Monitoring Protein Kinase A Activities Using Expressed Substrate in Live Cells

Jing Wang and X. Johne Liu

Summary

Protein kinase A (PKA) activity is regulated by intracellular cyclic adenosine monophosphate. Conventional protein kinase assays after cell lysis are hence not suitable for analyzing PKA activities. In this chapter, we describe a new method for monitoring PKA activity in live cells. A triparti substrate for PKA (Myr-HA-P2AR-C) is constructed that contains an N-terminal myristylation sequence followed by an antigenic hemagglutinin epitope tag and a substrate motif (the C-terminal tail of human P2 adrenergic receptor). The PKA phosphorylation status of the substrate in frog oocytes is determined either by two-dimensional electrophoresis followed by HA epitope immunoblotting or by direct SDS-PAGE followed by immunoblotting using anti-P-P2 adrenergic receptor antibodies specifically recognizing the PKA-phosphorylated C-terminus. We also describe the application of this strategy in mammalian somatic cells through DNA transfection. Myr-HA-P2AR-C should be widely adaptable as an in vivo PKA activity indicator.

Key Words: Antibodies; cAMP; expressed substrate; PKA activity indicator; protein kinase A; 2D gel electrophoresis.

1. Introduction

Protein kinase A (PKA), also known as cyclic adenosine monophosphate (cAMP)-dependent protein kinase, is a tetrameric complex consisting of two regulatory sub-units (PKAr) and two catalytic subunits (PKAc). In the absence of cAMP, the holoenzyme exists in its tetrameric form and is inactive. Upon the binding of cAMP to the regulatory subunits, the two catalytic subunits are released from the holoenzyme and become active (1). Because PKA activity is regulated by intracellular cAMP concentrations, any in vitro protein kinase assays after cell lysis would not accurately represent intracellular PKA activity.

To resolve this problem, several groups have developed protein substrates as PKA activity indicators in live cells. Nagai et al. (2) constructed a fluorescence-based expressed substrate to measure PKA activity in live cells. The substrate contains two

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

green fluorescence protein (GFP) variants of different excitation/emission spectra, linked by a PKA substrate motif. In the absence of PKA phosphorylation (or activation of PKA), the two GFP variants are in close contact and hence produce a phenomenon called fluorescence resonance energy transfer (FRET), which can be measured optically by sophisticated microscopy. When PKA is activated, the substrate motif becomes phosphorylated and assumes a more relaxed conformation; therefore, the two GFP variants become further apart, hence reducing FRET. The major drawback of this strategy is the very modest FRET change because of the intrinsic background FRET (in the absence of PKA activation) and the overlapping fluorescence spectra of the two GFP variants. This will seriously limit its application in the whole animal.

A similar FRET-based substrate (AKAR for A-kinase activity reporter) by Zhang et al. (3) improves the activation/background ratio. However, neither withdrawing dibutylryl (db)-cAMP from the culture medium nor the inclusion of PKA inhibitor (H-89) reverses the FRET change induced by db-cAMP, raising the possibility that the substrate motif within A-kinase activity reporter may be phosphorylated by other protein kinases in addition to PKA in live cells.

A third PKA indicator has been developed by Zarrine-Afsar and Krylov (4), who employed capillary electrophoresis and an expressed PKA substrate to monitor intra-cellular activation of protein kinase A. However, based on the evidence presented in their article, their substrate lacks the specificity and therefore is unlikely to be an acceptable indicator.

To circumvent these problems, we developed a novel assay to monitor endogenous PKA activity in live cells (5). The rationale of this assay is to introduce a specific PKA substrate into live cells with a PKA phosphorylation status that can be analyzed following cell lysis. We constructed a triparti substrate for PKA, Myr-HA-P2AR-C, with an N-terminal myristylation sequence derived from the c-Src protein (6), followed by a hemagglutinin (HA) epitope and the PKA substrate motif. We chose the C-terminal tail of human p2-adrenergic receptor (P2AR) as the PKA substrate (7). This construct, Myr-HA-P2AR-C, was transcribed in vitro, and the resulting messenger RNA(mRNA) was injected into frog oocytes. The phosphorylation status of Myr-HA-P2AR-C protein, and hence the intracellular PKA activity, was determined by two-dimensional (2D) gel electrophoresis followed by anti-HA immunoblotting. Alternatively, the PKA phosphorylation of the substrate can be determined by direct immunoblotting with antibodies recognizing the PKA-phosphorylated C-terminus of P2AR. In this chapter, we describe the application of this strategy for analyzing PKA activities in both frog oocytes and mammalian somatic cells.

2. Materials

1. Myr-HA-P2AR-C complementary DNA (cDNA) construct: the construction of this expression plasmid has been described in detail (5).

2. mMessageMachine kit with Sp6 polymerase (Ambion, Texas).

3. OR2: first prepare and autoclave Ca2+-free OR2: 83 mMNaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES at pH 7.8. To Ca2+-free OR2, add CaCl2 to 1 mM and gentamicin to 100 |lg/mL. All solutions are stored at room temperature. Ca2+-free OR2 is good for weeks, whereas OR2 is prepared within the week.

4. Extraction buffer: 20 mM HEPES, pH 7.2, 50 mM glycerophosphate, 2.5 mM MgCl2, 0.25 M sucrose, 0.1 M NaCl, 1% Triton X-100; store at 4°C. Before use, the following protease/phosphatase/kinase inhibitors are added:

a. Phenylmethylsulfonyl fluoride, dissolved in dimethyl sulfoxide (DMSO; 200 mM) and frozen in 20-|lL aliquots; 1/1000 dilution in ice-cold extraction buffer.

b. Leupeptin, dissolved in water (10 mg/mL) and frozen in 20-|L aliquots; 1/1000 dilution.

c. Sodium orthovanadate, dissolved in water (1 mM) and frozen in 20-|L aliquots; 1/1000.

d. H89 (Calbiochem), dissolved in DMSO (10 mM) and frozen in 20-|L aliquots; 1/1000 dilution.

e. Okadaic acid (Sigma), dissolved in DMSO (100 ||M) and frozen in 20-|L aliquots; 1/100 dilution.

5. Mini-protein II Tube Module for first-dimension gel electrophoresis (Bio-Rad).

6. 2X First-dimension sample buffer: 8 M urea, 2% Triton X-100, 5% P-mercaptoethanol, 1.6% 5/7 ampholyte, and 0.4% 3/10 ampholyte (Bio-Rad). This buffer is mixed with an equal volume of extracts before loading on the tube gels.

7. First-dimension overlay buffer: 4 M urea, 0.8% Bio-Lyte 5/7 ampholyte, 0.2% Bio-Lyte 3/10 ampholyte, and 0.0025% bromophenol blue.

8. 2X SDS sample buffer: 125mMTris-HCl, pH 6.8, 20% glycerol, 4.1% SDS, 0.005% bromo-phenol blue, 10% P-mercaptoethanol.

9. Anti-p-P2AR (S345, S346) antibody (Santa Cruz).

10. Complete Dulbecco's modified Eagle's medium (DMEM): DMEM (Invitrogen) containing fetal bovine serum (FBS; 10%), penicillin, and streptomycin (50 U/mL) and fungizone (0.05 |g/mL).

11. Serum-free DMEM: complete DMEM minus FBS.

12. LipofectAMINE 2000 (Invitrogen).

13. Opti-MEM (Invitrogen).

14. db-cAMP (Sigma) dissolved in water (150 mM) and frozen in 50-|L aliquots; 1/100 dilution in serum-free DMEM for activation of PKA in live cells.

15. Phosphate-buffered saline, 10 mM phosphate buffer, pH 7.5, 150 mM NaCl; autoclave and store at room temperature.

3. Methods

The methods described next outline: (1) expression of Myr-HA-P2AR-C in frog oocytes by mRNA injection and analyses of PKA phosphorylation by 2D electrophoresis; (2) expression of Myr-HA-P2AR-C in a mammalian somatic cell line (COS7) and analyses of PKA phosphorylation; (3) sample preparation for direct immunoblotting using anti-p-P2AR (S345, S346).

3.1. Analyzing PKA Activities in Frog Oocytes

We isolate plasmid DNA using Qiagen's Midi DNA columns. In vitro transcription (total volume 20 |L) is carried out using Ambion's mMessagemMachine kit, with 2 to 3 |g of linearized DNA per reaction. Final mRNA preparation is dissolved in 20 |L water and stored at -70°C in 3- to 5-|L aliquots (see Note 1). We inject 10 to 20 nL mRNA into the cytoplasm of each stage VI oocyte (manually defolliculated; see Chapter 3, this volume). Injected oocytes are incubated for at least overnight at 18 to 20°C

in OR2 before subjecting to any drug treatment. The following describes sample preparation and 2D analyses.

1. Place 10 to 20 oocytes in an Eppendorf tube and remove excess OR2.

2. Pipet the desired amount (2 |L/oocyte) of ice-cold extraction buffer; lyse oocytes by forcing them through a yellow pipet tip (a few times are sufficient) in the extraction buffer (Subheading 2., item 4, with all the supplements).

3. Centrifuge extracts for 15 min at 13,500 cpm (in a refrigerated Eppendorf centrifuge), transfer the supernatant to a clean tube.

4. Mix the clarified supernatant with an equal volume of 2X first-dimension sample buffer.

5. Incubate the mixture at room temperature for 10 to 15 min before loading 22 |L of each sample onto individual minitube gels (see Note 2).

6. Carefully place 30 |L of first-dimension overlay buffer on top of the sample. This buffer provides a cushion between the sample and the electrolyte (100 mM NaOH).

7. After filling the lower tank with 10 mM H3PO4, the tube gel adaptor is placed into the tank. Fill the chamber of the tube adaptor with 100 mM NaOH. Remove bubbles from the lower end of the tube gels with a bent needle attached to a syringe.

8. Carry out first-dimension electrophoresis at 750 V for 3.5 h.

9. Extrude the tube gel with the gel ejector and load the tube gel onto a mini-SDS (15%) polyacrylamide gel made with a "toothless" comb (see Note 3).

10. Overlay the tube gel with 1X SDS sample buffer (2X SDS sample buffer diluted 1/1 with water) sufficient to submerge the tube gel. Wait for 10 min before filling with SDS-PAGE running buffer and proceed to 2D run.

11. SDS-PAGE, transfer, and immunoblotting with antibodies against HA are carried out according to standard procedures (see Note 4).

Under these conditions, Myr-HA-P2AR-C appears as a single spot designated as spot 2 (Fig. 1A). Following 1-h incubation with progesterone, spot 2 diminishes, and correspondingly, a new spot (spot 1) becomes the predominant form (Fig. 1B). Spot 1 and spot 2 differ only in their isoelectrical points, with spot 2 more acidic and hence more phosphorylated. An unknown endogenous protein (indicated by an asterisk) that is recognized by anti-HA antibodies and that does not undergo progesterone-induced changes in its migration pattern serves as a convenient internal marker. To confirm that spot 2 represents the PKA-phosphorylated form of the substrate, we carry out similar analyses that indicate that Myr-HA-P2AR-C/PKA- (7), which lacks PKA phosphorylation sites, always appears as a single spot, spot 1 (Fig. 1C,D).

3.2. Analyzing PKA Activities in COS7 Cells

1. Cell culture is carried out using standard procedures.

2. Transfection is carried out in 6-cm plates when cells are 90 to 95% confluent.

4. Dilute 20 | L of LipofectAMINE 2000 reagent in 500-| L Opti-MEM. Mix gently and incubate for 5 min at room temperature.

5. After 5-min incubation, combine the diluted DNA with the diluted LipofectAMINE 2000. Mix gently and incubate for 20 min at room temperature to allow the DNA-Lipofect-AMINE 2000 complexes to form.

6. Add the 1000-|L DNA-LipofectAMINE 2000 complexes to each 6-cm plate containing cells and medium. Swirl gently to make sure the cells are covered with the solution.

Fig. 1. Two-dimensional analyses of Myr-HA-P2AR-C phosphorylation in frog oocytes. Oocytes are injected with mRNAs for wild-type Myr-HA-P2AR-C (A,B) or Myr-HA-P2AR-C/PKA- (C,D). After 24-h incubation, oocytes in (B,D) are treated with 1 |lM progesterone for 1 h. Then, the oocytes from all groups are lysed and analyzed by 2D gel electrophoresis followed by HA immunoblotting. The asterisk indicates the nonspecific oocyte protein (see text) that serves as an internal marker for the positions of spot 1 and spot 2.

Fig. 1. Two-dimensional analyses of Myr-HA-P2AR-C phosphorylation in frog oocytes. Oocytes are injected with mRNAs for wild-type Myr-HA-P2AR-C (A,B) or Myr-HA-P2AR-C/PKA- (C,D). After 24-h incubation, oocytes in (B,D) are treated with 1 |lM progesterone for 1 h. Then, the oocytes from all groups are lysed and analyzed by 2D gel electrophoresis followed by HA immunoblotting. The asterisk indicates the nonspecific oocyte protein (see text) that serves as an internal marker for the positions of spot 1 and spot 2.

7. Incubate cells at 37°C incubator for 6 h, replace with 5 mL complete DMEM.

8. After 24 h, rinse cells with phosphate-buffered saline; follow with the addition of serumfree DMEM and further incubation for 24 h (see Note 5).

9. Add db-cAMP (1.5 mM final) to the serum-free medium and return to incubator for an extra 40 min.

10. Aspirate medium, lyse cells by scraping in 1X first-dimension sample buffer (containing 200 ||M phenylmethylsulfonyl fluoride, 10 |g/mL leupeptin; 50 ||L) on ice.

11. Clarify cell extracts and analyze PKA phosphorylation exactly as described (Subheading 3.1., steps 5-9).

The results shown in Fig. 2A indicate that, after serum deprivation for 24 h, Myr-HA-P2AR-C appears as two spots of more or less equal intensity (Fig. 2A), corresponding to spot 1 and spot 2, respectively, in frog oocytes (Fig. 1). Brief incubation of these cells with db-cAMP resulted in disappearance of spot 1 such that Myr-HA-P2AR-C migrated as a single spot (spot 2). In contrast, Myr-HA-P2AR-C/PKA-migrated as a single spot 1 under any conditions (Fig. 2B).

3.3. Analyzing PKA Phosphorylation by Immunoblotting With Phosphor-Specific Antibodies

Myr-HA-P2AR-C contains multiple phosphorylation sites catalyzed by G proteincoupled receptor kinases, in addition to the known PKA phosphorylation site (RRSS346) (7,8). Phosphorylation of Myr-HA-P2AR-C by G protein-coupled receptor kinases or other protein kinases will complicate 2D gel analyses, as is the case in frog oocytes that have undergone maturation (5).

db-cAMP

*

1 2

1 2

1 *

o

OH"

OH"

1 2

1 2

Fig. 2. Two-dimensional analyses of Myr-HA-P2AR-C phosphorylation in COS7 cells. COS7 cells are transfected with (A) Myr-HA-P2AR-C or (B) Myr-HA-P2AR-C/PKA-. The transfected cells are starved for 24 h in serum-free DMEM, followed by treatment with 1.5 mM db-cAMP for 40 min. Cells are then directly lysed into first-dimension sample buffer and are analyzed as in Fig. 1.

To circumvent this problem, we have sought to develop phosphor-specific antibodies against the PKA-phosphorylated P2AR C-terminus. Fortunately, Santa Cruz has recently made available PKA phosphorylation site-specific antibody (anti-p-P2AR; S345, S346). Although our mutagenesis studies have indicated that Myr-HA-P2AR-C is mostly likely phosphorylated by PKA on a single serine residue (S346) (5), the Santa Cruz antibodies clearly and specifically recognized PKA-phosphorylated Myr-HA-P2AR-C from both frog oocytes and COS7 cells. The following describe procedures for sample preparation for direct immunoblotting with anti-p-P2AR (S345, S346).

To prepare oocyte samples:

1. Follow Subheading 3.1., steps 1 to 3 (see Note 6).

2. Mix clarified supernatant with an equal volume of 2X SDS sample buffer.

4. Load 10 ||L sample on a well in a 15% SDS polyacrylamide gel (see Note 7).

5. We normally run duplicate gels, each blotted with anti-HA (as expression/loading controls) and anti-p-P2AR (S345, S346).

To prepare COS7 cell samples:

1. Aspirate serum-free DMEM from the plate.

2. Add 100 |L SDS sample buffer (2X is fine) directly to a 6-cm plate and lyse the cells by scraping.

Fig. 3. Analyzing Myr-HA-P2AR-C phosphorylation by anti-p-P2AR (S345, S346). (A) Oocytes are injected with mRNA for Myr-HA-P2AR-C (lanes 2-5) or Myr-HA-P2AR-C/PKA-(lane 1). After 24 h incubation, oocytes are treated with 1 |lM progesterone for 1 h (lane 4). Oocytes in lane 5 have received a second injection of PKAc (0.8 U per oocyte) immediately before the addition of progesterone. All groups of oocytes were lysed and analyzed by SDS-PAGE, followed by immunoblotting using anti-HA and anti-p-P2AR (S345, S346). (B) COS7 cells are transfected with Myr-HA-P2AR-C and treated with or without db-cAMP (as described in Fig. 2). COS7 cell extracts are analyzed by SDS-PAGE, followed by immunoblotting using anti-HA and anti-p-P2AR (S345, S346).

Fig. 3. Analyzing Myr-HA-P2AR-C phosphorylation by anti-p-P2AR (S345, S346). (A) Oocytes are injected with mRNA for Myr-HA-P2AR-C (lanes 2-5) or Myr-HA-P2AR-C/PKA-(lane 1). After 24 h incubation, oocytes are treated with 1 |lM progesterone for 1 h (lane 4). Oocytes in lane 5 have received a second injection of PKAc (0.8 U per oocyte) immediately before the addition of progesterone. All groups of oocytes were lysed and analyzed by SDS-PAGE, followed by immunoblotting using anti-HA and anti-p-P2AR (S345, S346). (B) COS7 cells are transfected with Myr-HA-P2AR-C and treated with or without db-cAMP (as described in Fig. 2). COS7 cell extracts are analyzed by SDS-PAGE, followed by immunoblotting using anti-HA and anti-p-P2AR (S345, S346).

3. Transfer the lysate to Eppendorf tubes. To reduce viscosity (caused by the presence of chromosomal DNA), pass the lysates through a 27-gage needle attached to a 1-mL syringe. Be careful not to "overdraw" the plunger or you will have difficulties recovering the samples.

4. Heat the samples for 5 to 10 min at 85 to 100°C.

5. Load 25 to 30 | L of samples onto each well.

Figure 3A (lanes 2,3) shows that Myr-HA-P2AR-C expressed in G2 oocytes is prominently recognized by anti-p-P2AR (S345, S346), indicating that these oocytes contain activated PKA. Extracts derived from progesterone-treated oocytes have lost this band (lane 4) despite the presence of Myr-HA-P2AR-C protein, as indicated by HA immunoblotting. A prior injection of catalytic PKA (PKAc) prevents the loss of anti-p-P2AR (S345, S346) recognition (lane 5). In contrast, a mutant form of Myr-HA-P2AR-C that lacks the PKA phosphorylation site (PKA-) is never recognized by anti-p-p2AR (S345, S346) (lane 1 and data not shown). These results clearly indicate that Myr-HA-P2AR-C serves as a specific indicator of PKA activities in frog oocytes. Similarly, in COS7 cells, Myr-HA-P2AR-C can also function as an in vivo indicator of PKA activities (Fig. 3B).

4. Notes

1. We normally analyze 0.5 ||L of the mRNA sample on a 1% agarose gel to determine the quality of the mRNA (single band). Occasionally, we estimate mRNA quantities by comparing to mRNA standards of known concentrations. The 20-| L transcription reaction usually produces approx 20 | g of mRNA.

2. The tube gels (8.5-cm tube length with 1.3-mm inner diameter) can be prepared according to the Bio-Rad protocol provided with the Mini-Protein II Tube Gel Module a day earlier or on the same day (minimum polymerization time 30-min).

3. We often place two tube gels, both trimmed to reduce the length, head to tail on the same SDS-PAGE gel. This trick cuts the number of SDS-PAGE gels in half (an example is shown in Fig. 1C,D).

4. We use Amersham's ECL™ Western blotting detection reagents to develop immunoblots. We often need to expose the X-ray film for a few hours to see significant signals.

5. It is essential that cells are serum starved for at least 24 h. Cells that are continuously cultured in serum-containing medium contain only the PKA-phosphorylated form of the substrate, presumably because serum contains PKA-activating agents (such as growth factors).

6. It is not advisable to lyse oocytes directly in SDS sample buffer because the presence of large amounts of yolk proteins will adversely affect resolution of cellular proteins. In fact, we advise not to use any buffers that contain Tris base; in our experience, Tris base alone will extract yolk proteins and make it difficult to run "good" gels. The extraction buffer (Subheading 2., item 4 with all the supplements, 5 |L/oocyte) was used to prepare sample for direct SDS-PAGE.

7. Myr-HA-P2AR-C migrates on SDS-PAGE as less than 20 K relative molecular mass, so be careful not to run the protein off the gels. We normally run the gels until the dye front is 5 mm from the bottom of the gels.

Acknowledgments

We thank Robert J. Lefkowitz for providing the full-length cDNA constructs containing wild-type human P2AR and its mutant lacking PKA phosphorylation sites (S345AS346A). We also thank Veronique Montplaisir for performing experiments depicted in Fig. 3B. Work in the lab of X. J. L. is supported by operating grants from Canadian Institute of Health Research (CIHR) and Natural Sciences and Engineering Research Council (NSERC). J. W. is the recipient of a CIHR doctoral research award, and X. J. L. is the recipient of a Premier's Research Excellence Award.

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