©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of G by Protein Kinase C Blocks Interaction with the Complex (*)

(Received for publication, March 21, 1995; and in revised form, June 7, 1995)

Timothy A. Fields Patrick J. Casey (§)

From the Departments of Molecular Cancer Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710-3686

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

G is a G protein alpha subunit with biochemical properties that distinguish it from other members of the G protein alpha subunit family. One such property is its ability to be stoichiometrically phosphorylated by protein kinase C (PKC), both in vitro and in intact cells. The site of this phosphorylation has been mapped to a region near the N terminus of G, but no functional significance of the modification has been established. To investigate this question, we have developed a baculovirus/Sf9 cell expression system to produce G. The protein purified from Sf9 cells is functional as assessed by its ability both to bind guanine nucleotide in a Mg-sensitive fashion and to serve as a substrate for phosphorylation by PKC. Furthermore, addition of the G protein beta complex purified from bovine brain inhibits phosphorylation of G in a dose-dependent manner. Conversely, phosphorylation of G inhibits its ability to interact with beta subunits. These results establish a functional consequence for PKC-catalyzed phosphorylation of G and suggest a mechanism for regulation of signaling through G(z) by preventing reassociation of its subunits.


INTRODUCTION

G(z) is a member of the family of trimeric guanine nucleotide-binding regulatory proteins (G proteins) (^1)which generally function by coupling receptor(s) for extracellular ligands to intracellular effector(s)(1, 2, 3) . The mechanism through which G proteins link receptors to effectors involves a complex cycle. The activation phase of this cycle consists of receptor-catalyzed guanine nucleotide exchange (GTP for GDP) on the alpha subunit, resulting in dissociation of alpha-GTP from the beta complex; GTP hydrolysis and subsequent subunit reassociation are associated with the deactivation phase.

As with other heterotrimeric G proteins, G(z) is classified by the identity of its alpha subunit(4, 5) . Although its specific signaling function has not been established, several studies have supported the idea that G(z) can couple membrane receptors to intracellular effectors. Activated m2-muscarinic receptor can stimulate nucleotide exchange on G when co-reconstituted in lipid vesicles(6) . Cotransfection of G with either the A(1)-adenosine, alpha(2)-adrenergic, or D(2)-dopamine receptor into hEk 293 cells results in pertussis toxin-insensitive inhibition of adenylyl cyclase in response to activation of each of these receptors(7) . In the latter experiments, insensitivity to pertussis toxin confirmed the involvement of G, as this G protein is not a substrate for the toxin(8) . Additionally, activated G can directly inhibit G-stimulated adenylyl cyclase in vitro(9) . However, since adenylyl cyclase inhibition in tissues that express G is generally pertussis toxin-sensitive, and since the above receptors ordinarily induce pertussis toxin-sensitive effects, the role of G in adenylyl cyclase inhibition in vivo is uncertain(10) .

Although the physiologic function of G(z) remains obscure, what is clear is that G(z) is quite distinct from other G proteins with regard to the biochemical properties of its alpha subunit (8) , and these properties may provide some clue as to the function of G(z). G exhibits a very slow intrinsic rate of nucleotide exchange which is almost completely suppressed by physiological levels of Mg. The protein also hydrolyzes bound GTP quite slowly relative to other alpha subunits and thus may retain activity for many minutes following activation. Additionally, the expression of G is highly constrained. Northern and immunoblot analyses have shown that G is found predominantly in neuronal tissues and in platelets(4, 5, 8, 11, 12) .

Another potentially important property of G is that it is an effective substrate for phosphorylation catalyzed by protein kinase C (PKC). This phosphorylation has been demonstrated both in vitro and in intact platelets treated with PKC-activating agents such as thrombin, thromboxane A(2) analogs, and phorbol esters(13, 14) . Phosphorylation is rapid and nearly stoichiometric, suggesting a mechanism for cross-talk between PKC- and G(z)-mediated processes. The functional consequence of this phosphorylation, though, has remained elusive. The primary site for phosphorylation of G has been mapped through both biochemical and mutational analyses of G to Ser(15) . Since Ser lies within the region implicated as a contact site between the alpha subunit and the beta complex of G proteins(2) , we considered it possible that phosphorylation of G might influence its interaction with beta. To test this hypothesis and to produce protein for future studies aimed at identifying signaling processes controlled by G(z), we have expressed G by recombinant baculovirus infection of Spodoptera frugiperda (Sf9) cells. We chose the baculovirus-Sf9 expression system because this cell type is known to be able to myristoylate G protein alpha subunits(16) , and this N-terminal modification likely plays an important role in subunit interactions for G(z), as it does for other G proteins(17, 18) . Using purified G from this source, we have examined the effect of phosphorylation of G on its interaction with beta complexes. We report that the beta complex inhibits the PKC-catalyzed phosphorylation of G, and, furthermore, that phosphorylation of G directly interferes with its ability to bind beta. We discuss the potential relevance of these findings to signaling through G(z).


EXPERIMENTAL PROCEDURES

Cell Culture

Sf9 cells were grown and maintained at 27 °C in Grace's Insect Media (Life Technologies, Inc.) supplemented with lactalbumin, yeast hydrolysate, 10% fetal calf serum (Life Technologies, Inc.), 0.1% Pluronic F68 (Life Technologies, Inc.), and antibiotic/antimycotic mixture (0.25 µg/ml amphotericin B, 100 units/ml streptomycin sulfate, and 100 units/ml penicillin G sodium) (Life Technologies, Inc.).

Construction of GTransfer Vector for Recombinant Baculovirus Production

The methods used in this study for manipulating DNA have all been described(19) . The parent vector, pVL941, containing a polyhedron promoter with flanking viral sequences and an ampicillin resistance gene was obtained from Pharmingen. The plasmid containing the entire coding sequence for human G, pBS-Gz, has been described(4) . To construct the transfer vector, the G coding fragment was excised from pBS-Gz with EcoRI, and the ends were filled by treatment with the Klenow fragment and deoxyribonucleoside triphosphates. pVL941 was digested with BamHI, made blunt by treatment with Klenow as above, and treated with calf intestinal alkaline phosphatase. The vector product was then ligated with the blunt-end G fragment. The resultant plasmid obtained, termed pVL941/Gz, was confirmed as the correct ligation product by restriction mapping.

Preparation of Recombinant Baculoviruses and Infection of Sf9 Cells

The techniques employed for generating recombinant baculovirus and expression of heterologous protein in Sf9 cells have been described(20, 21) . Linearized viral DNA (BaculoGold) was obtained from Pharmingen. This viral DNA and the purified pVL941/Gz plasmid were cotransfected into Sf9 cells by calcium phosphate precipitation. After 4 days, the transfection supernatant was harvested, and recombinant baculovirus was plaque-purified and amplified. The identity of the virus was confirmed by infecting Sf9 cells with the amplified viral stocks at an approximate multiplicity of infection of 1. Infected cells were harvested 48 h postinfection and lysed, and the cell extracts were analyzed for the presence of G by immunoblot using a G-specific antisera, P-961(8) . The recombinant baculovirus for expression of the hexahistidine-tagged (2) subunit ((2)-H(6)) was a gift from Tohru Kasaza and Alfred Gilman (Southwestern Medical Center, Dallas, TX). The baculovirus for expression of the beta(1) subunit was a gift from Janet Robishaw (Weis Center for Research, Danville, PA).

Purification of Recombinant Gfrom Sf9 Cells

All procedures were performed at 2-4 °C unless otherwise stated. Cholate extracts were prepared essentially as described with some minor modifications(22) . Approximately 2 liters of Sf9 cells (1 times 10^6 cells/ml) were infected with the three recombinant baculoviruses, i.e. those encoding G, beta(1), and (2)-H(6), at multiplicity of infection values of 1. The cells were harvested at 50 h postinfection by centrifugation at 1000 times g. The cell pellet was suspended in 140 ml of 50 mM Hepes (pH 7.5), 10 mM beta-mercaptoethanol, 50 mM NaCl, 1 mM MgCl(2), 10 µM GDP, and a mixture of protease inhibitors (23 µg/ml phenylmethylsulfonyl fluoride, 11 µg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone, 11 µg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, 2 µg/ml leupeptin, and 2 µg/ml aprotinin). The cell suspension was subjected to nitrogen cavitation at 500 p.s.i. for 45 min. Nuclei and unbroken cells were removed by centrifugation at 500 times g for 10 min. Membranes were collected from the 500 times g supernatant by centrifugation at 100,000 times g for 45 min. The membrane pellet was suspended in 50 ml of the same buffer used for lysis and subjected to 10 strokes in a Teflon-glass homogenizer. This membrane suspension was diluted to 5 mg of protein/ml with the same buffer, and sodium cholate was added to a final concentration of 1%. After stirring on ice for 1 h, the mixture was centrifuged at 100,000 times g for 45 min; the supernatant obtained was designated the cholate extract.

The procedure for further purification was adapted from that used by Kozasa and Gilman (9) and is based on the ability to selectively adsorb (2)-H(6), along with the associated beta(1) and G in the G protein heterotrimer, onto immobilized Ni resin. The cholate extract was diluted 5-fold with 20 mM Hepes (pH 8.0), 100 mM NaCl, 1 mM MgCl(2), 10 µM GDP, 10 mM beta-mercaptoethanol, and 0.5% polyoxyethylene 10-lauryl ether (CE) (Buffer A). After incubation on ice for 20 min, the diluted extract was loaded onto 3 ml of the immobilized Ni resin Ni-NTA (Qiagen) pre-equilibrated with Buffer A. The column was washed consecutively with 20 volumes of Buffer A containing 300 mM NaCl and 5 mM imidazole (Buffer B) and then 5 volumes of Buffer A containing 20 mM Hepes (pH 7.0), 5 mM imidazole, and 0.1% CE (Buffer C). The column was warmed to room temperature for 10 min, and G eluted from the column with 3 volumes of 20 mM Hepes (pH 7.0), 100 mM NaCl, 10 µM GDP, 10 mM beta-mercaptoethanol, 30 µM AlCl(3), 50 mM MgCl(2), 10 mM NaF, 5 mM imidazole, and 1% sodium cholate (Buffer D). The inclusion of Al and F results in activation of the heterotrimer and thus release of alpha subunits (predominantly the expressed G) from the beta(1)(2)-H(6).

The elution from the Ni-NTA column was diluted 3-fold with 20 mM Hepes (pH 7.0), 1 mM EDTA, 3 mM DTT, 10 µM GDP, 5 mM MgCl(2), 0.7% CHAPS (Sigma) and injected onto a Mono S HR5/5 FPLC column (Pharmacia Biotech Inc.). The column was washed with 5 ml of the same buffer containing 50 mM NaCl and eluted with a 20-ml gradient of 50 to 1000 mM NaCl in the same buffer. The gradient conditions were as follows: 50 to 335 mM NaCl over 5 ml, 335 to 525 mM over the next 10 ml, and 525 to 1000 mM over 5 ml. G eluted at 400 mM NaCl as assessed by GTPS binding and immunoblot analysis. The peak fractions were pooled, supplemented with bovine serum albumin to a final concentration 2 mg/ml, and concentrated in a Centricon-30 concentrator (Amicon). The protein was divided into aliquots, flash-frozen in liquid nitrogen, and stored at -80 °C.

Synthesis of beta(1)(2)-H(6)Resin

Approximately 250 ml of Sf9 cells (1 times 10^6 cells/ml) were coinfected with the beta(1) and (2)-H(6) viruses at multiplicity of infection values of 1. The cells were harvested at 50 h postinfection, and membranes were prepared as described above. The membranes were extracted with 1% sodium cholate as above, and the extract was diluted 5-fold with Buffer A and chromatographed on 1 ml of Ni-NTA agarose. The column was washed extensively as described above. The resultant resin containing immobilized beta(1)(2)-H(6) was designated beta(1)(2)-H(6) resin and was stored in Buffer D at 4 °C until use.

Trypsin Protection Assay

G was incubated with either GDP or GTPS at 625 µM for 90 min at 30 °C in the presence of 50 mM Hepes (pH 7.5), 1 mM EDTA, 1 mM DTT, 0.05% Lubrol, and MgCl(2) such that free Mg was either 1 µM or 10 mM. Free Mg was calculated using a K(d) of EDTA for Mg of 1 µM(23) . Following the incubation, the samples with low free Mg were supplemented with MgCl(2) to a final concentration of 10 mM. Trypsin was added to a final concentration of 0.08 mg/ml, and the incubation continued at 30 °C for 10 min. The reaction was stopped by addition of soybean trypsin inhibitor. Laemmli sample buffer was added to each tube, and the samples were processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to nitrocellulose and immunoblot analysis with antisera 2921.

Phosphorylation of G

Phosphorylation of the recombinant G was performed as described (14) with minor modifications. For the experiments assessing beta inhibition of PKC-catalyzed phosphorylation, G was preincubated with purified beta for 2 h on ice in 5 µl of 50 mM Hepes (pH 7.5), 1 mM EDTA, 5 mM MgCl(2), 1 mM DTT, 0.1% Lubrol, and 25 µM GDP. Following incubation, the components required for phosphorylation were added such that the final concentrations were 50 mM Hepes (pH 7.5), 1 mM EDTA, 1 mM DTT, 5 mM MgCl(2), 8 mM CaCl(2), 12 µg/ml diolein, 120 µg/ml phosphatidylserine, 20 units/ml PKC, 10 µM GDP, and 0.25 mM [-P]ATP (1000 cpm/pmol) in a volume of 12.5 µl. Reactions were conducted at 30 °C for the times indicated in the respective figure legends. For the time course analysis (Fig. 3), the reaction volume was increased and aliquots were withdrawn at the time points indicated in the figure legends. Reactions were stopped by addition of sample buffer, heated at 60 °C for 5 min, and processed by SDS-PAGE and autoradiography.


Figure 3: beta-Mediated inhibition of phosphorylation of G: time course and reversal. Purified G (0.5 pmol) was preincubated with either 2.5 pmol of beta (+) or the appropriate volume of buffer(-) at 0 °C for 2 h as described under ``Experimental Procedures.'' The components necessary for phosphorylation were then added, and each reaction was incubated at 30 °C for the times indicated. Reactions were stopped by placing the tubes on ice and adding Laemmli sample buffer. The samples were processed by SDS-PAGE and autoradiography. A, time course of beta-mediated inhibition of phosphorylation. The autoradiogram from one representative experiment is shown. The film was exposed for 7 h. Excision of the G band in lane 9 and analysis by liquid scintillation spectrometry indicated a phosphorylation stoichiometry of 50% based on the quantity of G processed. B, quantitation of phosphorylation. Radioactivity in the bands representing phosphorylated G was quantitated densitometrically from a scanned image of the autoradiogram in A. The quantity for the band in lane 9 was arbitrarily assigned a value of 100, and the remaining densities are expressed as a percentage of this value. Phosphorylation of G either in the absence (circle) or presence (bullet) of beta is plotted versus time. C, reversal of beta-mediated inhibition of phosphorylation. The solid bars represent the phosphorylation of G. Reactions were performed for 25 min as described above, except that the preincubation contained either buffer (represented by bar 1), a 5-fold excess of beta over G (2), or the same quantity of beta plus an additional 5-fold excess of a mixture of G and G relative to beta (3). The control experiment (4) contained the mixture of G and G, but neither G nor beta. The amount of phosphorylation of G in condition 1 was arbitrarily assigned a value of 100, and conditions 2-4 were expressed as a percentage of this value. Data represent the mean of three separate determinations. The cross-hatched bars represent the phosphorylation of histone in the absence (5) or presence (6) of an approximate 5-fold excess of beta relative to histone. Reaction conditions and processing of samples were identical with those above except that incubations were 10 min. The amount of histone phosphorylation in the absence of beta was arbitrarily assigned a value of 100, and phosphorylation in the presence of beta was expressed as a percentage of this value.



For experiments examining the interaction of phosphorylated G with immobilized beta, recombinant G (1-2 pmol) in volume of 12.5 µl was subjected to phosphorylation by incubation for 30 min at 30 °C in 50 mM Hepes (pH 7.5), 0.4 mM EDTA, 0.4 mM DTT, 0.04% Lubrol, 10 mM MgCl(2), 8 mM CaCl(2), 50 µM GDP, 12 µg/ml diolein, 120 µg/ml phosphatidylserine, 100 units/ml PKC, and 0.25 mM [-P]ATP (1000 cpm/pmol). The reaction mixture was then diluted with 100 µl of ice cold Buffer A and mixed with 50 µl of beta(1)(2)-H(6) resin (pre-equilibrated in Buffer A). After a 1-h incubation at 4 °C, the resin was washed consecutively with 2 volumes of Buffer A, 4 volumes of Buffer B containing 600 mM NaCl, and 4 volumes of Buffer B containing 100 mM NaCl and 0.25% sodium cholate instead of 0.5% CE. The resin was then washed at room temperature with 4 volumes of Buffer D followed by 4 volumes of 20 mM Hepes (pH 7.0), 100 mM NaCl, 10 µM GDP, 10 mM beta-mercaptoethanol, 1 mM MgCl(2), 150 mM imidazole, and 1% sodium cholate (Buffer E). The imidazole elutes the beta(1)(2)-H(6) from the resin by competing for Ni binding. The control reaction was performed in an identical fashion, except that ATP was excluded. For phosphorylated G, equivalent volumes of each fraction were processed by SDS-PAGE and autoradiography. For nonphosphorylated G, the fractions were subjected to precipitation with trichloroacetic acid (24) and processed by SDS-PAGE and immunoblot analysis using the G-specific antisera, P961.

Sucrose Density Gradient Centrifugation

Recombinant G in a volume of 50 µl was subjected to phosphorylation by incubation for 30 min at 30 °C in 50 mM Hepes (pH 7.5), 1 mM EDTA, 1 mM DTT, 0.04% Lubrol, 10 mM MgCl(2), 8 mM CaCl(2), 10 µM GDP, 12 µg/ml diolein, 120 µg/ml phosphatidylserine, 500 units/ml PKC, and 9 mM ATP. The reaction was stopped by placing the mixture on ice. A 10-fold excess of beta in 10 µl of 50 mM Hepes (pH 7.5), 1 mM EDTA, 1 mM DTT, 0.05% Lubrol, 5 mM MgCl(2), and 100 µM GDP was then added. After 2 h on ice, the reaction mixture was applied to the top of a 2-ml linear gradient of 5%-20% sucrose prepared in 50 mM Hepes (pH 7.5), 1 mM EDTA, 1 mM DTT, 0.05% Lubrol, 5 mM MgCl(2), and 25 µM GDP. The sample was subjected to centrifugation at 54,000 rpm in a Beckman TLS-55 rotor for 8 h at 4 °C. The gradient was fractionated by withdrawing successive 100-µl aliquots. Fractions were processed by SDS-PAGE and immunoblot analysis using antisera P961.

Miscellaneous Materials and Methods

Protein concentrations were determined using the Amido Black dye binding method(25) . Nucleotide binding was quantitated by filtration through nitrocellulose as described(26) . SDS-PAGE was performed as described(27) . G protein beta subunits were purified from bovine brain membranes as described (28) . G and G were purified from bovine brain as described(28, 29) . Protein kinase C was partially purified from bovine brain as described(30) . One unit of activity is defined as the amount of PKC required for phosphorylation of 1 pmol of histone per min at 30 °C. For Fig. 3B, 4B, 5C, and 6, the bands representing G were quantitated densitometrically using Image® software version 1.38 on unmodified scanned images. The technique employed for immunoblot analysis has been described(31) . The antisera 2921, directed against a C-terminal decapeptide derived from the G sequence(13) , was a gift of David R. Manning. Antisera P961, a G-specific antisera, has been described(8) . The G-specific antisera was obtained from DuPont NEN.


RESULTS

Expression of G

Infection of Sf9 cells with the recombinant baculovirus encoding G resulted in high levels of the protein being expressed (data not shown). However, despite the ability of the recombinant G produced to incorporate [^3H]myristate, the bulk of the protein expressed was insoluble and resistant to detergent extraction (data not shown). A small quantity of detergent-extractable G could be detected by growing the cells after infection at lower temperatures (21 °C), but the amount was so small as to preclude this as a viable option for purification (data not shown). To produce larger quantities of active G, we took advantage of the finding by Hepler et al.(22) that coexpression of certain alpha subunits with beta increased the amount of active G protein produced. Coinfection of Sf9 cells with baculoviruses coding for G, beta(1), and (2)-H(6), the latter of which provides an epitope tag to facilitate purification of trimeric G(z)(9) , did in fact result in production of sufficient quantities of the protein for study (see below).

Purification of Recombinant G

Membranes were prepared from Sf9 cells infected with three recombinant baculoviruses encoding G, beta(1), and (2)-H(6), and recombinant G was purified as described under ``Experimental Procedures.'' The purification is summarized in Fig. 1and Table 1. The majority of the protein extracted from the Sf9 membranes either flows through the Ni-NTA resin or is removed by the extensive washing (Fig. 1A, lanes 1-4; Table 1). G, which is bound to the column through association with the adsorbed beta(1)(2)-H(6) complex, is eluted by washing the resin with buffer containing 1% cholate and Al and F; this treatment activates the alpha subunit and promotes dissociation from the adsorbed beta(1)(2)-H(6)(22) .


Figure 1: Purification of G. G was purified from Sf9 cells simultaneously infected with recombinant baculoviruses encoding G, beta(1), and (2)-H(6) as described under ``Experimental Procedures.'' A, aliquots from the Ni-NTA column were processed by 12% SDS-PAGE and stained with Coomassie Blue: lane 1, cholate extract; lane 2, flow-through; lane 3, high salt/low imidazole wash; lane 4, low salt/low imidazole; lane 5, 1% cholate, Al, and F elution. Lane 6 shows purified G obtained from Mono S chromatography of the material in lane 5. The arrows on the left represent the migration positions of molecular weight markers processed on the same gel. B, samples identical with those in A were processed by SDS-PAGE followed by immunoblot analysis with the G-specific antisera, P961.





The major protein obtained from the elution step migrates at 40 kDa by SDS-PAGE. The presence of G in this band was confirmed by immunoblot analysis (Fig. 1B), but it also contained a significant amount of a G(q)-immunoreactive protein (data not shown). However, further chromatography of the Ni-NTA elution on an anion exchange Mono S column resulted in an essentially homogenous protein of 40 kDa being obtained (Fig. 1A, lane 6) in which only G immunoreactivity could be detected (data not shown). Quantities of G sufficient for analysis were purified in this manner (see Table 1).

The guanine nucleotide binding properties of recombinant G were assessed by the trypsin protection assay. This procedure takes advantage of the fact that activation of G protein alpha subunits by the GTP analog, GTPS, partially protects them from cleavage by trypsin, such that only a small portion of their N terminus is removed. By contrast, in their basal (i.e. GDP-liganded) state, alpha subunits are rapidly degraded by trypsin. Thus, this assay provides a very sensitive means of assessing the nucleotide-bound state of a G protein. The assay is also very specific, as the protein is detected by immunoblot analysis. Purified G was incubated with either GTPS or GDP in the presence of either 1 µM free Mg or 10 mM free Mg. Following incubation with nucleotide, the samples were incubated either with or without trypsin. The data in Fig. 2show the results of these experiments. The binding of GTPS protected G from trypsin digestion (lane 2). However, in the presence of 10 mM free Mg this protection is not evident (compare lanes 2 and 4), which is expected since 10 mM free Mg suppresses G's intrinsic rate of nucleotide exchange to almost zero(8) . These data confirm that the protein which was purified is active.


Figure 2: Demonstration of magnesium suppression of nucleotide exchange by trypsin protection. Purified G (1 pmol) was incubated at 30 °C for 90 min in the presence of either GDP (lanes 1, 3, 4, and 6) or GTPS (lanes 2 and 5) as described under ``Experimental Procedures.'' In lanes 1-3, the free Mg was 1 µM, while in lanes 4-6 it was 10 mM. After 90 min, MgCl(2) to 10 mM was added to the samples in lanes 1-3, trypsin was added to the samples in lanes 2, 3, 5, and 6, and the incubation was continued for another 10 min at 30 °C. Half of each sample was processed by SDS-PAGE and transferred to nitrocellulose. This nitrocellulose membrane was processed by immunoblot analysis with antisera 2921, a G-specific antisera.



The beta Subunit Complex Inhibits Phosphorylation of G

Preliminary experiments confirmed that the G purified from the Sf9 expression system served as a very good substrate for phosphorylation by PKC (data not shown). To assess the ability of beta to influence the PKC-catalyzed phosphorylation of G, purified G was preincubated with beta prior to phosphorylation with PKC. Addition of a 5-fold excess of beta over G to the preincubation mixture significantly reduced the rate of phosphorylation (Fig. 3). Inclusion of a 5-fold excess of a mixture of G and G, which competes with G for binding of beta, completely reversed the inhibition of phosphorylation, demonstrating that the reduced rate of phosphorylation was in fact due to the presence of the beta complex (Fig. 3C). The inhibition of phosphorylation was not due to an effect of the beta on PKC itself, as beta did not inhibit the ability of PKC to catalyze phosphorylation of histone (Fig. 3C).

The beta-dependent inhibition of G phosphorylation was examined in further detail by determining the concentration dependence of this suppression (Fig. 4). This analysis revealed that inhibition of PKC-catalyzed phosphorylation by beta is dose-dependent, and that 50% inhibition was observed upon addition of a roughly equimolar amount of beta. Higher concentrations of beta resulted in almost complete suppression of phosphorylation, providing very strong evidence that association of beta with G prevents the phosphorylation of G by PKC.


Figure 4: Dose dependence of beta-mediated inhibition of phosphorylation of G. Purified G (0.5 pmol) was preincubated with either beta or buffer as described in the legend to Fig. 3, except that quantities of beta were varied as indicated. The approximate molar ratios of beta:G are indicated above the respective lanes. Reactions were conducted for 25 min at 30 °C and processed as described in the legend to Fig. 3. A, inhibition of G phosphorylation with increasing doses of beta. The autoradiogram from one representative experiment is shown. The stoichiometry of phosphorylation of G in lane 1 was determined to be 50%. The film was exposed for 8 h. B, quantitation of phosphorylation. Radioactivity in the bands representing phosphorylated G in A were quantitated densitometrically as described in the legend to Fig. 3. The density of the band in lane 1 was arbitrarily assigned the value of 100, and the remaining bands are expressed as a percentage of this value. The quantity of phosphorylated G is plotted versus the molar ratio of beta:G.



Phosphorylation of GPrevents Its Interaction with beta

Since binding of beta to G inhibited its phosphorylation, it seemed likely that phosphorylation would, in turn, interfere with the ability of G to associate with beta in trimer formation. To address this question, we took advantage of the Sf9 system again by coexpressing the beta(1) and (2)-H(6) subunits as a means for rapid production of immobilized beta. A cholate extract of Sf9 cells coexpressing beta(1) and (2)-H(6) was processed by chromatography on Ni-NTA agarose as described under ``Experimental Procedures'' to produce a resin containing adsorbed beta(1)(2)-H(6) through the hexahistidine-tagged subunit.

G was incubated with PKC both in the presence and absence of [P]ATP; the latter condition served as the control reaction. Following the phosphorylation reaction, both phosphorylated and nonphosphorylated proteins were incubated with the beta(1)(2)-H(6) resin, and the elution patterns of each were analyzed (Fig. 5). As expected, since this was the route to its initial purification, nonphosphorylated G bound quite tightly to the beta(1)(2)-H(6) resin; it did not flow through the resin nor was it washed off with either high salt or 0.25% cholate (Fig. 5A, lanes 2-4). Washing the column with buffer containing 1% cholate and Al and F results in activation of the heterotrimer and, thus, the release of nonphosphorylated G from the immobilized beta (Fig. 5A, lane 5). A small portion of G eluted when the resin was washed with 150 mM imidazole, a treatment which strips the beta(1)(2)-H(6) and any other associated protein from the resin by competing for Ni binding (Fig. 5A, lane 6). In contrast, the majority of the phosphorylated G incubated with beta(1)(2)-H(6) resin either flows through or is washed off in the high salt and 0.25% cholate washes (Fig. 5B, lanes 2-4), indicating that its binding to the immobilized beta is much weaker than for the nonphosphorylated protein. While a small, but significant, amount of the phosphorylated protein remains associated with the resin after the first two elution conditions and is eluted by 1% cholate and Al and F (Fig. 5B, lane 5), this fraction largely represents nonspecific interaction of phosphorylated protein with the resin, as phosphorylated G elutes in a near-identical pattern from resin which has been denatured by treatment with 8 M urea (data not shown). Quantitation of the amounts of both nonphosphorylated and phosphorylated G eluted from the beta resin under each of the conditions is shown in Fig. 5C.


Figure 5: Effect of phosphorylation of G on its interaction with immobilized beta. Identical aliquots of purified G (1 pmol) were incubated with PKC, Ca, and phospholipids either in the absence (A) or presence (B) of [-P]ATP for 30 min at 30 °C as described under ``Experimental Procedures.'' Samples were then incubated with beta(1)(2)-H(6) resin for 1 h at 4 °C, and the resin was sequentially subjected to the indicated treatments as described under ``Experimental Procedures.'' A, G treated in the absence of ATP. The resultant fractions were collected, concentrated by trichloroacetic acid precipitation, and processed by SDS-PAGE and immunoblot analysis with P961. The blot from one representative experiment is shown. B, G treated in the presence of ATP (i.e. phosphorylated G). Equivalent portions of each fraction were processed by SDS-PAGE and autoradiography. The autoradiogram from one representative experiment is shown. The film was exposed for 12 h. C, quantitation of the elution patterns for phosphorylated and nonphosphorylated G. G in the fractions eluted from each resin were quantitated as described in the legend to Fig. 3. Quantities are expressed as a percentage of the total G eluted in each condition, i.e. phosphorylated or nonphosphorylated.



To further investigate the interference of G-beta association by phosphorylation, we used sedimentation through sucrose density gradients. As for other G proteins(32) , association of G with beta is expected to shift its sedimentation constant relative to the monomeric G subunit. If phosphorylation interferes with the G-beta association, then this modification should prevent a beta-dependent shift in the sedimentation position of G in a density gradient. The results, shown in Fig. 6, demonstrate that this is in fact the case. Both phosphorylated and nonphosphorylated forms of G were incubated with beta and then subjected to centrifugation through a 5-20% sucrose gradient. For nonphosphorylated G, the addition of beta markedly shifts its sedimentation. However, addition of beta to phosphorylated G does not significantly shift its sedimentation; rather, it sediments at nearly the same position as free, nonphosphorylated G. These data provide convincing evidence that phosphorylation of G prevents its interaction with beta.


Figure 6: Sedimentation profile of G and phosphorylated G in the presence of beta. Purified G (6 pmol) was incubated with PKC, Ca, and phospholipids either in the presence (circle) or absence (bullet) of ATP for 30 min at 30 °C as described under ``Experimental Procedures.'' Samples were then incubated with 60 pmol of beta and subjected to centrifugation through a 5-20% sucrose gradient. Successive fractions were taken from the top of each gradient and 15% of each fraction was processed by SDS-PAGE and immunoblot analysis with P961. The bands representing G were quantitated as described in the legend to Fig. 3. Quantities are expressed as a percentage of the total G fractionated in each condition. The arrow represents the peak migration position for G treated in the absence of both ATP and beta and processed through an identical gradient.




DISCUSSION

One of the more intriguing properties of G is its ability to be rapidly phosphorylated by activated PKC both in vitro and in stimulated platelets. While previous studies had defined the primary site for this modification(14, 15) , no functional consequence of phosphorylation had been established. Since the location of the primary phosphorylation site lay within the N-terminal domain, a region known to be important for interaction of alpha subunits with beta, we hypothesized that phosphorylation may play a role in regulating G-beta interaction. In order to test this hypothesis, we required a source of purified G. Escherichia coli expression, however, was not feasible because G from this source does not efficiently interact with beta(8) . This is presumably due to the inability of bacteria to myristoylate the protein, since N-terminal myristoylation is known to be important for subunit interactions of G proteins(18) . Thus, we turned to expression in a eukaryotic system with documented ability to myristoylate G proteins, the baculovirus/Sf9 expression system(16) . Successful production and purification of recombinant G was greatly aided by the use of the hexahistidine-tagged (2), as coexpression of G with the beta(1) subunit and (2)-H(6) provided an efficient, rapid means of purification by affinity chromatography.

PKC-catalyzed phosphorylation of G was indeed influenced by the G protein-beta complex. The ability of the beta complex to suppress phosphorylation suggests that formation of the G-beta trimer prevents phosphorylation by blocking access of the enzyme to the phosphorylation site. Additionally, once phosphorylated, G has a greatly decreased ability to associate with beta. The most likely basis for the reduced association of G with beta following phosphorylation is that the presence of the phosphate group on the N-terminal domain of G could prevent oligomer formation through either steric interference or charge repulsion in the contact interface between G and beta.

Since phosphorylation influences the interaction between G and beta, this modification may play a role in modulating signaling through G(z). A model for this potential modulation is as follows. Activation of G(z) by an appropriately liganded receptor would result in GTP binding and subsequent subunit dissociation. The GTP-bound G could interact with an as yet unidentified effector, after which the GTPase activity of G could convert it to the GDP-bound form, which would then dissociate from the effector molecule. In this form (G-GDP), the protein can be readily phosphorylated by activated PKC(14) . Once phosphorylated, G could not reassociate with beta and therefore could not recycle to interact again with receptor, resulting in further signaling through G(z) being prevented. In summary, the prediction of this model is that simultaneous activation of G(z)- and PKC-controlled processes would result in rapid attenuation of signaling through the G(z) pathway.

This regulatory model may have some general applicability to signaling through other G proteins, as several reports indicate that another G protein, G(i), can also serve as a substrate for PKC. The G subunit is phosphorylated in hepatocytes in response to PKC activation(33, 34) . Houslay and colleagues (35) identified the specific G protein subtype phosphorylated in this system as G(2), and several groups observed a correlation between phosphorylation of G(2) and inactivation of signaling through this G protein(36, 37, 38) . Further, they observed that treatment with a phosphatase restored signaling through G(2)(37, 39) . Our model offers a possible biochemical explanation for their observations, in that phosphorylation of G(2) could also interfere with its subunit reassociation. Indeed, an amino acid that serves as a secondary site for PKC phosphorylation in G, that of Ser(15) , is also found in G(2) and several other G proteins, and this amino acid also lies within the N-terminal domain implicated in interaction with beta. Thus, prevention of subunit reassociation through PKC-catalyzed phosphorylation could be a mechanism for the observed inactivation of signaling through G(2).

In the model presented here, phosphorylation would directly attenuate signaling through G. In addition, it is certainly possible that signaling through G might directly or indirectly lead to activation of PKC, and that phosphorylation of G by this kinase is a means of feedback regulation. Such attenuation of G protein-mediated signaling by phosphorylation is well-documented in the case of desensitization at the level of the receptors involved in many of these processes(40) . These data provide new leads for developing experimental approaches to identifying components and events in G(z)-mediated signaling.


FOOTNOTES

*
This work was supported by a Basil O'Connor Scholar Award from the March of Dimes and by American Cancer Society Grant BE-117. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Molecular Cancer Biology, Duke University Medical Center, Box 3686, Durham, NC 27710-3686. Tel.: 919-613-8612; Fax: 919-613-8642.

(^1)
The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; GTPS, guanosine 5`-(3-O-thio)triphosphate; PKC, protein kinase C; Sf9, Spodoptera frugiperda; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; CE, polyoxyethylene 10-lauryl ether; FPLC, fast protein liquid chromatography.


ACKNOWLEDGEMENTS

We thank Tohru Kozasa and Alfred G. Gilman for the recombinant baculovirus encoding the (2)-H(6) subunit and for the communication of results prior to publication. We thank Janet Robishaw for the generous gift of the recombinant baculovirus encoding the beta(1) subunit and David Manning for the 2921 antisera and for helpful discussions. We also thank Joyce Higgins and Jennifer Glick for a critical reading of this manuscript.


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