Protein Kinase A-mediated Phosphorylation of the Galpha 13 Switch I Region Alters the Galpha beta gamma 13-G Protein-coupled Receptor Complex and Inhibits Rho Activation*

Jeanne M. ManganelloDagger§, Jin-Sheng Huang§, Tohru Kozasa, Tatyana A. Voyno-Yasenetskaya, and Guy C. Le Breton

From the Department of Pharmacology, University of Illinois, Chicago, Illinois 60612

Received for publication, September 9, 2002, and in revised form, October 18, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies mapped the protein kinase A (PKA) phosphorylation site of Galpha 13 and studied the consequences of its phosphorylation. Initial experiments using purified human Galpha 13 and the PKA catalytic subunit established that PKA directly phosphorylates Galpha 13. The location of this phosphorylation site was next investigated with a new synthetic peptide (G13SRIpep) containing the PKA consensus sequence (Arg-Arg-Pro-Thr203) within the switch I region of Galpha 13. G13SRIpep produced a dose-dependent inhibition of PKA-mediated Galpha 13 phosphorylation. On the other hand, the Thr-phosphorylated derivative of G13SRIpep possessed no inhibitory activity, suggesting that Galpha 13 Thr203 may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Galpha 13-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Galpha 13 phosphorylation stabilized coupling of Galpha 13 with platelet thromboxane A2 receptors but destabilized coupling of Galpha 13 to its beta gamma subunits. In order to determine the functional consequences of this phosphorylation on Galpha 13 signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells overexpressing human Galpha 13 wild type (Galpha 13-WT) or Galpha 13-T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%; p < 0.002) of Rho activation in Galpha 13 wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of Galpha 13 Thr203.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Protein phosphorylation is a well established and ubiquitous mechanism for regulating protein function. Such regulation can impact cellular signaling at multiple levels including enzymatic activity, protein structure, protein translocation, and protein-protein interactions, among others. Regarding seven-transmembrane receptors, evidence has been provided that phosphorylation of specific sites can serve to alter receptor-G protein coupling, initiate receptor internalization, and ultimately modulate cross-membrane receptor signaling (1). Whereas such effects of receptor phosphorylation have been well documented, considerably less is known regarding the prevalence or the possible signaling consequences of G protein phosphorylation (2-6). Nevertheless, several reports have provided evidence that G protein phosphorylation can alter heterotrimer complex formation and downstream signaling events. Specifically, Kozasa and Gilman (4) demonstrated that in vitro phosphorylation of Galpha 12 and Galpha z by PKC1 results in conformational changes that inhibit interaction of the Galpha and Gbeta gamma subunits. A similar PKC-mediated phosphorylation effect on Galpha -Gbeta gamma interaction was observed by Fields and Casey (3) for Galpha z and by Murthy et al. (6) for Galpha i1/2. Offermanns et al. demonstrated that a PKC-dependent phosphorylation of Galpha 12 and Galpha 13 occurred when human platelets were activated by thrombin, the TXA2 mimetic U46619, or the phorbol ester phorbol 12-myristate 13-acetate (PMA) (7). Collectively, the above results therefore suggest that different Galpha subunits can indeed serve as substrates for PKC. Whereas the functional consequences of such phosphorylation remain largely unknown, the strategic position of G proteins in the signaling cascade suggests that phosphorylation-mediated changes in G protein conformation or activity could serve as a significant regulatory point in the signaling process. In this connection, we recently provided evidence that TXA2 receptor-coupled Galpha 13 is phosphorylated through a PKA-mediated process (8). These results represented the first demonstration that a platelet Galpha subunit is phosphorylated by cAMP-dependent kinase and provided a possible explanation for the high sensitivity of TXA2 receptor signaling to increased cAMP levels. The present studies extend these findings by characterizing the specific PKA phosphorylation site in Galpha 13 and identifying the molecular and functional consequences of this phosphorylation. Our results demonstrate that PKA phosphorylates Galpha 13 at Thr203, which is situated within the functionally important switch I region of the Galpha subunit (9-14). This phosphorylation results in stabilization of Galpha 13 coupling to TXA2 receptors and destabilization of Galpha 13 coupling to its beta gamma subunits. Separate experiments also provided evidence that a functional consequence of this phosphorylation is decreased basal Rho activation levels. Thus, PKA-mediated Galpha 13 Thr203 phosphorylation appears to represent a novel mechanism for the regulation of signaling through the Galpha 13 pathway.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- G13SRIpep (CLLARRPTKGIHEY) and G13SRIpepP (CLLARRPpTKGIHEY (where pT represents phosphothreonine)) peptides were synthesized by Multiple Peptide Systems (San Diego, CA). An N-terminal cysteine was added to each peptide to aid in coupling to carrier protein for future antibody production. Outdated human platelet units were obtained from Heartland Blood Centers (Aurora, IL). G418 was obtained from Calbiochem; PKAcat (protein kinase A, catalytic subunit, bovine heart), protein A-Sepharose, aprotinin, leupeptin, coumeric acid, luminol, 8-Br-cAMP, NaF, and CHAPS were obtained from Sigma; [gamma -32P]ATP (4,500 Ci/mmol) was purchased from ICN Biochemicals, Inc.; G13-N, Myc, Galpha 12, and Gbeta common IgG and Rho A polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit polyclonal anti-peptide antibodies to TXA2 receptor (P2 Ab) and Galpha q (QL Ab) were prepared as previously described (15); the Wizard Plus Midiprep DNA Purification kit was from Promega (Madison, WI); LipofectAMINE Plus and LipofectAMINE 2000 were purchased from Invitrogen; the QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA); horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) was from Bio-Rad; the BCA protein assay kit was from Pierce; and the Rho activation assay kit was from Upstate Biotechnology, Inc. (Lake Placid, NY). Primers (T203A-DS, 5'-GGA TGC CTT TGG CGG GTC TTC TGG CAA GCA GAA TAT CTT G-3'; T203A-US, 5'-GCC AGA AGA CCC GCC AAA GGC ATC CAT GAA TAC GAC TTT G-3') were from Integrated DNA Technologies, Inc. (Coralville, IA).

In Vitro Phosphorylation of Purified Galpha 13-- Galpha 13 (16) was purified from Sf9 cells, as previously described (17). For phosphorylation of purified Galpha subunits, 5 pmol of Galpha subunit was added to 20 mM [gamma -32P]ATP (4,500 Ci/mmol), PKAcat (protein kinase A, catalytic subunit (50 ng; 60 units) or PKA vehicle (100 mM NaCl, 20 mM MES, pH 6.5, 30 mM beta -mercaptoethanol, 100 mM EDTA, 50% ethylene glycol) in phosphorylation buffer (25 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 125 mM CaCl2, 1 mM dithiothreitol) in a total reaction volume of 115 µl, and the mixture was incubated for 30 min at 30 °C. The reaction was terminated by adding Laemmli (18) sample buffer (62.5 mM Tris-HCl, pH 6.5, 3% SDS, 10% glycerol). Proteins were separated by SDS-PAGE and were visualized by silver staining (19) followed by autoradiography.

Cell Culture and Transfection-- COS-7 cells and CHO cells were maintained in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. COS-7 cells were transiently transfected when the cells were 80-90% confluent in 100-mm culture dishes. The medium was replaced with serum-free Dulbecco's modified Eagle's medium, and the cells were transfected using the LipofectAMINE Plus method (Invitrogen) according to the manufacturer's protocol, using 10 µg of DNA and 20 µl of LipofectAMINE per transfection. After 3 h at 37 °C, the medium was supplemented with 1 volume of 20% fetal bovine serum, Dulbecco's modified Eagle's medium. Experiments were performed 48 h after transfection. Transient transfection of COS-7 cells with cDNAs for Galpha 13, Gbeta 1, and Myc-tagged Ggamma 2 subunits was performed using LipofectAMINE 2000 reagent, according to the manufacturer's instructions. Transfections in CHO cells with human Galpha 13 wild type (Galpha 13-WT) or Galpha 13-T203A mutant plasmids were performed as described above. After 3 days of transfection, G418 (500 µg/ml) was added for selection. After 3-5 weeks, the stable cell lines were established and confirmed by Western blot.

Immunoprecipitation and Phosphorylation of Galpha 13-- The transfected cells were washed three times with cold PBS and were harvested with lysis buffer (50 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml leupeptin). The lysates were centrifuged to remove insoluble material, and the protein concentration of the supernatant was measured using the BCA protein assay method. The protein concentration of each lysate was adjusted to be equal within an experiment and was typically 0.50-2.0 mg/ml. Aliquots of 0.6 ml were incubated overnight at 4 °C with 10 µg/ml Galpha 13 antibody and were subsequently incubated with protein A-Sepharose beads (55 µl of a 10% (w/v) suspension) for 4 h at 4 °C. The immune complexed beads were washed once with lysis buffer and were supplemented with resuspension buffer (50 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.014% Tween 20, 1 mM dithiothreitol). 85 µl of protein-bound beads were then incubated with 50 µM [gamma -32P]ATP, 150 ng of PKA, 1 mM cAMP, and the indicated concentration of blocking peptide in a total volume of 125 µl. The mixture was incubated for 30 min at 30 °C, after which time the beads were pelleted by centrifugation and washed three times with resuspension buffer. The beads were then suspended in Laemmli sample buffer plus resuspension buffer, and immune complexes were eluted by boiling. The eluted proteins were subjected to SDS-PAGE, silver staining, and autoradiography. Phosphorylated proteins on autoradiograms were quantified using a densitometer (Protein Databases, Inc., Huntington Station, NY), and values were normalized for the amount of silver-stained protein on the gel.

DNA Constructs and Site-directed Mutagenesis-- Galpha 13 was cloned from a human adenocarcinoma LoVo cell line and was subcloned into a pcDNA3 vector. The Galpha 13 mutation with the substitution of Ala for Thr203 was performed using the Stratagene QuikChange site-directed mutagenesis kit according to the manufacturer's protocol. The final mutant was verified by DNA sequencing.

Ligand Affinity Chromatography-- 12 units of outdated human platelets were pooled and incubated with 3 mM aspirin for 45 min. Solubilized platelet membranes were then prepared as previously described (8), yielding a typical protein concentration of 4.5 mg/ml. Solubilized membranes were phosphorylated and purified by ligand affinity chromatography, as described (8). Briefly, solubilized proteins were incubated with 1 mM 8-Br-cAMP or vehicle (H2O), 100 µl of ATP, 60 mM CaCl2, and incubation medium (45 mM histidine HCl, 50 mM KH2PO4, 20 mM NaF, 120 mM KCl, pH 7.4) for 30 min at 30 °C. The phosphorylated samples were supplemented with 500 mM KCl, 0.5 mg/ml asolectin, 20% glycerol, 0.2 mM EGTA, 10 mM CHAPS and were incubated on the ligand affinity columns overnight at 4 °C. Unbound proteins were washed with buffer D (20 mM Tris base, 10 mM CHAPS, 20% glycerol, 500 mM KCl, 0.2 mM EGTA, 0.5 mg/ml asolectin, pH 7.4) supplemented with 20 mM NaF and 2 mM orthovanadate. TXA2 receptor-G protein complexes were eluted from the column with the TXA2 antagonist BM13.177 in buffer D at a flow rate of 0.125 ml/min. A 3-ml elution fraction was collected, dialyzed in 10 mM NH4HCO3, and then lyophilized. Proteins were reconstituted into 150 µl of H2O and were subjected to SDS-PAGE.

Pull-down Assay for GTP-Rho-- Rho activation was determined as previously described (20). CHO-Galpha 13-WT and CHO-Galpha 13-T203A mutant cells were seeded on six-well plates, grown to 80% confluence, and serum-starved for 24 h. Following treatment with 8-Br-cAMP (1 mM) at 37 °C for 15 min, the cells were washed once with PBS and harvested with 300 µl of lysis buffer (50 mM Tris-HCl, pH 7.5 containing 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, 50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml each of leupeptin and pepstatin, and 0.2% Nonidet P-40). The cell lysates were clarified by centrifugation at 15,000 × g for 1 min and incubated with 20 µg of glutathione S-transferase-Rho binding domain fusion protein conjugated with glutathione beads at 4 °C for 1 h. The beads were washed three times with lysis buffer and subjected to SDS-PAGE on a 12% gel. Bound RhoA was detected by Western blot using a polyclonal antibody against RhoA.

Statistical Analysis-- Data were analyzed according to the analysis of variance using Dunnett's multiple comparison post-test or Student's t test, as indicated, using GraphPad PRISM statistical software (San Diego, CA). Statistical significance is defined as p < 0.05, p < 0.01, or p < 0.002, as indicated.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In Vitro PKA phosphorylation of Purified Galpha 13-- Whereas we previously demonstrated that cAMP induces phosphorylation of TXA2 receptor-coupled Galpha 13 in human platelets (8), these results did not determine whether the observed phosphorylation is directly mediated by PKA or whether additional kinases are involved. In order to address this question, additional studies were performed using human Galpha 13 purified from Sf9 cells (16) with [gamma -32P]ATP in the presence or absence of the PKA catalytic subunit (PKAcat). Electrophoretic separation and silver staining of these samples revealed the presence of a single protein at 44 kDa corresponding to Galpha 13 (Fig. 1a, lanes 1 and 2) but no such band in the presence of PKA alone (Fig. 1a, lane 3). Analysis of this gel by autoradiography revealed substantial phosphorylation of Galpha 13 (at 44 kDa) when PKA is added (Fig. 1b, lane 1) but no phosphorylation (Fig. 1b, lane 2) in the absence of PKA. These experiments therefore establish that PKA alone is sufficient to phosphorylate Galpha 13. The specificity of this Galpha 13 phosphorylation was examined in parallel experiments by assaying a separate platelet G protein, Galpha i (purified from Sf9 cells as previously described) (17). Fig. 1a illustrates a single protein band (lanes 4 and 5) corresponding to Galpha i. However, it can also be seen (Fig. 1b, lanes 4 and 5) that no phosphorylation of Galpha i was observed in either the presence or the absence of PKA. Collectively, these results demonstrate that PKA directly phosphorylates Galpha 13 and that this reaction does not require intermediate kinases or other co-factors.


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Fig. 1.   In vitro PKA phosphorylation of purified Galpha 13. a, silver-stained gel of purified Galpha 13 and Galpha i in the presence and absence of PKA. b, autoradiogram of phosphorylated Galpha 13 and Galpha i in the presence or absence of PKA. Results are representative of three separate experiments.

Inhibition of Galpha 13 Phosphorylation by a Galpha 13 Peptide Containing a PKA Consensus Sequence-- A 14-amino acid synthetic peptide (G13SRIpep) corresponding to the specific Galpha 13 amino acid sequence Leu197-Tyr209 was initially used to locate the PKA phosphorylation site of Galpha 13. This amino acid region was selected because analysis of the primary structure of the Galpha 13 subunit revealed only one PKA consensus sequence represented by Arg-Arg-Pro-Thr203. On this basis, G13SRIpep, which encompasses this region, was used to probe the Leu197-Tyr209 region for potential phosphorylation. Thus, it was reasoned that PKA-mediated phosphorylation of Galpha 13 would be blocked or reduced by a competing peptide substrate (G13SRIpep) containing an amino acid sequence that is identical to that contained within Galpha 13. In these experiments, human wild type Galpha 13 was immunoprecipitated from transiently transfected COS-7 cell using protein A-Sepharose beads coupled to an anti-peptide antibody directed against the N terminus of Galpha 13 (G13-N IgG). The alpha  subunit-antibody-bead complex was then incubated with PKAcat and [gamma -32P]ATP in the presence or absence of G13SRIpep. Following incubation, the proteins were eluted from the beads and were subjected to gel electrophoresis and autoradiography. It can be seen that the efficiency of immunoprecipitation was comparable for each Galpha 13-transfected COS-7 cell sample (Fig. 2a, lanes 1-6). It can also be seen that PKA induced a substantial Galpha 13 phosphorylation (Fig. 2b, lane 1). Furthermore, upon the addition of G13SRIpep, there was a dramatic dose-dependent inhibition of this PKA-mediated effect (Fig. 2b, lanes 2-5). Quantitation of this peptide inhibition is illustrated in Fig. 2c, where it can be seen that Galpha 13 phosphorylation was significantly blocked at a peptide concentration of as little as 50 µM, and was completely inhibited at a peptide concentration of 500 µM. Fig. 2a, lanes 7 and 8, demonstrate that the vector-transfected COS-7 cells do not reveal detectable amounts of Galpha 13 under these conditions, and consistent with this finding, there was no observed PKA-induced phosphorylation in the 44-kDa region of the immunoprecipitated protein samples (Fig. 2b, lanes 7 and 8). These findings using transfected COS-7 cells therefore confirm our previous results in platelets showing PKA-mediated phosphorylation of Galpha 13 and suggest that the PKA phosphorylation site of Galpha 13 is contained within the Leu197-Tyr209 sequence. However, since it is possible that the ability of G13SRIpep to block phosphorylation was due to a nonspecific peptide effect, a control peptide was evaluated in subsequent experiments. Specifically, it was reasoned that the most appropriate control would employ a peptide with an amino acid sequence identical to that of G13SRIpep, the only difference being substitution of a phosphorylated Thr in the peptide sequence. Thus, the phosphorylated peptide should serve as a poor substrate for PKA. On this basis, G13SRIpepP was synthesized and tested for its ability to affect PKA-mediated phosphorylation under the same experimental conditions as those used for G13SRIpep. As can be seen in Fig 3a, lanes 1-4, equal amounts of Galpha 13 protein were immunoprecipitated under each experimental condition. It can also be seen (Fig. 3b, lane 1) that PKA addition again resulted in substantial Galpha 13 phosphorylation. However, in contrast to the effects of G13SRIpep, the addition of G13SRIpepP was completely ineffective in blocking this PKA-mediated phosphorylation (Fig. 3b, lanes 2 and 3), even at a concentration (500 µM) that resulted in complete inhibition by G13SRIpep. The quantitative analysis of these phosphorylation profiles is illustrated in Fig. 3c. Since Galpha 13 contains only a single PKA consensus sequence within Leu197-Tyr209, these findings therefore suggest 1) that G13SRIpep specifically acts to block PKA-induced phosphorylation of Galpha 13; 2) that the location of the PKA phosphorylation site is contained within the Leu197-Tyr209 sequence of Galpha 13; and 3) that Thr203 serves as the phosphorylation site within this sequence.


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Fig. 2.   Inhibition of Galpha 13 phosphorylation by a Galpha 13 peptide sequence. a, silver stain of immunoprecipitated Galpha 13 protein. b, autoradiogram of phosphorylated Galpha 13. c, dose-dependent inhibition of PKA-induced phosphorylation of Galpha 13 by G13SRI peptide; n = 6. Statistical analysis was performed by analysis of variance. *, p < 0.05; **, p < 0.01.


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Fig. 3.   No Inhibition of PKA-induced phosphorylation of Galpha 13 by G13SRI-P peptide. a, silver stain of immunoprecipitated Galpha 13 protein. b, autoradiogram of phosphorylated Galpha 13; c, quantitative analysis of PKA-induced phosphorylation; n = 4. Statistical analysis was performed by analysis of variance. *, p < 0.05; **, p < 0.01.

PKA Does Not Phosphorylate Galpha 13-T203A Mutant Protein-- In order to provide independent confirmation that Thr203 is in fact the residue phosphorylated by PKA, a mutant Galpha 13 (Galpha 13-T203A) was produced in which Thr203 was changed to Ala by site-directed mutagenesis. In these experiments, the Galpha 13-T203A mutant (or wild type Galpha 13) was immunoprecipitated from transiently transfected COS-7 cells using the G13-N antibody, and the alpha  subunit-antibody-bead complex was subjected to phosphorylation, as described earlier. It can be seen (Fig. 4a, lanes 1-4) that the efficiency of immunoprecipitation was comparable for both wild type and mutant protein. However, a comparison of the phosphorylation profiles reveals that substitution of Thr203 with Ala resulted in a complete loss of PKA-mediated Galpha 13 phosphorylation (Fig. 4b). In this regard, some experiments revealed a minor phosphorylation in the mutant with or without PKA. However, this apparent phosphorylation was not found to be significantly different from vector control, and no detectable phosphorylation was observed in the mutant autoradiogram (Fig. 4b, lanes 3 and 4). The complete absence of PKA-induced phosphorylation in the T203A mutant, in combination with the previous results, therefore provides evidence that PKA phosphorylates a single site at Thr203 contained within switch I region of Galpha 13.


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Fig. 4.   No PKA-induced phosphorylation of Galpha 13-T203A mutant protein. a, phosphorylation of Galpha 13 WT and Galpha 13-T203A mutant protein by PKA; n = 6. b, autoradiogram of phosphorylated Galpha 13 protein. c, silver stain of immunoprecipitated Galpha 13 protein.

Binding of beta gamma to Wild Type Galpha 13 and Galpha 13-T203A Mutant Protein-- The next series of experiments investigated the molecular consequences of this PKA-mediated Thr203 phosphorylation. Because phosphorylation is known to alter protein conformation and function, it is possible that Thr203 phosphorylation may change the conformational or chemical characteristics of Galpha 13 and thereby alter its normal function in the receptor-coupled signaling process. Indeed, this possibility might be anticipated for two reasons: 1) Thr203 resides within switch I region of the Galpha 13 subunit, and 2) the switch I region is considered important for modulating activation of the alpha  subunit and for regulating its affinity for the beta gamma subunits (16). Based on these considerations, we examined whether PKA phosphorylation of the switch I region Thr203 residue affects the stability of the Galpha beta gamma 13 heterotrimeric complex.

In these studies, COS-7 cells were transfected with either wild type or mutant Galpha 13 (Galpha 13-T203A) as well as with beta  and Myc-tagged gamma 2. The beta gamma subunits were then immunoprecipitated with anti-Myc antibody. It can be seen that co-transfection of beta  and Myc-tagged gamma 2 cDNAs alone (without Galpha 13) showed no Galpha 13 protein in either the lysate or the immunoprecipitate (Fig. 5, row a, lane 3, and row b, lane 3). As expected, Galpha 13 was immunoprecipitated (Fig. 5 row a, lane 2) and present in the lysate (Fig. 5, row b, lane 2) when the cells were co-transfected with beta , Myc-tagged gamma 2 cDNAs, and wild type Galpha 13. On the other hand, no such Galpha 13 immunoprecipitation was observed (Fig. 5, row a, lane 1) when the co-transfection was done with Galpha 13-T203A mutant. Furthermore, Western blotting of total cell lysates demonstrated that both constructs of Galpha 13 were expressed at the same levels (Fig. 5, row b, lanes 1 and 2), suggesting that the inability of mutated Galpha 13 to complex with beta 1gamma 2 was not due to insufficient expression. These findings therefore suggest that mutation of Galpha 13 at Thr203 disrupts heterotrimer stability.


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Fig. 5.   beta gamma binding to wild type Galpha 13 and Galpha 13-T203A mutant protein. The figure illustrates an immunoblot of Galpha 13 that co-precipitated with Myc-tagged beta gamma from transfected COS-7 cells. Wild type Galpha 13 co-immunoprecipitated with beta gamma subunits (lane 2), whereas the mutant Galpha 13 protein did not (lane 1).

Ligand Affinity Co-purification of TXA2 Receptor-G Protein Complexes-- The next series of experiments extended the above finding and investigated possible consequences of Galpha 13 Thr203 phosphorylation on stability of the receptor-Galpha 13 complex. To this end, affinity purification of the receptor-G protein complex was employed. This technique has previously been used to co-purify native TXA2 receptors with their associated Galpha proteins (21, 22) and was used to demonstrate PKA phosphorylation of TXA2 receptor-coupled Galpha 13 (8). In the present experiments, solubilized platelet membranes were prepared and incubated in the presence or absence of 1 mM 8-Br-cAMP plus [gamma -32P]ATP. The TXA2 receptor-G protein complexes were then purified by affinity chromatography (8), and the column eluate was assayed for TXA2 receptor, Galpha 13, Galpha q, and beta  subunits by immunoblotting. It can be seen that in the absence of 8-Br-cAMP (Fig. 6a, lane 1) TXA2 receptors co-purified with their associated G protein heterotrimeric complexes. Thus, the TXA2 receptor elute fractions yielded positive immunoreactivity for the Galpha 13, Galpha q, and Gbeta subunits. It can also be seen, however, that 8-Br-cAMP treatment produced a significant shift in this elution profile (i.e. a substantial increase in co-eluted Galpha 13 and a substantial decrease in co-eluted Gbeta ) (Fig. 6a, lane 2). On the other hand, 8-Br-cAMP treatment produced no change in the amounts of either Galpha q or TXA2 receptor (Fig. 6a, lanes 1 and 2). Quantitation of these results is illustrated in Fig. 6b.


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Fig. 6.   Effect of PKA phosphorylation on TXA2 receptor-G protein complex formation. a, immunoblot of TXA2 receptor and G proteins eluted from the ligand affinity column in the absence (lane 1) or presence (lane 2) of 1 mM 8-Br-cAMP. The illustrated blot represents an experiment that yielded one of the largest differences between experimental and control conditions. b, quantitation of average protein elution levels from four different experiments (determined by densitometric scan); *, p < 0.05.

The ability of 8-Br-cAMP to cause such a selective effect on the G13 heterotrimeric complex is consistent with previous results, which have shown that PKA is capable of phosphorylating Galpha 13 (8) but incapable of phosphorylating either Galpha q (23) or platelet TXA2 receptors.2 Furthermore, since it is known that co-elution of complexed proteins is proportional to the affinity between these proteins, the observed shifts in the elution profiles for both Galpha 13 and beta  subunits suggest that PKA-mediated Thr203 phosphorylation of Galpha 13 has two separate effects on Galpha 13. First, this phosphorylation appears to destabilize its association with Gbeta gamma subunits, resulting in beta gamma dissociation. This finding is consistent with the results in Fig. 5 using Myc-tagged gamma 2. Second, Thr203 phosphorylation appears to stabilize the association between TXA2 receptors and Galpha 13. Thus, PKA-mediated phosphorylation within the switch I region of Galpha 13 appears to impact the interaction of this Galpha subunit with each of its protein partners.

PKA-mediated Inhibition of Rho Activation-- Since Galpha 13 is known to signal through the Rho pathway (16, 24-27), the final series of experiments investigated the effects of Galpha 13 Thr203 phosphorylation on the basal Rho activation state. In these studies, CHO cell lines overexpressing human Galpha 13-wild type (Galpha 13-WT) or Galpha 13-T203A mutant (which cannot undergo PKA phosphorylation; Fig. 4) were assayed for Galpha 13-Rho activation. Fig. 7 illustrates the fraction of activated Rho A relative to total Rho A in the cell homogenate. Using Galpha 13-WT cells, it was found that the addition of 1 mM 8-Br-cAMP caused a substantial decrease in basal Rho activation levels (Fig. 7, lanes 1 and 2). On the other hand, 8-Br-cAMP did not significantly decrease Rho activation in the Galpha 13-T203A mutant (Fig. 7, lanes 3 and 4). The average of seven separate experiments revealed that 8-Br-cAMP caused a 50 ± 14% inhibition (p < 0.002) in the Galpha 13-WT cells but had no significant effect (5 ± 13%; p > 0.4) in the mutant cells. These results therefore demonstrate that Galpha 13 T203A mutation decreases the ability of 8-Br-cAMP to block Rho activation, suggesting that Galpha 13 phosphorylation at Thr203 interferes with the Galpha 13 signaling process.


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Fig. 7.   PKA-mediated inhibition of Rho activation. The figure illustrates Rho activation in Galpha 13-WT-transfected CHO cells (lanes 1 and 2) and Galpha 13-T203A mutant (lanes 3 and 4). Effects of 8-Br-cAMP on basal Rho activation levels are shown (lanes 2 and 4).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heterotrimeric G proteins are ubiquitously expressed across different cell lines and serve as important mediators in the initiation and maintenance of cellular function. As such, these proteins are particularly well poised to serve as targets for modulation of the separate signaling pathways. One mechanism by which the functional activity of G proteins might be regulated is through the process of phosphorylation. For example, results from studies by Aragay and Quick (5) suggested that thyrotropin-releasing hormone responses require PKC phosphorylation of Galpha 16. This suggestion was based on the finding that mutation of the Galpha 16 PKC phosphorylation sites blocked the thyrotropin-releasing hormone-induced responses (5). Other studies have indicated that PKC-mediated phosphorylation can down-regulate G protein activity. Specifically, Glick and Casey (28) reported that phosphorylation of Galpha z by PKC substantially reduced its activation. Furthermore, Murthy et al. reported that PKC activation leads to phosphorylation of Galpha i1/Galpha i2, and blocks downstream signaling events mediated through both Galpha i1- and Galpha i2-coupled receptors. Moreover, they suggested that phosphorylation of Galpha subunits could also indirectly affect responses mediated by beta gamma subunits. Thus, it appears that PKC-mediated Galpha subunit phosphorylation may play a significant role in regulating G protein function and hence its downstream signaling processes.

In addition to PKC, evidence has been provided that PKA is also capable of phosphorylating Galpha subunits in vitro (29, 30). Furthermore, we recently demonstrated that TXA2 receptor-coupled Galpha 13 is phosphorylated by PKA in vivo (8). The present studies extended these findings by characterizing the specific PKA phosphorylation site in Galpha 13 and identifying the molecular and functional consequences of this phosphorylation. Our initial results showed that PKA phosphorylation of Galpha 13 does not require intermediate kinases or other co-factors. Our subsequent studies using site-specific peptides and site-directed mutagenesis provided evidence that PKA phosphorylates Galpha 13 at a single site (i.e. Thr203 contained within the switch I region of the Galpha subunit). Because this phosphorylation occurs at a conformationally sensitive site of the of the Galpha subunit, it is possible that such phosphorylation could potentially change the structural or chemical characteristics of Galpha 13 and thereby alter its interaction with receptor-G13 beta gamma partners or its downstream signaling capacity. In this connection, switch I and II regions are structurally and functionally analogous to those first described in the small GTPase Ras (9), whereas the switch III region is unique to heterotrimeric G proteins. These switch regions are domains of the alpha  subunit that have been found to undergo conformational changes during G protein activation induced by GDP-GTP exchange (9-14). Furthermore, crystallization studies have revealed that the switch I region contains critical sites for not only binding GTP (13, 22) but also for binding the beta gamma subunits (13). Consequently, this region is thought to be important for modulating activation of the alpha  subunit as well as for regulating the affinity of the alpha  subunit for the beta gamma subunits (13).

On this basis, we next examined whether PKA phosphorylation of the switch I region Thr203 residue of Galpha 13 affects the stability of the Galpha beta gamma 13 heterotrimeric complex or the interaction of this complex with TXA2 receptor protein. Using co-elution affinity chromatography and co-immunoprecipitation, it was found that Galpha 13 phosphorylation stabilized the coupling of Galpha 13 to platelet TXA2 receptors but destabilized the coupling of Galpha 13 to its beta gamma subunits. Thus, phosphorylation of Galpha 13 at Thr203 appeared to produce structural effects on the receptor-G protein heterotrimer complex.

Our final series of experiments investigated the effects of Galpha 13 Thr203 phosphorylation on downstream signaling through Rho. It was found that 8-Br-cAMP caused a substantial reduction of the basal Rho activation state in CHO cells overexpressing wild type Galpha 13. On the other hand, mutation of Galpha 13 Thr203 to Ala significantly blocked the cAMP-mediated effects. Since separate experiments demonstrated that the T203A mutant is incapable of PKA-mediated phosphorylation, these results suggest that Thr203 phosphorylation serves as a mechanism for PKA modulation of Galpha 13 downstream signaling. This finding is consistent with previous results that demonstrated cAMP-dependent inhibition of Rho activation (27).

Based on the above considerations, we propose a model by which PKA may regulate G13 signaling (Fig. 8). In the resting state, an equilibrium exists between the intact receptor-G protein complex and the dissociated complex. This dissociation-reassociation cycling process defines basal GTPase activity, which is normally low because most receptor-G protein exists in the complexed form. This basal activity also defines the resting activation state of the downstream effectors. On the other hand, the cycling rate can be markedly accelerated in the presence of agonist. Thus, according to the classical model (31), the agonist induces a shift in the equilibrium in favor of heterotrimer dissociation, which is characterized by increased GDP-GTP exchange on the Galpha subunit (17). This process in turn causes a rapid dissociation of Galpha from the beta gamma subunits, enabling both the alpha  and the beta gamma to increase their activation of downstream effectors. Upon hydrolysis of GTP to GDP, the receptor-Galpha beta gamma complex reforms and is thereby poised to undergo additional cycles of activation. On the other hand, if this cycling process is interrupted (e.g. by Galpha subunit phosphorylation), downstream effector activation could be significantly altered. Specifically, our results have shown that PKA-mediated phosphorylation of Galpha 13 at Thr203 has two structural effects: 1) it stabilizes the Galpha 13-G protein-coupled receptor complex, and 2) it inhibits Galpha beta gamma 13 association. Whereas the mechanisms of these effects are unknown, they may involve steric considerations or phosphorylation-induced conformational changes. For example, Thr203 is situated within the beta gamma interacting surface of Galpha 13. Consequently, phosphorylation of this site could prevent binding between the alpha  and beta gamma subunits and thereby inhibit formation of the heterotrimeric complex. At the same time, Thr203 phosphorylation may lead to conformational changes that favor stabilization of the Galpha 13-G protein-coupled receptor binding interaction.


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Fig. 8.   Proposed model of the mechanism by which cAMP/PKA modulate Galpha 13 signaling. a, cAMP activates PKA, which mediates Thr203 phosphorylation of Galpha 13 within the switch I region. b, Thr203 phosphorylation of Galpha 13 stabilizes coupling of Galpha 13 to G protein-coupled receptor and/or prevents binding of switch I region to effector(s). c, Thr203 phosphorylation of Galpha 13 decreases the stability of the Galpha 13 heterotrimer complex, leading to beta gamma subunits release and inhibition of heterotrimer complex reassociation. beta gamma release may transiently influence downstream effectors.

Regarding functional effects, stabilization of the Galpha -receptor complex by phosphorylation of Galpha 13 Thr203 would presumably lead to direct inhibition, since the Galpha subunit would be unavailable for downstream signaling. Alternatively, steric or charge-related effects of phosphorylation may also decrease binding of switch 1 region to the downstream effector (32), again leading to inhibition. Our results showing decreased Rho activation by Galpha 13 Thr203 phosphorylation are consistent with either of these possibilities. On the other hand, the consequences of inhibited beta gamma reassociation by phosphorylation are less clear. For example, one might think that initial dissociation of beta gamma would lead to downstream activation. However, this activation phase may be brief and self-limiting due to inhibition of cycling and G protein adaptation, which has recently been shown to occur in the face of persistent heterotrimeric dissociation (31). On this basis, the beta gamma downstream signaling response may be biphasic, and indeed there is precedent for a transient platelet activation phase (33) caused by protein kinase G (which presumably would also phosphorylate Thr203).

In summary, the present findings identify a novel mechanism for modulating G protein-coupled receptor signaling. Our demonstration of PKA-mediated Galpha 13 Thr203 phosphorylation provides evidence that a conformationally sensitive switch region of Galpha 13 can serve as a potential target for cAMP modulation. This phosphorylation appears to have both structural and functional effects in that it causes increased stability of Galpha 13 coupling to G protein-coupled receptors, decreased stability of the Galpha 13 heterotrimer complex, and decreased basal Rho activation state. Whereas the relative importance of this regulatory process is presently unknown, it is noteworthy that phosphorylated Galpha 13 exists even at basal cAMP levels (8). This finding raises the possibility that cAMP may exhibit tonic influences on Galpha 13 signaling even at low cellular cAMP levels. Finally, sequence analysis reveals that Galpha 12 also contains a PKA consensus sequence in switch I region, whereas other G proteins (e.g. Galpha q, Galpha i, Galpha z, or Galpha s) do not. Thus, the proposed ability of cAMP to modulate Galpha conformation/function may represent a regulatory mechanism that is unique to the ubiquitously expressed Galpha 12/13 family. Clearly, additional studies will be required to investigate this interesting possibility.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL-24530 (to G. L.), GM 54159 and GM65160 (to T. V-Y.), and GM59427 (to T. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Millennium Pharmaceuticals, Inc., 256 E. Grand Ave., South San Francisco, CA 94080.

§ These two authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave. (M/C 868), Chicago, IL, 60612. Tel.: 312-996-4929; Fax: 312-996-4929; E-mail: gcl@uic.edu.

Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M209219200

2 G. C. Le Breton, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; TXA2, thromboxane A2; PKA, protein kinase A; 8-Br-cAMP, 8-bromo-cyclic AMP; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; CHO, Chinese hamster ovary; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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