at 37 °C. Start a new reaction every 30 s by adding 10 µl of Kinase buffer. Keep track of the sequential order of all tubes. This is crucial in order to stop the reactions exactly at the specified time.
   7. Incubate tubes at 37 °C for 30 min.
   8. Stop the reaction after 30 min by addition of 10 µl 40% TCA. Note that the total volume in each tube now is 40 µl.
   9. Incubate tubes on ice for 20 min.
   10. Centrifuge at 10,000 x g for 3 min, at RT.
   11. For each tube, take 10 µl of the supernatant, and spot onto the center of a P81 piece of paper, while holding it with tweezers (Figures 2A and 2B). Each piece of P81 paper should be labeled with pencil in order to identify them later. Wait 5 s for the drop to be absorbed, and release it in a 5 L beaker containing 1 L of 5 mM ortho-phosphoric acid (Figure 2C).
   12. Wash together all papers 6 x 5 min using 1 L of 5 mM ortho-phosphoric acid each time, in a beaker placed on an orbital shaker.
   13. While the washing in Step D12 proceeds, spot 1 µl of Kinase buffer on a P81 paper. Do this in triplicate using 3 different papers, and then directly insert each one in a different plastic vial, without washing. This step is necessary for data analysis to calculate the total amount of total ATP-³²Pγ, initially present in the reaction media.
   14. After washing, place the P81 papers on a plastic tray, without contact between each other, and let them air dry for at least 2 h (Figure 2D). At this step, you can discontinue the protocol until the next day. Papers are identified according to the pencil mark written on them before spotting the reaction mixture.
   15. Once dried, place papers individually in a plastic vial, containing 1 ml of scintillation counter liquid, and register counts per minute (CPM) a vial counter.
       
       
       Figure 2. P81 paper spotting. A. Prepare 2 x 2 cm P81 papers labeled with pencil marks. B. Spot each paper with 10 µl the supernatant of the reaction mixture after centrifugation (Step D11). Notice that the washing is being performed behind the protecting shield in a radioisotopes room. C. Place one by one all the papers in the same beaker with 1 L of 5 mM ortho-phosphoric acid (Step D11). D. Let all pieces to air dry without touching each other.

Data analysis

On an Excel file, write down counts per minute (CPM) values of each condition, obtained in step D15 (Table 2). These measures correspond to the 10 µl of the supernatant of the reaction mixture spotted on P81 paper.

Table 2. In vitro PKA kinase assay data analysis. Schematic view of steps A-H for data processing.

1. Calculate the mean value (mean) for each condition considering the experimental triplicates.
2. Obtain the corrected mean by subtracting the negative control mean from all means, except for the Kinase buffer mean.
3. Adjust the mean CPM value of each condition to the total volume of the assay (40 µl) by multiplying the corrected mean by 4, since you put 10 µl on the P81 papers out of 40 µl.
4. Use the Kinase buffer mean CPM to determine the total amount of ATP-³²Pγ present in each reaction tube. This will be used to address the amount of ³²P incorporated into the Kemptide. To do this, first multiply the mean CPM value of the Kinase buffer by the dilution factor (in this case it is a factor of 10 since you spotted 1 µl of Kinase buffer out of 10 µl present in the reaction).
5. To determine how many [CPM/ρmol ATP], noted as “b” in Table 2, are in the mixture, divide by the total ATP moles added (in this case the reaction mixture contained 800 pmol of ATP, noted as “a” in Table 2).
6. To determine how many ρmoles of ATP were incorporated in each condition, divide total CPM of each condition by [CPM/ρmol ATP] of the mixture.
7. To determine how many ρmoles of ATP were incorporated in each condition per minute, divide this last value by the time of the reaction. In this case divide by 30, since the reaction lasted 30 minutes.
8. Report this value considering the amount of sperm used for each condition. Please see Table 3 for an example of results.
   
   Table 3. Example of results. The table exemplifies results obtained using the PKA inhibitor H-89 (Sigma #B1427, diluted in DMSO), at the concentrations specified.
   Note: In this example, all tubes contained cAMP and IBMX (see Table 1).
   
   

Recipes

1. H-TYH medium
   General composition:
   119.3 mM NaCl
   4.7 mM KCl
   0.8 mM Na-Pyruvate
   1.71 mM CaCl₂
   1.2 mM KH₂PO₄
   1.2 mM MgSO₄
   5.4 mM glucose
   20 mM HEPES
   pH = 7.2-7.4
   To prepare 25 ml of buffer, weight:
   NaCl                  174.3 mg
   KCl                     8.76 mg
   CaCl₂·2H₂O      6.28 mg
   KH₂PO₄            4.08 mg
   MgSO₄·7H₂O   7.39 mg
   HEPES               119.15 mg
   
   1. Dissolve all reagents in Mili-Q water to a final volume of 25 ml
   2. Sterilize by filtration using a 0.45 µm filter and store at 4 °C up to 1 month
   3. On the day of use, add 2.20 mg Sodium Pyrutave and 24.78 mg Glucose
   4. Bring to a final pH of 7.2-7.4 with freshly prepared 5 N NaOH
2. Lysis buffer (prepare day of use)
   General composition:
   25 mM Tris pH 7.4
   150 mM NaCl
   1x Cocktail protease inhibitor (EDTA-free, SIGMA)
   1% Triton X-100
   To prepare 900 µl of buffer:
   1 M Tris, pH 7.4                                  22.5 µl
   3 M NaCl                                              45 µl
   25x Cocktail protease inhibitor        36 µl
   Triton X-100                                         9 µl
   Distilled water                                    787.5 µl
3. 100 mM IBMX
   Dissolve 22.22 mg in 1 ml of DMSO
4. Kinase buffer
   Prepare 3x Kinase buffer as it will be diluted 3 times by addition of 1 volume (10 µl) of PKA fraction plus 1 volume of buffer solution (10 µl), accounting for 30 µl of reaction tube (see Table 1). The following volumes are calculated to prepare the amount of buffer needed for 30 reaction tubes (10 different conditions in triplicates), considering that 10 µl of Kinase buffer is added to each tube.
   

Acknowledgments

The work was funded by Agencia Nacional de Promoción Científica y Tecnológica, PICT 2015-3164 and 2017-3217 awarded to DK. The heptapeptide known as Kemptide was synthesized for the first time by Kemp et al, to study the specificity of the PKA towards its substrate (Kemp et al., 1977). The adapted protocol described in that manuscript has used for the first time by Visconti et al. (1997)

Competing interests

The authors declare no competing interests.

Ethics

Cauda epididymides were collected from C57BL/6 young adult male mice (8-20 weeks old) and sacrificed under supervision of the Animal Care and Use Committee of the Facultad de Ciencias Bioquímicas y Farmacéuticas de Rosario (UNR) (approved protocol numbers 7298/532).

References

1. Akamine, P., Madhusudan, Wu, J., Xuong, N. H., Ten Eyck, L. F. and Taylor, S. S. (2003). Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure. J Mol Biol 327(1): 159-171.
2. Buffone, M. G., Wertheimer, E. V., Visconti, P. E. and Krapf, D. (2014). Central role of soluble adenylyl cyclase and cAMP in sperm physiology. Biochim Biophys Acta 1842(12 Pt B): 2610-2620.
3. Kemp, B. E., Graves, D. J., Benjamini, E. and Krebs, E. G. (1977). Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem 252(14): 4888-4894.
4. Krapf, D., Arcelay, E., Wertheimer, E. V., Sanjay, A., Pilder, S. H., Salicioni, A. M. and Visconti, P. E. (2010). Inhibition of Ser/Thr phosphatases induces capacitation-associated signaling in the presence of Src kinase inhibitors. J Biol Chem 285(11): 7977-7985.
5. Stival, C., Puga Molina Ldel, C., Paudel, B., Buffone, M. G., Visconti, P. E. and Krapf, D. (2016). Sperm capacitation and acrosome reaction in mammalian sperm. Adv Anat Embryol Cell Biol 220: 93-106.
6. Stival, C., Ritagliati, C., Xu, X., Gervasi, M. G., Luque, G. M., Baro Graf, C., De la Vega-Beltran, J. L., Torres, N., Darszon, A., Krapf, D., Buffone, M. G., Visconti, P. E. and Krapf, D. (2018). Disruption of protein kinase A localization induces acrosomal exocytosis in capacitated mouse sperm. J Biol Chem 293(24): 9435-9447.
7. Visconti, P. E., Moore, G. D., Bailey, J. L., Leclerc, P., Connors, S. A., Pan, D., Olds-Clarke, P. and Kopf, G. S. (1995). Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 121(4): 1139-1150.
8. Visconti, P. E., Johnson, L. R., Oyaski, M., Fornes, M., Moss, S. B., Gerton, G. L. and Kopf, G. S. (1997). Regulation, localization, and anchoring of protein kinase A subunits during mouse sperm capacitation. Dev Biol 192(2): 351-363.
9. Zhang, P., Smith-Nguyen, E. V., Keshwani, M. M., Deal, M. S., Kornev, A. P. and Taylor, S. S. (2012). Structure and allostery of the PKA RIIbeta tetrameric holoenzyme. Science 335(6069): 712-716.