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Originally published In Press as doi:10.1074/jbc.M410242200 on November 8, 2004

J. Biol. Chem., Vol. 280, Issue 4, 2750-2758, January 28, 2005
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Enhanced Dephosphorylation of cAMP-dependent Protein Kinase by Oxidation and Thiol Modification*

Kenneth M. Humphries, Michael S. Deal, and Susan S. Taylor{ddagger}

From the Howard Hughes Medical Institute, Department of Chemistry and Biochemistry and Department of Pharmacology, The University of California, San Diego, La Jolla, California 92093-0654

Received for publication, September 7, 2004 , and in revised form, October 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase C{alpha} activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
cAMP-dependent protein kinase (PKA)1 is a ubiquitously expressed signaling molecule. PKA is essential for numerous metabolic processes and has been implicated in a wide range of cellular activities (1). It is not surprising then that PKA activity is regulated on many levels (2). In the absence of cAMP, the kinase exists as an inactive tetramer comprised of two catalytic (C) and two regulatory (R) subunits. Upon cAMP binding to the R-subunits, the active C-subunit is released. The free C-subunit can in turn be inactivated by binding the heat-stable PKI (3). More recently it has been shown that in addition to the classical regulatory mechanisms, kinase activity is modulated by localization of the holoenzyme to subcellular locations via A-kinase anchoring proteins and by the presence of microdomains of high concentrations of cAMP (4, 5). Finally, the free C-subunit can be inactivated by oxidation of the highly reactive cysteine (Cys199) located in the activation loop (6). This cysteine is capable of forming a mixed disulfide with glutathione or an internal disulfide with Cys343 located in the C terminus, thereby inactivating the kinase in a redox-sensitive manner.

Many kinases, including other members of the AGC family to which PKA belongs, are also regulated themselves by phosphorylation (7). Although many protein kinases are activated by the transient phosphorylation of their activation loops, PKA is unusual in that it appears to be assembled in an active, fully phosphorylated state and kept inactive by the bound R-subunits (8–10). The C-subunit contains two major phosphorylation sites, one at Thr197 in the activation loop and the other at Ser338 near the C terminus (8). Phosphorylation of Thr197 is critical, as this phosphate in the activation loop is essential for coordinating the active conformation and for optimal enzymatic activity (2). Removal of the Thr197 phosphate has not been widely appreciated as a regulatory mechanism, as early studies demonstrated that it is not only constitutively phosphorylated, but it is also very resistant to phosphatase treatment (8, 11, 12). However, although the kinase is indeed unusually resistant to phosphatase-mediated inactivation, there is now evidence that a protein phosphatase-2A-like enzyme can catalyze the removal (13, 14). In addition, dephosphorylated kinase can be rephosphorylated by PDK1 (15–17), further suggesting a reversible mode of regulation.

We reported previously (6) that the C-subunit is sensitive to oxidation-mediated inactivation, and that this was because of the presence of a highly reactive cysteine (Cys199) in the activation loop. This nucleophilic residue, conserved among the AGC family of kinases (18), is part of the P + 1 loop that interacts with protein substrates. Modification of Cys199 inactivates the enzyme (6, 19–22), presumably because of steric hindrance. Although Cys199 is not involved with the catalytic mechanism, as demonstrated by the full activity of a Cys199 to alanine mutant, we wanted to determine whether this residue's proximity to phospho-Thr197 may be an important factor in keeping the kinase protected from phosphatase-mediated inactivation. We hypothesized that modification of this residue, such as through the formation of a protein-glutathione mixed disulfide, may alter the dynamics of the activation loop in such a manner that the phosphate on Thr197 is now presented more favorably as a substrate for dephosphorylation. We show here that modification of Cys199 by mutagenesis, oxidation, or alkylation, both in vitro and in vivo, makes the enzyme sensitive to dephosphorylation. Rephosphorylation by PDK1 creates a cycle whereby the inactive kinase can be reactivated (18).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All chemicals and reagents were obtained from Sigma Aldrich unless otherwise noted. Phosphatase {lambda} was purchased from Calbiochem. Antibodies against PKA C-subunit (3) and phospho-Ser338 were described previously.2 Antibody raised against the phosphothreonine in the C-subunit activation loop (24) was a generous gift of Dr. Alexandra Newton (University of California, San Diego). Constitutively active His-tagged PDK1 and PDK1 substrate peptide were a generous gift of Dr. C. C. King (University of California, San Diego).

PKA C-subunit Purification—The recombinant murine C-subunit of PKA was expressed and purified as described previously (25). Isoform III, containing 2 phosphates, was used for in all experiments. The Cys199 to alanine mutation was introduced as described by Kunkel et al. (26) and isoform III purified in the same manner as the wild-type C-subunit.

Oxidation and Alkylation of PKA C-subunit—Prior to oxidation and alkylation, it was necessary to remove 2-mercaptoethanol from the C-subunit environment. Dialysis (SpectraPor, 10,000 molecular weight cutoff) was performed overnight at 4 °C into a buffer containing 40 mM KCl and 10 mM KPO4, pH 7.2 (Buffer A). C-subunit containing a Cys199–Cys343 intramolecular disulfide was prepared as described previously (6). Briefly, wild-type C-subunit (0.3 mg/ml) was incubated for 10 min at 23 °C, pH 7.2, with 1.0 mM diamide. An aliquot of reaction mixture was then removed and analyzed for activity using the coupled assay system described by Cook et al. (27). Less than 10% of kinase activity remained under these experimental conditions. Glutathionylation of the C-subunit was carried out as described previously (6). Briefly, the C-subunit (0.3 mg/ml) was incubated for 15 min with 100 µM diamide and 125 µM GSH at 23 °C in Buffer A followed by measurement of kinase activity. Less than 10% of kinase activity remained under these experimental conditions. Alkylation of the kinase was performed by incubation of the kinase (0.3 mg/ml) with 250 µM N-ethylmaleimide (NEM) for 20 min at 23 °C in Buffer A followed by the addition of 5.0 mM DTT. Less than 5% of kinase activity remained following alkylation. Following all kinase treatments, samples were again dialyzed into Buffer A to remove excess reagents.

Dephosphorylation of PKA C-subunit and PDK1 Kinase Assay—The PKA C-subunit was diluted to 3.0 µg/ml in a buffer containing 50 mM NaCl, 50 mM Tris, pH 7.2, 0.1 mM EGTA, and 0.2 mM MnCl2. Phosphatase {lambda} was added to 300 ng of the C-subunit, and samples were incubated for the indicated times at 23 °C. Reactions were stopped by the addition of either 5.0 mM vanadate or 10 mM NaF. Kinase activity was then assayed using the PepTag nonradioactive PKA assay system (Promega) in the presence or absence of 20 mM DTT. For rephosphorylation, phosphatase reactions were stopped by the addition of 10 mM NaF, and then 10 mM MgCl2, 1.0 mM ATP, 20 mM DTT, and 50 ng of PDK1 were added as indicated. Samples were incubated for an additional 60 min at 23 °C and then diluted into an SDS-PAGE sample buffer containing 25 mM DTT and boiled for 10 min.

PDK1 activity was assayed by incubation of PDK1 (800 ng) in a buffer containing 20 mM HEPES, pH 7.5, 5.0 mM MgCl2, 0.1 mM ATP, 2 µCi of [32P]ATP, and 3.3 µg of PDK1 substrate peptide (SKQARANS-FVGTAQYVSRRKR). Reactions (80 µl) were carried out in the presence or absence of 20 mM DTT for 0, 5, and 10 min at 23 °C and then stopped by the addition of 25 µl of a solution containing 100 mM ATP and 100 mM EDTA, pH 8.0. Reaction samples (80 µl) were then spotted on p81 paper (Whatman), washed three times in 0.4% phosphoric acid, and analyzed by scintillation counting. The reaction rate was determined to be linear under these experimental conditions.

Inactivation of PKA by PC12 Cell Lysates—Rat neuroblastoma PC12 cells (ATCC, CRL-1721) were grown in Dulbecco's modified Eagle's medium supplemented with 6.5% fetal bovine serum (CellGro), 6.5% horse serum (Invitrogen), and containing penicillin and streptomycin. Cells were then washed with PBS, scraped and collected in 1.0 ml of PBS, and pelleted by spinning 5 min at 500 x g. Pellets were then lysed by resuspending the pellet in 200 µl of a buffer containing 150 mM NaCl, 50 mM Tris, pH 7.2, and 1.0% Nonidet P-40 (Buffer B). After incubating on ice for 10 min, the lysate was spun for 5 min at 9300 x g, and the soluble fraction was collected. Protein concentrations of cell lysates were determined using the Coomassie Blue protein assay reagent (Pierce) with bovine serum albumin as the standard. PKA C-subunit samples (3.0 µg/ml) were added to cell lysates (1.0 mg/ml), incubated for 45 min at 30 °C, and then analyzed by Western blot analysis.

NEM-mediated Inactivation and Dephosphorylation of PKA in PC12 Cells—Prior to treatment, cells (~75% confluent) were washed with PBS and then placed in serum-free Dulbecco's modified Eagle's medium. As indicated, PKA was activated by the addition of 250 µM 3-isobutyl-1-methylxanthine, 250 µM 8-bromo-cyclic AMP, and 25 µM forskolin to the cells, and then the cells were incubated for 10 min at 23 °C. NEM (0.5 mM) was then added to the cells for 30 min at 37 °C. Cells were then washed with PBS, scraped and collected in 1.0 ml of PBS, and pelleted by spinning 5 min at 500 x g. Cell pellets were then resuspended in 0.5 ml of PBS and spun again. Pellets were then lysed by resuspending the pellet in 200 µl of Buffer B containing 10 mM DTT. After incubating on ice for 10 min, the lysate was spun for 5 min at 9300 x g, and the soluble fraction was collected. Western blot analysis indicated nearly complete solubilization of PKA under these conditions. Protein concentrations of cell lysates were determined using the Coomassie Blue protein assay reagent (Pierce) with bovine serum albumin as the standard. cAMP-dependent protein kinase activity was determined using the PepTag nonradioactive PKA assay system (Promega) (28). For this assay, 7.0 µg of total cell lysate was added to the PepTag mixture containing 5.0 µM cAMP for 10 min, and then the reaction was stopped by the addition of the PKA-specific inhibitor PKI (50 µM final). All kinase activities were determined to be both cAMP-dependent and PKI-sensitive.

Western Blot Analysis—Western blot analysis of pure proteins (30 ng/well) or PC12 cell lysates (40 µg/well) was performed by separating the proteins by SDS-PAGE (10% polyacrylamide, NuPAGE gel (Invitrogen)). Proteins were then transferred to nitrocellulose (Bio-Rad), blocked 1 h at 23 °C with 5% milk in TTBS (Tris-bufferd saline containing 0.1% Tween 20), and then incubated for 1 h with polyclonal primary antibody. Blots were then washed 5 times for 5 min with TTBS and incubated with goat anti-rabbit horseradish peroxidase-conjugated antibody (Amersham Biosciences, 1:10,000 dilution) for 1 h. Following three washes for 5 min with TTBS, blots were incubated with Super-Signal West Pico chemiluminescent substrate (Pierce) for 5 min and then exposed to HyperFilm (Amersham Biosciences) for 0–5.0 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cysteine 199 Contributes to PKA Resistance to Dephosphorylation—The C-subunit of PKA contains an essential phosphorylation site on Thr197. This phosphorylation site, located in the activation loop and required for optimal kinase activity, is largely resistant to dephosphorylation. To demonstrate this resistance, wild-type recombinant C-subunit (300 ng) containing two phosphorylation sites, one at Thr197 and the other at Ser338, was incubated with phosphatase {lambda} (50 ng) for indicated times at 22 °C. As shown in Fig. 1A, phosphatase {lambda}, a recombinant phosphatase that indiscriminately acts upon phosphothreonine, phosphoserine, and phosphotyrosine residues, induces minimal kinase inactivation (20% ± 6.7%) under our experimental conditions. Proximal to phospho-Thr197 is a highly reactive cysteine (Cys199). We hypothesized that this nucleophilic cysteine may contribute to the stability of phospho-Thr197. Indeed, mutant kinase in which Cys199 was mutated to an alanine (C199A) was much more susceptible to phosphatase {lambda}-mediated inactivation (Fig. 1A). Under identical experimental conditions, 79% ± 2.5% of C199A kinase activity was lost after 45 min.



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  		FIG. 1.
Cysteine 199 plays a role in protecting the PKA catalytic subunit from dephosphorylation. A, either wild-type (WT) ({blacksquare}) or C199A mutant (•) PKA catalytic subunit (300 ng) was incubated with phosphatase {lambda} (50 ng) for the indicated times, and the reaction was stopped by the addition of 5.0 mM vanadate. Kinase activity was then assayed as described under "Experimental Procedures." Values are means ± S.E. (n = 3). B, following incubation with phosphatase {lambda}, samples were subjected to Western blot (WB) analysis under reducing conditions using antibodies specific for the PKA C-subunit, phospho-Thr197 (Thr197-P), and phospho-Ser338 (Ser338-P).

 
To confirm that the observed loss of activity was because of dephosphorylation of the kinase at Thr197, samples were subjected to Western blot analysis and probed with phospho-specific antibodies. As shown in Fig. 1B, Western blot analysis confirmed the general resistance of wild-type kinase to dephosphorylation, whereas the C199A kinase was readily dephosphorylated. Interestingly, the phosphorylation state of Ser338 paralleled that of Thr197, also being resistant to removal in wild-type kinase but readily removed in C199A. This result suggests that dephosphorylation of Thr197 likely destabilizes the protein, thereby allowing the secondary phosphate on Ser338 to be removed. From this experiment, we deduce that Cys199, which exists as a thiolate anion, either acts to repulse phosphatase or is essential in coordinating or stabilizing the activation loop in such a conformation that the phosphate is not an accessible substrate.

Oxidation of the PKA C-subunit Enhances Dephosphorylation—We reported previously (6) that the reactive nature of Cys199 renders the kinase susceptible to oxidation-dependent inactivation. Based on the results described in Fig. 1, we hypothesized that oxidation or modification of Cys199 may also alter the ability of the C-subunit to be dephosphorylated. As previously described (6), incubation of the C-subunit with diamide, a sulfhydryl-specific oxidant, inhibits the kinase through the formation of an internal disulfide bond between Cys199 and Cys343. This inhibition is nearly complete (90%) but largely reversible by DTT (Fig. 2A). Alternatively, in the presence of reduced glutathione and diamide, the kinase is inhibited by the formation of a protein-glutathione mixed disulfide (6). This also results in near complete loss of kinase activity (92% decrease), but again it is largely reversible by reducing agent (Fig. 2A). Finally, modification of kinase with the alkylating agent NEM results in nearly complete loss of kinase activity (95%) and is not reversible by reducing agent (data not shown) (11).



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  		FIG. 2.
Oxidation or alkylation of the C-subunit of PKA enhances its ability to be dephosphorylated by phosphatase {lambda}. A, oxidized C-subunit samples were prepared as described under "Experimental Procedures." Kinase activity was then assayed in the presence or absence of 20 mM DTT to demonstrate the reversible nature of the oxidation. Oxidized and reduced C-subunits (300 ng) were then incubated with phosphatase {lambda} (50 ng, 45 min at 23 °C), the reaction was stopped by the addition of 5.0 mM vanadate, and kinase activities were measured in the presence of 20 mM DTT. Values are means ± S.E. (n = 3). B, reduced C-subunit, C-subunit disulfide, glutathionylated C-subunit, and alkylated C-subunit samples were analyzed by Western blot (WB) under reducing conditions before and after treatment with phosphatase {lambda} using C-subunit-specific, phospho-Thr197 (Thr197-P)-specific, and phospho-Ser338 (Ser338-P)-specific antibodies.

 
Oxidized kinase, containing either the internal disulfide bond or protein-glutathione mixed disulfide, or reduced kinase, was incubated with phosphatase {lambda} (45 min, 22 °C). Following this incubation, oxidized kinase activity was no longer recovered by the addition of DTT (Fig. 2A). Glutathionylated C-subunit had an activity prior to phosphatase treatment of 12.9 ± 1.4 nmol x min–1 x mg–1 as compared with 5.3 ± 0.6 nmol x min–1 x mg–1 after treatment. Similarly, diamide-treated C-subunit containing an internal disulfide had an activity of 10.9 ± 1.3 nmol x min–1 x mg–1 prior to phosphatase treatment and 2.4 ± 0.3 nmol x min–1 x mg–1 after. This suggested that, in its oxidized state, the C-subunit was now susceptible to dephosphorylation. Western blot analysis was performed, and samples were probed with phospho-Thr197- and phospho-Ser338-specific antibodies. The results of the Western blot analysis (Fig. 2B) confirmed that oxidized kinase was dephosphorylated at both phosphorylation sites, whereas reduced kinase was largely resistant to dephosphorylation. We noted that upon dephosphorylation of oxidized kinase, there was a slight decrease in the total amount of the C-subunit (Fig. 2B). This was likely because of precipitation of the destabilized, unphosphorylated protein.

To further demonstrate that blocking the nucleophilic Cys199 was sufficient for enhancing dephosphorylation, kinase was alkylated with NEM, incubated with phosphatase {lambda}, and then analyzed by Western blot. Fig. 2B demonstrates that alkylation of the C-subunit also greatly enhanced its ability to be dephosphorylated. This experiment further demonstrates that neutralizing the nucleophilic Cys199 decreases the stability of the phosphate on Thr197.

Reduced C-subunit Can Be Rephosphorylated by PDK1—As reported previously, Thr197 is a substrate of PDK1 (15–17). It was therefore of interest to see if oxidized, dephosphorylated C-subunit could be rephosphorylated by PDK1. In these experiments, oxidized C-subunit (glutathionylated or containing an internal disulfide) was dephosphorylated with phosphatase {lambda}, and reactions were then stopped by the addition of NaF. PDK1, DTT, and MgATP were then added as indicated (Fig. 3). Samples were then subjected to Western blot analysis, utilizing phospho-specific antibodies. The results of our experiment demonstrate that dephosphorylated kinase is not rephosphorylated upon addition of DTT and MgATP alone (Fig. 3, lane 3), which was true for both forms of oxidized C-subunit examined. Furthermore, addition of PDK1 in the absence of DTT resulted in little phosphorylation of Thr197 (Fig. 3, lane 4). In contrast, addition of PDK1, MgATP, and DTT resulted in rephosphorylation of Thr197 (Fig. 3, lane 5) with concurrent PKA reactivation (data not shown). This was true for both forms of the oxidized C-subunit examined. Furthermore, control assays examining the ability of PDK1 to phosphorylate a peptide substrate in the presence or absence of DTT demonstrated that reducing agent alone did not enhance kinase activity (data not shown), suggesting that DTT was acting to reduce the C-subunit and not simply acting to enhance PDK1 activity. Consistent with previous findings (16),2 PDK1 preferentially phosphorylated Thr197, whereas Ser338 remained unphosphorylated (Fig. 3). It is believed that Ser338 is autophosphorylated (12), suggesting that under our experimental conditions this process occurred slowly. The results of these experiments demonstrate that in contrast to phosphatase, which preferentially acts upon oxidized kinase, PDK1 utilizes the reduced C-subunit to specifically phosphorylate Thr197.



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  		FIG. 3.
Oxidized, dephosphorylated C-subunit is preferentially rephosphorylated by PDK1 in the presence of DTT. Oxidized C-subunit samples were treated with phosphatase {lambda} as described in Fig. 2. After 45 min, the dephosphorylation reaction was stopped by the addition of 10 mM NaF, and then ATP (1.0 mM) and MgCl2 (10 mM) were added in the presence or absence of DTT (20 mM) and/or PDK1 (50 ng). Samples were incubated for 60 min at 23 °C followed by the addition of SDS NuPAGE sample buffer, 20 mM DTT, and then the samples were subjected to Western blot (WB) analysis as in Fig. 1. Thr197-P, phospho-Thr197; Ser338-P, phospho-Ser338.

 
Cell Lysates Contain a Phosphatase That Preferentially Dephosphorylates Oxidized and Alkylated C-subunits—Having determined in vitro that oxidized and alkylated C-subunits are preferentially dephosphorylated by phosphatase {lambda}, it was essential to determine whether eukaryotic phosphatases also showed this preference. For these experiments, PC12 cells were lysed and the soluble extract incubated with reduced, oxidized (glutathionylated or containing an internal disulfide) or alkylated PKA C-subunit. Samples were then subjected to Western blot analysis utilizing the phospho-specific antibodies. Under our Western blot conditions, exogenous C-subunit was in a large excess over the endogenous PKA found in the lysates. Therefore, our Western blot analysis conditions readily detected the exogenous C-subunit without background bands from the lysate (not shown). Furthermore, incubation of the exogenous C-subunit with cell lysates did not result in loss of protein as seen by constant total kinase levels under the experimental conditions (Fig. 4).



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  		FIG. 4.
PC12 cell lysates preferentially dephosphorylate PKA C-subunit that has been either oxidized or thiol-alkylated. C-subunit (300 ng) was incubated with soluble PC12 cell extract (100 µg) for 60 min at 30 °C in the presence or absence of either 20 mM NaF or 25 nM okadaic acid. At the end of the reaction, SDS-PAGE sample buffer containing 25 mM DTT was added, and samples were boiled and then analyzed by Western blot (WB) as described in Fig. 1.

 
As shown in Fig. 4, incubation of the reduced C-subunit resulted in minimal dephosphorylation of Thr197 and Ser338. In contrast, incubation of oxidized or alkylated C-subunit resulted in nearly complete dephosphorylation of Thr197. This dephosphorylation was prevented by the addition of the nonspecific phosphatase inhibitor NaF (20 mM) or okadaic acid (25 nM). The amount of okadaic acid needed to inhibit dephosphorylation was constant with a previous report that determined Thr197 was dephosphorylated by a protein phosphatase-2A-like phosphatase (13). Ser338 was also preferentially dephosphorylated in alkylated and oxidized C-subunits containing an internal disulfide but to a lesser extent when glutathionylated.

Treatment of Cells with NEM Results in Kinase Inhibition and Dephosphorylation—We have demonstrated that the reduced thiol on Cys199 is critical in protecting the kinase from dephosphorylation. Oxidation or alkylation of this site in vitro by both phosphatase {lambda} and cell lysates enhances the ability of the kinase to be dephosphorylated. We believe that such a novel mechanism could exist in cells whereby modification of this highly reactive cysteine leads to kinase dephosphorylation. To test our model, PC12 cells were treated with PKA activators (the adenylate cyclase activator forskolin, the cell-permeable cAMP analog 8-bromo-cyclic AMP, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine) for 10 min at 22 °C or left untreated as a control. Cells were then treated with the cell-permeable alkylating agent NEM (29) (500 µM, 30 min at 37 °C) as indicated, washed, harvested, and lysed in the presence of DTT. Kinase activity was then evaluated in the presence of cAMP. As shown in Fig. 5, NEM had minimal effect on PKA activity (10% ± 0.9% decrease) when cells were treated in the absence of PKA activators. This was an expected result, as we have previously shown that the kinase is largely resistant to thiol modification in the absence of kinase activators because the reactive Cys199 is shielded by the bound regulatory subunit. In contrast, cells treated with PKA activators prior to NEM showed a dramatic and nearly complete loss (90 ± 1.5% decrease) of kinase activity.



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  		FIG. 5.
NEM inhibits PKA activity in PC12 cells but only after PKA is activated. PC12 cells were incubated in the presence or absence of forskolin (50 µM), 8-bromo-cyclic AMP (250 µM), 3-isobutyl-1-methylxanthine (100 µM), and okadaic acid (250 nM) as indicated for 10 min at 23 °C. NEM was then added at a final concentration of 500 µM, and cells were incubated for an additional 30 min at 37 °C. Cells were collected, lysed, protein concentrations standardized, and kinase activity determined in the presence of 5.0 µM cAMP as described under "Experimental Procedures." Values are means ± S.E. (n = 3).

 
To determine whether alkylation-mediated inactivation of PKA resulted in dephosphorylation, samples were analyzed by Western blot analysis and probed with the phospho-specific antibodies that were utilized in previous experiments (Figs. 1, 2, 3, 4). The activation loop antibody that recognizes Thr197 of the C-subunit was raised against phospho-Thr500 of PKC{beta} as characterized previously (24, 30). This antibody has cross-reactivity with other AGC kinases, each of which has a highly conserved and critical phosphothreonine in their activation loops, but does not recognize phosphorylation sites outside of this region (24). Under our Western blot conditions, this antibody specifically recognized three bands in PC12 cell lysates (Fig. 6A). PKC{alpha} is abundantly expressed in cells and likely represented the 76-kDa band. This was supported by the cross-reactivity of the 76-kDa band with a PKC{alpha}-specific antibody and its ability to be immunoprecipitated with a PKC{alpha}-specific antibody. The PKA C-subunit is also abundantly expressed in PC12 cells and likely represented the 40-kDa protein. This also was confirmed by cross-reactivity of the 4-kDa band with PKA C-subunit-specific antibody and its ability to be immunoprecipitated with a C-subunit-specific antibody. The upper band at ~95 kDa could not be readily identified but likely represents another AGC kinase.



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  		FIG. 6.
Treatment of PC12 cells with NEM results in the dephosphorylation of the PKA C-subunit and PKC{alpha}. The dephosphorylation of PKA is dependent upon the presence of PKA activators. A, PC12 cells were treated as described in Fig. 5 and then subjected to Western blot (WB) analysis using the phosphothreonine (Thr197-P) antibody that had been used in Figs. 1, 2, 3, 4 and that had been described under "Experimental Procedures." Under our experimental conditions, this antibody specifically recognized three bands in PC12 cell lysates: 40 kDa (PKA C-subunit), 76 kDA (PKC{alpha}), and 95 kDa (unidentified). B, phospho-Ser338 levels of the PKA C-subunit were determined using an anti-phospho-Ser338 (Ser338-P)-specific antibody as described in Fig. 1. C, total PKA C-subunit and PKC{alpha} levels were determined using kinase-specific antibodies.

 
As shown in Fig. 6A, the phosphorylation state of the PKA C-subunit was unaffected after forskolin stimulation (lane 2) or NEM treatment (lane 3) alone. In contrast, when PC12 cells were treated with PKA activators followed by NEM, conditions that resulted in PKA inactivation (Fig. 5), the kinase was completely dephosphorylated (Fig. 6A, lane 4). This dephosphorylation was blocked by pretreatment with okadaic acid (250 nM) (Fig. 6A, lane 5). Okadaic acid itself did not affect alkylation of the kinase, as PKA was still completely inactive under these conditions (Fig. 5). In contrast to alkylation-dependent dephosphorylation of Thr197, the phosphorylation state of Ser338 was largely unaffected by the addition of NEM. There was a slight decrease in phospho-Ser338 content upon PKA stimulation (Fig. 6B, compare lanes 1 and 2), but this was not further enhanced by the addition of NEM (Fig. 6B, lane 4). This slight decrease of phospho-Ser338 was blocked by the inclusion of okadaic acid (Fig. 6B, lane 5). The total C-subunit content was unaffected under our experimental conditions (Fig. 6C).

In a manner similar to the PKA C-subunit, PKC{alpha} was also dephosphorylated at its activation loop by treatment with NEM (Fig. 6A, lane 3). However, unlike the PKA C-subunit, PKC{alpha} was dephosphorylated in an NEM-dependent manner regardless of the presence of PKA or PKC activators (Fig. 6A, compare lanes 3 and 4). This dephosphorylation was also blocked by the addition of okadaic acid (250 nM). Total PKC{alpha} content was unaffected under the experimental conditions (Fig. 6C). In contrast to PKC{alpha} and the PKA C-subunit, the 95-kDa protein was largely dephosphorylated regardless of the presence of NEM. The addition of okadaic acid greatly enhanced the phosphorylation of this protein above basal levels (Fig. 6, lane 5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The sequence of the activation loop surrounding Thr197 reveals a remarkable chemical environment with the cysteine thiol anion poised in a central location between Arg165 and the phosphate of Thr197 (Fig. 7). Tyr215 also contributes to this site by hydrogen bonding to the backbone carboxyl of Thr197 and the side chain of Arg165. It is thus likely the close proximity of Arg165, which could stabilize a thiolate anion (31), accounting for the nucleophilic properties of Cys199 that render it so reactive. We know that phosphorylation of Thr197 orchestrates the site so that the enzyme is in a conformation that favors catalysis (2). We also know, based on a recent structure of the holoenzyme,3 that phosphorylation of Thr197 generates a stable binding surface for RI{alpha} where Trp196 plays a critical role at the interface. Here we show not only that the cysteine is a sensor for oxidation in the free C-subunit, but that it may also provide a shuttle for inactivation via dephosphorylation.



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  		FIG. 7.
A, the PKA C-subunit contains two cysteines (Cys199 and Cys343), noted as yellow circles, and two major phosphorylation sites (Thr197 and Ser338), noted as red circles. The reactive Cys199 is readily modified at physiological pH and is capable of forming an internal disulfide with Cys343 (6). B, a detail of the environment around Cys199, noted by the bold arrow, reveals its proximity to Thr197 and Arg165. It is likely that the nucleophilic properties of Cys199 are mediated by the charge on Arg165. C, a schematic illustrates the preferential dephosphorylation of oxidized or modified C-subunit, with glutathionylated C-subunit depicted. Preferential rephosphorylation of the reduced kinase creates a redox cycle whereby the kinase phosphorylation state may be regulated.

 

The importance of Cys199 in preventing dephosphorylation is illustrated by our in vitro experiments. We demonstrate that altering this site, either through mutagenesis, oxidation to form a Cys199-Cys343 disulfide, glutathionylation, or alkylation enhances the ability of the kinase to be dephosphorylated. Such modifications also enhanced the ability of the secondary phosphorylation site on Ser338 to be dephosphorylated in vitro but not by alkylation in PC12 cells (Fig. 6). We believe that removal of the primary phosphorylation site on the activation loop destabilizes the protein, thereby allowing for dephosphorylation of the secondary site. This discrepancy in cells could be due to differences in stability of the recombinant kinase used in our in vitro experiments versus the kinase found in eukaryotic cells. That is, the C-subunit in cells may be stabilized by myristoylation, other post-translation modifications, or by interactions of the C terminus of the kinase with other proteins, thus preventing dephosphorylation of Ser338.

It has been shown previously that PDK1 is capable of phosphorylating the C-subunit, an event that is likely to occur during protein translation (15–17). Interestingly, we found that although the kinase is more readily dephosphorylated when Cys199 is oxidized or modified, the kinase is preferentially rephosphorylated by PDK1 when reduced (Fig. 3, Fig. 7C). The competition between the phosphorylation/dephosphorylation reactions could thus be limited depending upon the redox status of the kinase.

We demonstrate that treatment of PC12 cells with NEM results in PKA inactivation. This inhibition can occur only when the kinase is first activated by cAMP. Under the conditions in which PKA is inhibited by NEM, it is also dephosphorylated. There are reports that protein phosphatase 2A itself can either be activated (33) or inhibited by NEM-mediated alkylation or glutathionylation (34). Thus, under our experimental conditions it may be possible that NEM is also acting to enhance cellular phosphatase activity. However, our in vitro experiments conclusively demonstrated that incubation of the alkylated C-subunit with cell lysates enhanced its ability to be dephosphorylated (Fig. 4). Furthermore, treatment of cells with lower concentrations of NEM, which resulted in a minimal loss of PKA activity, had no effect on C-subunit phosphorylation (data not shown). These results support our model in which direct thiol modification of the PKA C-subunit is necessary and sufficient for its dephosphorylation.

Although the cytosol is a reducing environment, the thiol status of proteins may be more dynamic than appreciated previously (35). Oxidative stress can lead to the formation of a large number of protein disulfides (36–38). Furthermore, thiol oxidation may have a role in normal cellular processes. For example, extracellular stimuli, such as by epidermal growth factor and insulin, lead to increased intracellular hydrogen peroxide production (39, 40). It is believed that oxidants produced during these signaling events act as second messengers that can affect downstream targets, such as thiols, that are keenly sensitive to oxidation (31, 41). Protein thiols can also be modified by other means, such as through nitrosylation (42) or through reactions with the products of lipid peroxidation, such as 4-hydroxy-2-noneal (43, 44). Based on our results, it would be expected that other such thiol modifications would also lead to a phosphatase-sensitive C-subunit. Alternatively, there is evidence that under certain pathological conditions, such as prostate cancer, PKA may be found extracellularly (45, 46). Extracellular PKA would have a much higher preponderance to form a Cys199-Cys343 or mixed disulfide and thus be primed for dephosphorylation.

Based on our results, we believe that thiol modification or oxidation of members of the AGC family of kinases, each of which contain a highly conserved cysteine in their activation loop, may be a general mechanism for enhancing their dephosphorylation. We demonstrate here that treatment of PC12 cells with NEM results in PKA and PKC{alpha} dephosphorylation. Because both kinases have been reported previously to be susceptible to glutathionylation (6, 32), it suggests that they are normally poised for thiol oxidation and dephosphorylation. In addition, a recent report demonstrated that hydrogen peroxide induces an internal disulfide in Akt, and this also enhanced its dephosphorylation (23), further suggesting a common mechanism. Future work will look at the prevalence of this mechanism as a means of normal cellular signaling and under conditions of oxidative stress.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM19301 (to S. S. T.) and GM64991 (to K. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Howard Hughes Medical Inst., Dept. of Chemistry and Biochemistry, CMM-W 125, 9500 Gilman Dr., La Jolla, CA 92093-0654; Tel.: 858-534-3677; Fax: 858-534-8193; E-mail: staylor{at}ucsd.edu.

1 The abbreviations used are: PKA, cyclic AMP-dependent protein kinase; NEM, N-ethylmaleimide; DTT, dithiothreitol; PBS, phosphate-buffered saline; PKC, protein kinase C. Back

2 G. H. Iyer, M. J. Moore, and S. S. Taylor, submitted for publication.. Back

3 Kim, C., Xuong, N. H., and Taylor, S. S. (2005) Science, in press.. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Tianyan Gao and Dr. Ganesh Iyer for helpful discussions, Dr. Elzbieta Radzio-Andzelm for assistance with figures, Dr. Michael J. Moore for providing the C199A construct, and Dr. Jennifer Giorgione for technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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