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[Cancer Research 64, 9131-9138, December 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

The Human Glutathione S-Transferase P1 Protein Is Phosphorylated and Its Metabolic Function Enhanced by the Ser/Thr Protein Kinases, cAMP-Dependent Protein Kinase and Protein Kinase C, in Glioblastoma Cells

Hui-Wen Lo, Gamil R. Antoun and Francis Ali-Osman

Department of Surgery and the Comprehensive Cancer Center, Duke University Medical Center, Durham, North Carolina


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We report here that the human glutathione S-transferase P1 (GSTP1) protein, involved in phase II metabolism of many carcinogens and anticancer agents and in the regulation of c-Jun NH2-terminal kinase-mediated cell signaling, undergoes phosphorylation by the Ser/Thr protein kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), resulting in a significant enhancement of its metabolic activity. GSTP1 phosphorylation by PKA was glutathione (GSH)-dependent, whereas phosphorylation by PKC did not require but was significantly enhanced by GSH. In the presence of GSH, the stoichiometry of phosphorylation was 0.4 ± 0.03 and 0.53 ± 0.02 mol incorporated phosphate per mole of dimeric GSTP1 protein. The GSTP1 protein was phosphorylated, in the presence of GSH, by eight different PKC isoforms ({alpha}, ßI, ßII, {delta}, {epsilon}, {gamma}, {eta}, and {zeta}), belonging to the three major PKC subclasses, albeit with various efficiencies. The catalytic efficiency, kcat/Km, of the phosphorylated GSTP1 was more than double that of the unphosphorylated protein. In MGR3 human glioblastoma cells, PKA and PKC activation resulted in a significant increase in the level of phosphorylation of the GSTP1 protein and was accompanied by a 2.1- and 2.7-fold increase, respectively, in specific GSTP1 activity in the cells. Peptide phosphorylation analyses and both phosphorylation and enzyme kinetic studies with GSTP1 proteins mutated at candidate amino acid residues established Ser-42 and Ser-184 as putative phospho-acceptor residues for both kinases in the GSTP1 protein. Together, these findings show PKA- and PKC-dependent phosphorylation as a significant post-translational mechanism of regulation of GSTP1 function. The GSH-dependence of the phosphorylation suggests that under high intracellular GSH conditions, such as is present in most drug-resistant tumors, the GSTP1 protein will exist in a hyper-phosphorylated and enzymatically more active state. In normal cells, the functional activation of the GSTP1 protein by PKA- and PKC-dependent phosphorylation could represent a potentially important mechanism of cellular protection, whereas in tumors, increased phase II metabolism of anticancer drugs by the more active phosphorylated GSTP1 protein could contribute to the drug resistance and therapeutic failure frequently associated with increased activities of these Ser/Thr kinases.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human glutathione S-transferase P1 (GSTP1) protein, encoded in a polymorphic gene locus (1 , 2) , is expressed at high levels in many human cancers including gliomas. In these tumors, the high GSTP1 expression is associated with tumor drug resistance, a more aggressive clinical course, and poor patient survival (3, 4, 5, 6) . A major role of the GSTP1 protein is in cellular phase II metabolism and xenobiotic detoxification, in which it catalyzes the S-conjugation of a wide variety of endogenous and exogenous compounds (7) , including many carcinogens and anticancer agents, with glutathione (GSH). In tumor cells, the enhanced detoxification of chemotherapeutic agents is a primary basis for the frequently observed GSTP1-associated tumor drug resistance and failure of cancer chemotherapy (4, 5, 6) . In normal tissues, on the other hand, GSTP1-catalyzed metabolic inactivation of endogenous and exogenous electrophilic and free radical generating compounds is a major mechanism of cellular protection against the toxicity and carcinogenicity of these compounds (8) . The proteins encoded by the different GSTP1 alleles (1 , 9) have been shown to differ in their ability to metabolize GST substrates, anticancer agents, carcinogens, and other genotoxins (1 , 9, 10, 11, 12) , and increasingly, GSTP1 genetic polymorphism is being shown to be an important determinant not only of response to cancer chemotherapy but also of individual susceptibility to cancer (1314, 15, 16, 17, 18, 19, 20, 21) .

In addition to its central role in cellular metabolism, an accumulating body of evidence is unraveling novel functions for the GSTP1 protein in many important cellular processes, including, stress and growth factor-induced signaling, cell proliferation, immune response, differentiation, cellular transformation, and apoptosis (22, 23, 24, 25) . A common feature of many of these processes is that they involve both protein phosphorylation and dephosphorylation, particularly, of regulatory proteins and transcription factors. Phosphorylation often significantly alters multiple properties of the proteins, including their function (activation/inactivation), sub-cellular localization, DNA binding ability, stability, interactions with other proteins, enzymatic activity, and substrate specificity (26, 27, 28, 29) . For example, the transduction of extracellular signals through the cytoplasm and into the cell nucleus is driven by phosphorylation cascades, involving mitogen-activated protein kinases (MAPKs), MAPK kinases (MAPKKs), and MAPKK kinases, as well as various Ser/Thr protein kinases such as cAMP-dependent protein kinase (PKA), protein kinase C (PKC), casein kinase I (CKI), and casein kinase II (CKII; refs. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 ). Similarly, the processes of cell proliferation, including, cell cycle progression and mitosis are regulated, in large part, by the phosphorylation and/or dephosphorylation of specific proteins, including p53, the retinoblastoma protein (pRB), and various cyclins, many of which are kinases themselves (31 , 32) . Although GSTs are involved in several cellular processes in which protein phosphorylation is a key regulatory mechanism, relatively little focus has been directed at the phosphorylation of this class of proteins. The notion that phosphorylation may represent a critical post-translational mechanism of regulation of GST function is underscored by the recent observation that the murine GSTA4-4 protein undergoes phosphorylation by both PKA and PKC, resulting in a significant enhancement of its affinity to HSP70 chaperone and subsequent translocation from the cytoplasm to mitochondria (33) . Although with respect to the GSTP1 protein, evidence exists that both the rat and human proteins may be substrates for phosphorylation (34 , 35) , the nature and physiologic significance of GSTP1 phosphorylation and its effects on GSTP1 function remain unknown. In this study, we sought to establish conclusively that the human GSTP1 protein undergoes phosphorylation; investigate the extent, nature, and conditions of such phosphorylation; characterize the kinases involved; and gain insights into the putative phospho-acceptor residues in the protein. Using human glioblastoma cells, we examined the extent of intracellular GSTP1 phosphorylation after activation of two key Ser/Thr protein kinases, PKA and PKC. To determine the physiologic significance of the GSTP1 phosphorylation, we investigated the effects of the phosphorylation on the phase II metabolic function of the GSTP1 protein.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Biochemicals and Other Reagents.
The MGR1 and MGR3 cell lines were established in our laboratory from primary specimens of an anaplastic astrocytoma and a human glioblastoma multiforme, respectively, as we described previously (36) . The cell lines were maintained in DMEM supplemented with 10% fetal calf serum. Recombinant GSTP1 protein was purchased from Calbiochem (La Jolla, CA). PKA (catalytic subunit of bovine heart PKA) was purchased from Roche Biochemicals (Indianapolis, IN). Rat brain PKC, which contains multiple PKC isoforms, was obtained from Stratagene (La Jolla, CA), whereas the individual PKC isoforms were purchased from Upstate Biotechnologies (Lake Placid, NY). Rat liver CKII, containing both CKII-{alpha} and -ß subunits was from Life Technologies, Inc. (Rockville, MD). [{gamma}-32P]ATP was obtained from Dupont/NEN (Boston, MA). The positive phosphorylation controls, histone H1, and phosphorylated heat- and acid-stable protein-1 were from Life Technologies, Inc. and Stratagene, respectively. Unless otherwise stated, all other chemicals and biochemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Computer-Assisted Analysis of Phosphorylation Sites in the GSTP1 Protein.
The computer application, PhosphoBase1 was used to examine the human GSTP1 protein for the presence of consensus phosphorylation motifs for ten different Ser/Thr protein kinases, namely, PKA, PKC, cyclic GMP-dependent protein kinase, CKI, CKII, calmodulin-dependent protein kinase II (CaMK II), glycogen synthase kinase 3 (GSK3), myosin light chain kinase, 34 kDa cell division cycle protein kinase (p34cdc2), and 70 kDa S6 kinase (p70s6k).

Analysis of Phosphorylation of GSTP1 by Ser/Thr Protein Kinases.
PKA-dependent GSTP1 phosphorylation was assayed in a 30 µL of reaction mixture containing 1 µg recombinant human GSTP1 protein, 40 units of PKA, 10 µCi [{gamma}-32P]ATP (6,000 Ci/mmol), 100 µmol/L cold ATP, 100 mmol/L MgCl2 and 1.7 mmol/L CaCl2 in 20 mmol/L HEPES (pH 7.5). Histone H1 was used as a positive phosphorylation control. The reactions were incubated at 30°C, and at the desired time points, 30 µL aliquots were removed and electrophoresed on a discontinuous (3 over 12%) SDS-PAGE. The gels were then rinsed in water, dried under vacuum, and autoradiographed. The phosphorylated bands were quantitated by densitometry.

GSTP1 phosphorylation by PKC was determined as described above for PKA, except that PKA was replaced with 20 ng of rat brain PKC in the reaction mixture. Histone H1 and phosphorylated heat- and acid-stable protein-1 were used as substrates in positive PKC phosphorylation control reactions. Phosphorylation of GSTP1 by CKII was similarly determined, except that 10 units of CKII (Life Technologies, Inc.) were used and the reactions were carried out at 37°C. C-jun was used as a positive phosphorylation control for CKII.

Effects of GSH on GSTP1 Protein Phosphorylation by Ser/Thr Kinases.
To mimic intracellular conditions in which the GSTP1 protein exists in equilibrium with GSH bound to its GSH-binding (G-) site (30 , 31) , we examined phosphorylation of GSTP1 by the Ser/Thr kinases after its incubation/equilibration with GSH. For this, 1 µg of recombinant GSTP1 protein was preincubated with and without 5 mmol/L GSH in a 30 µL reaction volume for 30 minutes at 37°C and then added to kinase reaction mixtures containing PKA, PKC, and CKII. The level of phosphorylation of the GSTP1 protein was then determined, as described above.

Determination of Kinetics and Stoichiometry of GSTP1 Phosphorylation.
For these studies, 1 µg of recombinant GSTP1 protein was preincubated with 5 mmol/L GSH for 20 minutes at 37°C to mimic the physiologic GSH-bound state of GSTP1. Phosphorylation reactions with PKA and PKC were set up as we described previously, containing saturating ATP concentrations (100 µmol/L), and over a time course of 0 to 4 hours, 30 µL aliquots of the reaction mixture were removed and subjected to SDS-PAGE. The phosphorylated GSTP1 bands were excised from the gel, and the incorporated 32P was quantitated by ß scintillation counting. The molar amount of incorporated phosphate was computed based on the specific activity of the [{gamma}-32P]ATP, expressed per mole of the dimeric GSTP1 protein and plotted against time.

GSTP1 Phosphorylation by PKC Isoforms.
We examined the phosphorylation of GSTP1 by eight different PKC isoforms (i.e., PKC-{alpha}, PKC-ßI, PKC-ßII, PKC-{delta}, PKC-{epsilon}, PKC-{gamma}, PKC-{eta}, and PKC-{zeta}) using the standard GSTP1 protein phosphorylation assay described in the section on protein phosphorylation above. Briefly, 30 µL reaction mixtures were set up, each containing 1 µg recombinant GSTP1 (with and without preincubation with 5 mmol/L GSH), 20 ng of PKC isoform, 100 µmol/L ATP and 10 µCi [{gamma}-32P]ATP (6,000 Ci/mmol). After a 30-minute incubation, 30 µL aliquots were removed and subjected to SDS-PAGE and autoradiography, as described previously. Band intensities were quantitated densitometrically.

Analysis of Phospho-Acceptor Residues in GSTP1 Peptides.
Three oligopeptides corresponding to regions of the GSTP1 peptide with the consensus PKA and PKC phosphorylation motifs and containing the putative phospho-acceptor amino acid residues Ser-27, Ser-42, and Ser-184 were used. The peptides with the amino acid sequences, Asp-Gln-Gly-Gln-Ser-Trp-Lys-Glu (Ser-27Pep), Trp-Gln-Glu-Gly-Ser-Leu-Lys-Ala (Ser-42Pep), and Val-Gly-Arg-Leu-Ser-Ala-Arg-Pro (Ser-184Pep) were custom synthesized by Sigma-Genosys (The Woodlands, TX). Two other peptides, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide) and Phe-Leu-Glu-Glu-Leu (P-5523), obtained from Sigma-Aldrich, served as positive and negative phosphorylation controls, respectively. Phosphorylation of the peptides by PKA, rat brain PKC, and eight different PKC isoforms, was examined in 30-µL reaction mixtures containing 600 µmol/L of each peptide and 1 mmol/L ATP. After a 1-hour incubation at 30°C, the reactions were terminated by adding 470 µL of 30% acetic acid, and the phosphorylated peptides were separated from the unincorporated ATP by anion exchange chromatography on an AG 1 x 8 resin (Bio-Rad Laboratories, Hercules, CA), as described previously (37) . The method is based on the ability of the AG 1 x 8 resin to bind ATP but not the phospho-peptides. Briefly, the terminated phosphorylation reaction mixture was applied to a polypropylene chromatographic column containing 2 mL of AG 1 x 8 that had been equilibrated with 30% acetic acid. The first 500 µL of flow-through, containing the unbound phosphorylated peptides, were collected directly into a scintillation vial after which the column was washed with an additional 4 mL of 30% acetic acid and collected into the same vial. The radioactivity in the pooled eluant was quantitated by ß scintillation counting and used to compute the level of [{gamma}-32P]phosphorylation of each peptide.

Site-Directed Mutagenesis of Putative Phospho-Acceptor Residues in GSTP1 Protein.
This was done with the GeneTailor site directed mutagenesis system (Invitrogen, Carlsbad, CA) on the template plasmid vector pBK-CMV/GSTP1C, which contains the cDNA encoding the wild-type GSTP1C (1) . Two sets of GSTP1 mutants were created, one in which Ser-42 and Ser-184 were mutated to alanine and the other in which the two residues were mutated to aspartic acid. Single mutants as well as double mutants containing mutations of both serines in a single cDNA were created. Briefly, the pBK-CMV/GSTP1C vector was methylated and 30 PCR cycles were done under the manufacturer’s recommended conditions. The forward PCR primers were 5'-GACGTGGCAGGAGGGCGCACTCAAAGCCTC-3' and 5'-GACGTGGCAGGAGGGCCATCTCAAAGCCTC-3' for the S42A and S42D mutants, respectively, and 5'-TATGTGGGGCGCCTCGCCGCCCGGCCCA-3' and 5'-TATGTGGGGCGCCTCCATGCCCGGCCCA-3' for the S184A and S184D mutants, respectively (the mutated codons in each primer are bold). The PCR products were used to transform mcrBC wild-type Escherichia coli, and colonies containing the unmethylated mutants (the methylated template DNA was cleaved by host endonuclease) were isolated, purified, and sequenced to confirm the presence of the required mutations. The Ser-42A and Ser-42D mutants were then used as templates to create the double mutants with the S42A/S184A and the S42D/S184D mutations. To create vectors for expressing hemagglutinin-tagged (HAT) GSTP1 mutant proteins in glioma cells, the vectors carrying the S42A, S184A, and S42A/S184A mutant cDNAs were subcloned into the BamHI and KpnI sites of the pHAT vector (BD Clontech, Franklin Lakes, NJ). The inserts were then released with the HAT sites and recloned into the pBK-CMV vector for subsequent eukaryotic expression.

To examine the contribution of the negative charge of the phosphate residue in phospho-GSTP1 on the observed changes in catalytic activity, S42D, S184D, and the double S42D/S184D GSTP1 mutant proteins were subcloned into pBK-CMV expression vectors, and the proteins were expressed in E. coli and purified as we described previously (1) . For the S->D mutant proteins, the computed kcat/Km values were used to compare the effects of the mutations on their catalytic activities, with and without phosphorylation by PKA and PKC.

Effects of PKA- and PKC-Dependent Phosphorylation on Catalytic Activity of Wild-Type and Mutant GSTP1 Proteins.
To investigate the effects of phosphorylation on the metabolic function of the wild-type and mutant GSTP1 proteins, 0.5 µg of each of the recombinant proteins was phosphorylated with PKA and rat brain PKC, as described earlier. Control reactions were set up similarly, but without the addition of the protein kinases. After a 1-hour incubation at 30°C, the reactions were terminated and used to determine the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) to GSH, as we described previously (1 , 7) . Reaction mixtures in 100 mmol/L potassium phosphate buffer (pH 6.5) contained 1 to 10 mmol/L CDNB and 2.5 mmol/L GSH (for CDNB enzyme kinetics) and 0.5 to 4 mmol/L GSH and 1 mmol/L CDNB (for GSH enzyme kinetics). Thirty microliters of reaction mixtures containing the phosphorylated and unphosphorylated GSTP1 proteins were added, and the change in absorbance at 340 nm was monitored over 2 minutes in a Beckman DU 60 spectrophotometer (Beckman Coulter, Fullerton, CA) equipped with an enzyme kinetic module. The computed reaction rates of spontaneous reactions of GSH with CDNB were subtracted from the rates of the GSTP1-catalyzed reactions and used to generate double reciprocal plots. Km, Vmax, kcat, and kcat/Km for unphosphorylated, PKA- and PKC-phosphorylated GSTP1 protein were determined for each substrate, as we described previously (1) and expressed as the mean ± 1 SD of triplicate experiments.

Analysis of Endogenous GSTP1 Protein Phosphorylation in Glioma Cells.
Exponentially growing MGR3 cells were plated at 2 x 106 cells per T75 tissue culture flask in phosphate- and serum-free MEM (Life Technologies, Inc.). After overnight incubation at 37°C, the medium was removed and replaced with 10 mL fresh phosphate- and serum-free-MEM containing either 25 µmol/L of the adenylate cyclase activator, forskolin (to activate the PKA pathway) or 50 nmol/L 12-O-tetradecanoylphorbol-13-acetate (TPA; to activate the PKC pathway), followed 15 minutes later by 1mCi/mL of [32P]Pi. After 3 hours, the cells were washed twice with PBS, harvested, and ultra-sonicated in 700 µL of 10 mmol/L Tris-HCl (pH 7.5) containing 1 mmol/L dithiothreitol and 1 mmol/L phenylmethylsulfonyl fluoride. The resulting cell lysates were centrifuged at 10,000 x g for 10 minutes, and the supernatants were subjected to immunoprecipitation. Briefly, 150 µL of the supernatants were precleared with 0.8 µg of normal rabbit serum and immunoprecipitated at 4°C (overnight) with 2 µg of polyclonal antihuman GSTP1 antibody (Biotrin Co., Dublin, Ireland). Protein A-agarose (20 µL) was added and the reaction mixtures incubated for 30 minutes at 4°C. Immunoprecipitates were collected by centrifugation, washed, and analyzed for phosphorylated GSTP1 protein by SDS-PAGE and autoradiography, as described earlier.

Analysis of Mutant GSTP1 Protein Phosphorylation in Glioma Cells.
To further characterize Ser-42 and Ser-184 as phospho-acceptor residues in the GSTP1 protein, pBK-CMV vectors containing the histidine-tagged GSTP1 S42A, S184A, and the double S42A/S184A GSTP1 mutant cDNAs were transfected into cells of the MGR1 cell line, which expresse low levels of endogenous GSTP1, as we described earlier (1) . The transfected cells were then treated with TPA and forskolin followed by [32P]Pi, as described above for the wild-type protein. After 3 hours, the cells were harvested, extracts prepared, and the histidine-tagged GSTP1 proteins were purified on a Talon cobalt resin column according to the manufacturer’s instructions. After SDS-PAGE electrophoresis, the phosphorylated proteins were solubilized in a scintillation mixture, and the amount of radioactivity was determined by scintillation counting. The level of phosphorylation in each of the GSTP1 mutants was determined relative to that of the wild-type GSTP1 protein after subtracting background radioactivity.

Effect of PKA and PKC Activation on GSTP1 Activity in Glioblastoma Cells.
MGR3 cells in mid-exponential growth were treated with 25 µmol/L forskolin (to activate PKA) or 50 nmol/L TPA (to activate PKC). After 6 hours, the cells were harvested and homogenized as described earlier. We centrifuged the cell homogenates at 15,000 x g for 20 minutes at 4°C and used the supernatant to assay for total GST and specific GSTP1 activity using CDNB and ethacrynic acid, respectively, as substrates. The enzyme activities were determined as we described previously (1 , 7 , 8) . Briefly, the protein concentrations of the cell extracts were determined by the Lowry method and adjusted to 2 µg/µl. The reaction mixture contained 50 µL of the cell extract, 2.5 mmol/L GSH and 1 mmol/L CDNB or 2 mmol/L ethacrynic acid. The reactions were followed over 3 minutes at the optimum wavelengths (8) . A unit of GST activity was defined as the amount of enzyme catalyzing formation of 1 µmol of GSH-conjugate per minute at 25°C. Specific GST activity was expressed as units per milligram of protein. The molecular extinction coefficients of the reaction products were used as published previously (8) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human GSTP1 Protein Contains Multiple Putative Ser/Thr Phosphorylation Sites.
The results of the computer-assisted analysis of the GSTP1 protein for the presence of potential serine/threonine phosphorylation sites in the GSTP1 protein are summarized in Table 1Citation . The identified phosphorylation motifs included one for PKA, three for PKC, three for GSK3, five for CKI, and four for CKII, all of which shared complete amino acid sequence homology with the consensus phosphorylation motifs of the respective protein kinases. Five of the putative phospho-acceptor Ser and Thr residues were located within motifs of multiple protein kinases, i.e., Ser-27 (PKC and CKII), Ser-37 (CKI and CKII), Ser-42 (PKC and GSK3), Thr-109 (CKI and CKII), and Ser-184 (PKA and PKC). The sites for CKI and GSK3 are unique because their phosphorylation requires prephosphorylation of the NH2-terminal Ser/Thr (CKI) or the COOH-terminal +4 Ser (GSK3) residues. No phosphorylation sites for the Ser/Thr protein kinases PKG, CaMII, myosin light chain kinase, p34cdc2, and p70s6k were found in the GSTP1 protein. On the basis of these results, we focused our studies of GSTP1 protein phosphorylation on PKA, PKC, and CKII.


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  		Table 1 Phosphorylation motifs of Ser/Thr kinases in the human GSTP1 protein

 
Phosphorylation of the GSTP1 Protein in Cell-Free System.
The results of the analysis of the cell-free phosphorylation of recombinant GSTP1 protein by PKC, PKA, and CKII, summarized in Fig. 1Citation , shows a significant and time-dependent GSTP1 protein phosphorylation by PKC. In all reactions, significant autophosphorylation of PKC was observed as a 84 kDa band. The positive control, histone H1, yielded a phosphorylated product of ~33 kDa. In contrast to PKC, there was no detectable phosphorylation of the GSTP1 protein by PKA. PKA autophosphorylation, however, occurred in all reactions, yielding the expected 40 kDa phosphorylation product. Similar to PKA, no GSTP1 phosphorylation was observed with CKII, although c-jun, a natural CKII substrate, was significantly phosphorylated, appearing as a 40 kDa band. Auto-phosphorylation of CKII-{alpha} and -ß yielded bands of 40 kDa and 33 kDa in size, respectively.



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  		Fig. 1. Phosphorylation of the GSTP1 protein by Ser/Thr kinases in a cell-free system. Recombinant GSTP1 protein was incubated in the protein kinase assays, with and without PKA, rat brain PKC, or CKII. Control substrates were histone H1 for PKA and PKC and c-jun for CKII. After 45 minutes (controls) or 7.5, 15, 30, and 45 minutes, aliquots were removed and analyzed for GSTP1 protein phosphorylation by electrophoresis and autoradiography, as described in the "Materials and Methods" section. The auto-phosphorylated products of the three protein kinases are indicated by arrows.

 
GSH Enhances GSTP1 Protein Phosphorylation by PKA, PKC, and PKC Isoforms.
Figure 2Citation summarizes the results of these studies. In the presence of 5 mmol/L GSH, GSTP1 phosphorylation by both PKA and PKC (Fig. 2A)Citation was rapid and increased time-dependently up to 60 minutes. In contrast to PKC, which phosphorylated the GSTP1 protein, even in the absence of GSH, Fig. 2BCitation showed an absolute requirement of GSH for GSTP1 protein phosphorylation by PKA. Maximum GSH-dependent enhancement of GSTP1 phosphorylation reached a maximum at 2.5 mmol/L GSH for PKA and 1.25 mmol/L for PKC. Phosphorylation of phosphorylated heat- and acid-stable protein-1 or histone H1, both of which lack a GSH-binding site, was unaffected by GSH (results not shown), indicating that the observed enhancement of PKA- and PKC-dependent GSTP1 phosphorylation by GSH is the result of complex formation of GSTP1 with GSH, rather than a nonspecific interaction of glutathione with the substrate or with PKC or PKA. CKII did not phosphorylate the GSTP1 protein even in the presence of GSH (data not shown).



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  		Fig. 2. Effect of GSH on GSTP1 phosphorylation by PKA and PKC. A, GSTP1 protein was preincubated with 5 mmol/L GSH and the level of its phosphorylation determined over 0–60 minutes. B, GSTP1 protein was incubated with increasing GSH concentrations (0 to 5 mmol/L) for 30 minutes, and the level of GSTP1 and control substrate phosphorylation was examined. PHAS-1, phosphorylated heat- and acid-stable protein.

 
The results of the analysis of the effect of GSH on GSTP1 phosphorylation by the PKC isoforms are summarized in Fig. 3Citation and Table 2Citation and show that in the presence of GSH, all eight PKC isoforms belonging to the three PKC classes [i.e., conventional (PKC-{alpha}, -ßI, -ßII, and -{gamma}), novel (PKC-{delta}, -{epsilon}, and -{eta}), and atypical (PKC-{zeta})] phosphorylate the GSTP1 protein efficiently. In the presence of GSH, there was a 4.9- to 52.2-fold increase in GSTP1 phosphorylation, compared with the levels without GSH. Only PKC-{alpha} and PKC-{gamma} phosphorylated the GSTP1 protein significantly in the absence of GSH, the latter to a greater extent than the former. As would be expected, rat brain PKC, which contains primarily PKC-{alpha}, -ßI, -ßII, and -{gamma} isoforms, also phosphorylated the GSTP1 protein significantly, both in the presence and absence of GSH.



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  		Fig. 3. Phosphorylation of the GSTP1 protein by PKC isoforms. The standard phosphorylation reaction was set up containing GSTP1 protein that had been preincubated with and without 5 mmol/L GSH for 30 minutes, and 20 ng of each of the PKC isoforms (PKC-{alpha}, PKC-ßI, PKC-ßII, PKC-{delta}, PKC-{epsilon}, PKC-{gamma}, PKC-{eta}, and PKC-{zeta}). After 30 minutes at 37°C, aliquots were removed and subjected to electrophoresis and autoradiography.

 

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  		Table 2 Phosphorylation of GSTP1 protein by different PKC isoforms

 
Phosphorylation of Amino Acid Residues in GSTP1 Protein Kinase Phosphorylation Motifs.
Figure 4Citation summarizes the results of the analysis of the putative phospho-acceptor amino acid residues in the GSTP1 peptide phosphorylated by PKA, PKC, and the different PKC isoforms. Ser-184Pep, which contains Ser-184 in the GSTP1 phosphorylation motifs of both PKA and PKC, was highly phosphorylated by PKA and PKC, whereas Ser-42Pep present in the GSTP1 PKC phosphorylation motif was phosphorylated by PKC but not PKA. The PKC isoforms varied significantly in their ability to phosphorylate both Ser-42Pep and Ser-184Pep. Ser-184Pep was highly phosphorylated by PKC-{alpha} and PKC-{eta}, modestly by PKC-ßII and PKC-{delta}, and only minimally by PKC-ßI, PKC-{epsilon}, and PKC-{zeta}. In contrast, of the eight PKC isoforms, only PKC-{eta} phosphorylated Ser-42Pep to a significant degree. Neither PKA nor PKC, and none of the PKC isoforms, phosphorylated the GSTP1 peptide containing Ser-27, although the Phosphobase computer application identified this as a putative PKC phosphorylation motif in the GSTP1 protein. As expected, the negative control peptide, P-5523, was not phosphorylated by any of the kinases. Together, these results indicate Ser-42 and Ser-184 (for PKC) and only Ser-184 (for PKA) as putative phospho-acceptor residues in the GSTP1 protein.



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  		Fig. 4. Phosphorylation of GSTP1 peptides containing consensus PKA and PKC phosphorylation sites. Each oligopeptide was homologous to a region of the GSTP1 peptide containing either the PKA or the PKC phosphorylation motif with a single putative phospho-acceptor amino acid residue. Standard protein kinase reactions were set up containing the GSTP1 peptides, PKA, rat brain PKC, or various individual PKC isoforms. After 60 minutes, the reactions were terminated and the phosphorylated peptides separated from ATP by anion exchange chromatography. The radioactivity in the phosphorylated peptides was quantitated by ß-scintilation, normalized against controls and used to compute the level of phosphate incorporated into the peptides.

 
Kinetics and Stoichiometry of GSTP1 Phosphorylation.
In preliminary analysis, we determined that 40 units of PKA and 20 ng of PKC were required for maximum phosphorylation of 1 µg of GSH-bound recombinant GSTP1 in a 30-µL reaction volume containing 100 µmol/L ATP. These conditions were thus used to examine the stoichiometry of GSTP1 phosphorylation. The results, summarized in Fig. 5Citation , show that PKA and rat brain PKC phosphorylated the GSTP1 protein with stoichiometries of 0.4 ± 0.03 and 0.53 ± 0.02 mol phosphate per mol GSTP1. The results of the time course studies of GSTP1 phosphorylation, examined over a 4-hour period, showed that for both PKA and PKC, the level of [32P]phosphate incorporation in the dimeric GSTP1 protein reached a maximum after 60 minutes.



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  		Fig. 5. Stoichiometry of GSTP1 phosphorylation. GSTP1 phosphorylation by PKA and PKC was examined over 0 to 4 hours in a reaction mixture containing 100 µmol/L ATP. After SDS-PAGE, the phosphorylated GSTP1 bands were excised, and the level of 32P-incorporation was determined by ß scintillation counting.

 
Phosphorylation Enhances GSTP1 Enzymatic Activity.
The results of the study of the effects of phosphorylation on the function of the GSTP1 protein in phase II metabolism are summarized in Table 3Citation and show a significant enhancement of the catalytic activity of the GSTP1 protein, after its phosphorylation by both PKA and PKC. For GSH, Km values decreased by 2.8- and 3-fold, whereas the catalytic efficiency, kcat/Km, increased 2- and 2.6-fold for PKA- and PKC-phosphorylated GSTP1 protein, respectively. This observed increased activity of the GSTP1 protein after phosphorylation by both PKA and PKC was statistically significant (P = 0.05). The effects of phosphorylation on GSTP1 function were also significant for the cosubstrate CDNB. For CDNB, Km decreased by 2.6- and 3-fold for GSTP1 phosphorylated by PKA and PKC, respectively, whereas kcat/Km increased by 1.8- and 2.0-fold, respectively.


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  		Table 3 Enhancement of GSTP1 catalytic activity after phosphorylation by PKA and PKC

 
PKA and PKC Activation Enhances GSTP1 Phosphorylation and Enzymatic Activity in Glioma Cells.
The results of these studies, summarized in Fig. 6Citation , show that the GSTP1 protein undergoes phosphorylation in MGR3 cells, after activation of both PKA and PKC signaling pathways. Lysates of MGR3 cells treated with forskolin (Fig. 6A)Citation or TPA (Fig. 6B)Citation and precipitated with anti-GSTP1 antibodies showed a distinct GSTP1 phosphorylation band (Lane 1). The level of phosphorylation was, however, significantly greater (~3.4-fold) when cells were treated with TPA than when treated with forskolin. No GSTP1 phosphorylation was observed with lysates of forskolin- or TPA-treated cells after precipitation with normal rabbit serum (Lane 2), or with lysates of untreated cells immunoprecipitated with anti-GSTP1 antibodies (Lane 3) or normal rabbit serum (Lane 4).



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  		Fig. 6. GSTP1 protein phosphorylation in human glioblastoma cells, after PKA and PKC activation. MGR3 cells were grown for ~16 hours in phosphate- and serum-free MEM and then treated with fresh medium containing 1 mCi/mL [32P]Pi and 50 nmol/L TPA or 25 µmol/L forskolin, to activate the PKA and PKC pathways, respectively. After 3 hours, the cells were lysed, centrifuged, and the supernatants immunoprecipitated with GSTP1 antibody or normal rabbit serum. The immunoprecipitates were analyzed by SDS-PAGE. In Lanes 1 and 3, the lysates were immunoprecipitated with GSTP1 antibody, and in Lanes 2 and 4 with normal rabbit serum. Forskolin- and TPA-treated cells are in Lanes 1 and 2, and untreated control cells in Lanes 3 and 4 of A and B, respectively.

 
Bulk GST activity, determined with the GST substrate, CDNB, in the cells after activation of PKA and PKC were 42 ± 4.37 and 53 ± 6.93 units/mg protein, respectively, compared with 32.3 ± 5.7 units/mg protein in the untreated control. Specific GSTP1 activity, determined with ethacrynic acid as substrate, was 11 ± 3.3, 23.1 ± 4.87, and 29.7 ± 4.61 units/g protein for the control-, forskolin- and TPA-activated MGR3 cells, respectively.

Effects of Mutations of Ser-42 and Ser-184 on GSTP1 Phosphorylation and Catalytic Activity.
The results of the studies examining the effects of mutating Ser-42 and Ser-184 to aspartate on the activity of the GSTP1 protein are summarized in Tables 4Citation and 5Citation . Both the S42D and S184D mutations, with and without phosphorylation of the protein with PKA and PKC, resulted in a significant reduction in catalytic activity (kcat/Km), compared with the wild-type protein (Table 3)Citation . Overall, however, as with the wild-type protein, the activities of the phosphorylated mutant proteins were higher than those of their unphosphorylated counterparts. The activity of the double mutant protein (S42A/S184A) was particularly affected, and after the PKA- and PKC-phosphorylation, reactions were reduced to 91 and 82% of control, respectively. The results of the studies with the Ser->Ala mutations at residues 42 and 184 showed a significant reduction in the level of phosphorylation of the mutant GSTP1 protein in glioblastoma cells with activated PKA and PKC pathways. The level of phosphorylation of the pulled-down histidine-tagged S42A and S184A mutant proteins in cells with activated PKA and PKC was decreased by 67 and 64%, respectively. In cells transfected with the S42A/S184A double mutant, the reduction in GSTP1 phosphorylation was even greater, 91 and 89% of controls, in PKA- and PKC-activated cells. Together, these results further support S42 and S184 as major phosphor-acceptor residues for PKA and PKC and suggest that the increased activity of the phospho-GSTP1 was not simply a consequence of the negative charge introduced in the GSTP1 protein by the phosphate group.


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  		Table 4 Effect of mutations of serine residues 42 and 184 to aspartic acid on the catalytic activity of the GSTP1 protein, without and after phosphorylation by PKA and PKC

 

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  		Table 5 Effect of mutations of serine residues 42 and 184 on the level of phosphorylation of the GSTP1 protein in glioblastoma cells after activation of the PKA and PKC pathways

 
Statistical Analysis.
All data points were determined in triplicate and expressed as the mean ± 1 SD. We analyzed differences in catalytic activity between the unphosphorylated and phosphorylated GSTP1 protein for statistical significance using ANOVA.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The findings in this study provide conclusive evidence that the human GSTP1 protein is phosphorylated by Ser/Thr protein kinases, PKA and PKC, and that the phosphorylation enhances the metabolic function of the GSTP1 protein. The former results are consistent with previous observations of phosphorylation of both murine and human GSTs (33, 34, 35) . In this study, we show that GSTP1 protein phosphorylation by PKA and PKC is GSH-dependent and, in glioma cells, is significantly enhanced after activation of PKA- and PKC-dependent signaling pathways. Both PKA and PKC are ubiquitous cellular Ser/Thr protein kinases that regulate many important physiologic functions in cells by phosphorylating serine and threonine residues in unique motifs of key regulatory proteins. Members of the PKC family function as signal transducers and are characterized by a dependence on the lipid second messenger, diacylglycerol and Ca+2, or phorbol esters for their activation, auto-phosphorylation and trans-phosphorylation, and on phosphoinositide-dependent protein kinases and tyrosine kinases for their maturation (38 , 39) . They are high affinity receptors for the phorbol ester tumor promoters and are involved in multiple stages of carcinogenesis and tumor progression. PKAs belong to another important family of Ser/Thr kinases, the activation of which is dependent on the second messenger cAMP (26 , 40) . After activation of the membrane-bound adenylate cyclase (e.g., by forskolin) intracellular cAMP is increased, a result of the conversion of ATP to cAMP by the activated adenylate cyclase (40) . The increased intracellular cAMP then activates PKA by dissociating its regulatory subunits from the catalytic subunits. Recently, we reported that the GSTP1 gene is transcriptionally activated by cAMP via a novel consensus cAMP-responsive element (CRE) in the 5'-region of the gene (41) and showed that the activation is mediated by the binding of PKA-phosphorylated CRE-binding protein-1 to the CRE, after activation of the adenylate cyclase pathway.

The findings of a post-translational phosphorylation of the GSTP1 protein in this study, together with those we previously reported to involve CRE-binding protein-1/CRE-mediated transcriptional activation of the GSTP1 gene (41) indicate two levels of cellular PKA-dependent GSTP1 regulation, one transcriptional and the other post-translational. Under our experimental conditions, PKC was more efficient than PKA in phosphorylating the GSTP1 protein. Intracellularly, PKC may thus be more important than PKA in the post-translational regulation of GSTP1 function via phosphorylation. This is supported by our observation of a higher level of GSTP1 protein phosphorylation in glioblastoma cells after activation of PKC than after PKA activation.

The observation that GSTP1 protein phosphorylation by the two Ser/Thr kinases was highly dependent on reduced GSH has important implications, because many human tumors are characterized by elevated GSH levels (up to 10 mmol/L; refs. 5 , 6 , 42, 43, 44 ). The significant enhancement of PKC phosphorylation of GSTP1 by GSH and the apparent absolute requirement of GSH for PKA-dependent GSTP1 phosphorylation suggests that the GSTP1 protein in cells of tumors with high GSH content will exist in a hyper-phosphorylated and enzymatically more active state. This combined with the high levels of PKC often present in glioma and other tumor cells will result in an enhanced drug conjugation/inactivation leading to drug resistance. In normal cells, an antioxidant state associated with elevated GSH will result in a functionally enhanced GSTP1 and, consequently, in increased cellular protection against carcinogens and genotoxins. The molecular basis for the enhancing effect of GSH on GSTP1 phosphorylation by PKC and PKA is unclear. On the basis of X-ray crystallographic studies showing that the G-site of the GSTP1 peptide, located within an {alpha}-helix region containing amino acids 35–46, assumes a less flexible form after GSH binding (45, 46, 47) , we postulate that conformational changes induced in the GSTP1 protein after GSH binding may position the GSTP1 protein more favorably for phosphorylation by the Ser/Thr kinases.

Another major finding in this study is that both in the cell-free system and in human glioblastoma cells with activated PKA and PKC, the enzymatic activity of the phosphorylated GSTP1 protein increased significantly compared with the unphosphorylated counterpart. In glioblastoma cells, activation of the PKC pathway was associated with an increase in GSTP1 activity by almost 3-fold. The utilization ratio, kcat/Km of the phosphorylated protein was almost 2.5-fold higher than that of the unphosphorylated protein. The effect of phosphorylation was slightly higher for the cosubstrate GSH than for CDNB, suggesting that the phosphorylation may have a greater effect on the G-site than on the H-site of the GSTP1 protein. The increased activity of the GSTP1 protein after its phosphorylation is consistent with similar alterations in enzymatic activity observed in a number of other important cellular proteins, including, 5-lipoxygenase (29) , telomerase (48) , and topoisomerase II (49) , after their phosphorylation. Although a number of studies (45 , 50 , 51) have shown Trp-38, Gly-41, Lys-44, Cys-47, and Gly-50 to be critical residues that determine GSH affinity to the G-site of the GSTP1 protein, our data showing that Ser-42 and Ser-184 are among the putatative residues, the phosphorylation results of which in altered Km and kcat/Km of GSTP1 for GSH suggest that these two amino acids may also be critically involved in GSH binding to the G-site and ultimately in determining GSTP1 enzymatic function.

The results of the studies of PKA and PKC phosphorylation sites in the GSTP1 protein using GSTP1 oligopeptides indicated Ser-42 and Ser-184 to be potential phosphor-acceptor residues for both kinases. To gain further insight into these as among the phospho-acceptor amino acids, we mutated the two serines to alanine, individually and jointly, and showed that the level of phosphorylation of the mutant proteins in glioblastoma cells was significantly decreased for both the S42A and S184A mutants and almost undetectable in the double mutant. The two serines were also mutated to aspartic acid to examine the extent to which the negative charge introduced at the phosphorylation site was involved in the changes in catalytic activity observed after phosphorylation of the GSTP1 protein. In both cases, the mutations actually decreased the catalytic efficiency of the GSTP1 protein, with the S184D having a greater impact on catalytic function of the GSTP1 protein than the S42D mutation. This observation is consistent with the observed higher level of phosphorylation of the S184Pep than the S42Pep GSTP1 peptide.

All eight PKC isoforms, PKC-{alpha}, PKC-ßI, PKC-ßII, PKC-{epsilon}, PKC-{gamma}, PKC-{eta}, and PKC-{zeta} phosphorylated the GSTP1 protein efficiently, and with the exception of PKC-{alpha} and PKC-{gamma}, the phosphorylation was GSH-dependent. Interestingly, PKC-{alpha}, which is highly expressed in malignant gliomas (52) , was among the most potent of the PKC isoforms in its ability to phosphorylate the GSTP1 protein, both in the presence and in the absence of GSH. PKC-targeted antisense oligodeoxyribonucleotides and small molecule inhibitors that suppress PKC expression and/or its activity, are currently being developed as novel anticancer therapeutics (53 , 54) . Our findings in this study suggest that the increased drug sensitivity observed in tumors treated with such PKC inhibitors (53 , 54) may, at least in part, be because of a decrease in GSTP1-mediated drug metabolism, resulting from decreased GSTP1 phosphorylation.

In summary, we provide conclusive evidence of phosphorylation of the GSTP1 protein as a post-translational mechanism of regulation of GSTP1 function in xenobiotic metabolism that can contribute to normal tissue protection and to tumor growth and that can cause tumor resistance to chemotherapeutic agents metabolized by GSTP1. The phosphorylation could also affect other important cellular processes in which GSTP1 plays a role, including stress response, signaling, and apoptosis. These findings establish a strong link between cellular Ser/Thr kinase-dependent signaling pathways and GSTP1-mediated phase II metabolism and thus advance our understanding of the role of the GSTP1 gene and its encoded protein in normal and neoplastic biology.


   FOOTNOTES
 
Grant support: RO1 CA91438 and RO1 CA79644 to FA-O from the National Cancer Institute, National Institute of Health.

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.

Note: Present address for H-W. Lo is Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030.

Requests for reprints: Francis Ali-Osman, 199 Medical Science Research Building Durham, NC 27710; Phone 919-681-5769; Fax: 1-919-684-8757; E-mail: francis.aliosman{at}duke.edu

1 www.cbs.dtu.dk/databases/PhosphoBase. Back

Received 2/ 6/04. Revised 8/30/04. Accepted 10/11/04.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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