©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase C Phosphorylates G and Inhibits Its Interaction with G(*)

(Received for publication, December 22, 1995; and in revised form, March 5, 1996)

Tohru Kozasa Alfred G. Gilman (§)

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Of nine G protein alpha subunits examined, only alpha and alpha(z) served as substrates for phosphorylation by various isoforms of protein kinase C in vitro. A close homolog of alpha, alpha, was not phosphorylated. Exposure of NIH 3T3 cells that stably express alpha to phorbol 12-myristate 13-acetate also resulted in phosphorylation of the protein. Phosphorylation in vitro occurred near the amino terminus (probably Ser), and approximately 1 mol of phosphate was incorporated per mol of alpha. Although G protein heterotrimers containing either alpha or alpha(z) were poor substrates for phosphorylation, the isolated alpha subunits were phosphorylated equally well in their GDP- or GTPS-bound forms. The guanine nucleotide binding properties of purified alpha and alpha(z) were unaltered by phosphorylation, as was the capacity of alpha(z) to inhibit type V adenylyl cyclase. However, phosphorylation of either protein greatly reduced its affinity for G protein beta subunits, consistent with the newly determined crystal structure of a G protein heterotrimer. We suggest that protein kinase C regulates alpha- and alpha(z)-mediated signaling pathways by preventing their association with beta.


INTRODUCTION

Heterotrimeric guanine nucleotide-binding proteins (G proteins) (^1)transduce regulatory signals from cell surface receptors to effectors such as adenylyl cyclases, phosphodiesterases, phospholipases, and ion channels(1, 2, 3) . Each G protein oligomer contains a guanine nucleotide-binding alpha subunit and a high-affinity dimer of beta and subunits. There are many isoforms of each subunit and thus a very large number of distinct G protein oligomers. G protein alpha subunits are commonly described as members of four subfamilies: alpha(s) and alpha (stimulators of adenylyl cyclases); alpha, alpha, alpha, alpha(o), alpha, alpha, alpha(z), and alpha(g) (functionally diverse group of pertussis toxin substrates, with the exception of alpha(z)); alpha(q), alpha, alpha(14), and alpha (activators of phospholipase C-betas); and alpha and alpha.

The two members of the alpha and alpha subfamily, discovered most recently, are expressed ubiquitously (4) and share interesting biochemical characteristics, including relatively slow guanine nucleotide exchange and hydrolysis(5, 6) . Although the receptors and effectors that interact with these G proteins have not yet been identified, overexpression of wild type or mutationally activated alpha or alpha transforms fibroblasts (7, 8, 9) . Furthermore, overexpression of constitutively activated alpha or alpha stimulates Na/H exchange activity(10, 11) . Of interest, Dhanasekaran et al.(10) showed that this stimulatory effect of alpha, but not that of alpha, is lost after prolonged exposure of cells to PMA. These results suggest that alpha and alpha transduce similar regulatory signals related to cell growth or transformation and that there is further regulation of the alpha pathway by PKC.

There are other interactions between G protein-regulated pathways and PKC. Treatment of cells with PMA has a variety of often confusing effects on their capacity to synthesize cyclic AMP in response to various activators or inhibitors; certain adenylyl cyclases are activated following phosphorylation by PKC in vitro(12, 13) . Activation of phospholipase C by muscarinic or alpha(1)-adrenergic agonists is blocked by treatment of astrocytoma cells or hepatocytes, respectively, with PMA(14, 15) . The inhibitory effects of substance P on an inward rectifier K channel appear to be mediated by a pertussis toxin-insensitive G protein and protein kinase C(16) . With regard to direct effects of PKC on G protein subunits, there are descriptions of phosphorylation of G and G both in vitro and in vivo(17, 18, 19, 20) , but the functional significance of such modification has been unclear. We describe here the phosphorylation of alpha by PKC in vitro and, in addition, in cells exposed to PMA. We further demonstrate that phosphorylated alpha and alpha(z) have reduced affinity for G protein beta subunits compared to the unmodified alpha subunits. Similar results with alpha(z) have just been reported by Fields and Casey(21) .


EXPERIMENTAL PROCEDURES

Purification of G Protein Subunits from Sf9 Cells

alpha and alpha(z) were purified from Sf9 cells infected with appropriate baculoviruses after their coexpression with beta(1) and His(6)-(2) subunits as described by Kozasa and Gilman(5) , with the following modifications. A novel His(6)-(2)-encoding virus (amino acid sequence MAHHHHHHGG-(2)-(3-71)) was utilized. The resulting protein binds with higher apparent affinity to Ni-NTA than does the protein without the two glycine residues inserted after the hexahistidine tag. After application of the Sf9 cell membrane extract to the Ni-NTA column (Qiagen), the resin was washed extensively with buffer containing 15 (instead of 5) mM imidazole. alpha was eluted from the Ni-NTA column with buffer containing AMF (30 µM AlCl(3), 50 mM MgCl(2), and 10 mM NaF) and was purified further on a Mono S HR5/5 column (Pharmacia Biotech Inc.) with solutions containing 10% glycerol to prevent precipitation during concentration of peak fractions.

alpha, alpha(q), and beta(1)(2) were purified as described previously (5) ; alpha and alpha were purified by the same procedure used for alpha. alpha was purified with the Ni-NTA column described for alpha and then according to Singer et al.(6) . beta(1)(2)C68S was purified as described (22) and generously provided by Dr. Bruce Posner (this laboratory).

Purification of PKC from Sf9 Cells

Sf9 cells (1 liter, 1.5 times 10^6 cells/ml) were infected with a recombinant baculovirus encoding rabbit PKCalpha(23) . Cells were harvested 48 h later and suspended in 120 ml of ice-cold lysis buffer (20 mM Tris-HCl (pH 7.5), 5 mM EGTA, 1 mM EDTA, 10 mM beta-mercaptoethanol, and protease inhibitors). After cell lysis (nitrogen cavitation at 500 psi for 30 min) and centrifugation (100,000 times g for 30 min), PKCalpha was purified chromatographically using DEAE-Sephacel, hydroxyapatite, Phenyl-Superose HR10/10, and Mono Q HR5/5 (23) and assayed as described by Yasuda et al.(24) . The yield was 1 mg (from a 1-liter culture), the specific activity was 800 units/mg, and the protein was more than 90% pure based on silver staining after gel electrophoresis. PKC, , and were purified by the same method and were kindly supplied by Dr. William D. Singer (UT Southwestern Medical Center) and Dr. Shigeo Ohno (Yokohama City University).

Phosphorylation of G Subunits by PKC

Candidate PKC substrates (in buffer containing 0.7% CHAPS) were incubated at 30 °C with rat brain PKC (Calbiochem) or purified recombinant PKCalpha in 100 µl of 25 mM Tris-HCl (pH 7.5), 5 mM MgCl(2), 125 µM CaCl(2), 1 mM DTT, 10 µM [-P]ATP (5000 cpm/pmol), and 10 µg/ml phosphatidylserine-diolein (Sigma). The final concentration of CHAPS was less than 0.07%. PKC (1 milliunit/pmol of G) was added to start the reaction. When included, G was first incubated with G subunits for 10 min on ice. Reactions were stopped by addition of 500 µl of 1% SDS, 0.2 mM ATP and 500 µl of 30% trichloroacetic acid. The mixtures were filtered to collect precipitated protein (BA85 filters; Schleicher & Schuell), and the filters were washed (12 ml of 5% trichloroacetic acid) and counted.

For preparation of phosphorylated alpha subunits, 2 nmol of alpha or alpha(z) were incubated in 4-ml reaction mixtures with PKC (2 units) and 10 µM ATP (500 cpm/pmol) for 40 min at 30 °C. Phosphorylated alpha subunits were purified (Mono S HR5/5) and processed as described(5) . The stoichiometry of phosphorylation was approximately 0.5 for alpha and 1-1.5 for alpha(z); protein concentrations were determined by staining with Amido Black(25) .

Trypsin Protection

Phosphorylated alpha was incubated with GDP (100 µM) or GDP + AMF for 10 min at 0 °C prior to treatment with TPCK-treated trypsin (20% of the mass of alpha) for 20 min at 30 °C. After addition of an equal volume of 2 times sample buffer, the products were analyzed by SDS-PAGE, followed by autoradiography or Western blotting with alpha antiserum J169(5) .

Inhibition of GTPS Binding to alpha by beta

G and beta subunits were mixed at 0 °C and incubated for 10 min in 50 mM NaHepes (pH 8.0), 50 mM NaCl, 10 mM (for alpha) or 0.5 mM (for alpha(z)) MgSO(4), 1 mM EDTA, 1 mM DTT, 0.3% CHAPS, and 0.1% CE (final volume 25 µl). GTPS binding solution was added (25 µl of 50 mM NaHepes (pH 8.0), 1 mM EDTA, 1 mM DTT, and 10 µM [S]GTPS (8000 cpm/pmol)), and the mixture was incubated at 30 °C for 60 min. After addition of 2 ml of ice-cold 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 20 mM MgCl(2), bound GTPS was quantified by filtration as described(26) .

Gel Filtration of alpha and beta(1)(2)

alpha (250 pmol) was incubated on ice for 10 min with or without 750 pmol of beta(1)(2) in 180 µl of 50 mM NaHepes (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 3 mM MgCl(2), and 0.7% CHAPS prior to application to a Superdex 200 HR 10/30 gel filtration column (Pharmacia) equilibrated with 50 mM NaHepes (pH 8.0), 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 3 mM MgCl(2), and 1% octyl beta-glucoside (Calbiochem). Fractions of 0.4 ml were collected at a flow rate of 0.3 ml/min and were analyzed by SDS-PAGE and silver staining.

Expression, Labeling, and Immunoprecipitation of alpha

NIH 3T3 cells that stably express alpha (NIH 3T3-G12) were obtained by transfection with plasmids pCMValpha and pSV2Neo and selection in medium containing 600 µg/ml G418 (Life Technologies, Inc.). A mixture of G418-resistant colonies was collected 20 days after transfection. Expression of alpha was confirmed by immunoblotting of cell membrane extracts with antiserum J169.

For labeling with either [S]methionine or [P]P(i), cells were incubated with methionine- or phosphate-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) for 1 h, followed by incubation with medium supplemented with [S]methionine (50 µCi/ml; 3 h) or [P]P(i) (0.5 mCi/ml; 2 h). Cells were washed twice with 20 mM NaHepes (pH 7.5) and 150 mM NaCl, harvested, suspended in 500 µl of hypotonic buffer (20 mM NaHepes (pH 7.5), 1 mM EDTA, 1 mM DTT), frozen, and thawed three times, and centrifuged at 125,000 times g at 4 °C for 20 min to prepare cytosolic and crude membrane fractions. NaF (5 mM) and beta-glycerophosphate (10 mM) were included in the lysis buffer for cells labeled with [P]P(i). Membrane extracts were prepared with 500 µl of 20 mM NaHepes (pH 7.5), 150 mM NaCl, 1% sodium cholate, 1% Triton X-100, and 0.5% SDS (RIPA buffer) prior to centrifugation at 125,000 times g for 20 min.

For immunoprecipitation, 25 µl of membrane extract was incubated with 2.5 µl of 10% fixed Staphylococcus aureus (Pansorbin; Calbiochem) on ice for 30 min. After centrifugation at 15,000 times g for 5 min, supernatants were incubated overnight at 4 °C with 7.5 µg of anti-alpha IgG or control rabbit IgG. Pansorbin (5 µl; 10%) was added for an additional 30 min prior to collection of immunoprecipitates by centrifugation and suspension in 100 µl of RIPA buffer. The suspension was layered over 1 ml of RIPA buffer containing 20% sucrose (w/v) and centrifuged at 15,000 times g for 5 min. Pellets were extracted with SDS-PAGE sample buffer, heated (90 °C; 3 min), and subjected to SDS-PAGE followed by autoradiography. Gels containing [S]methionine-labeled proteins were treated with EN^3HANCE (DuPont NEN).

Miscellaneous Procedures

SDS-PAGE was performed as described by Laemmli(27) ; gels were stained with silver according to Wray et al.(28) . Protein concentrations were estimated by staining with Amido Black (25) or by the method of Bradford(29) . Immunoblotting was performed using the ECL chemiluminescence detection system (Amersham). GTPS binding and adenylyl cyclase assays were performed as described(5) . An IgG fraction of antiserum J169 was prepared by chromatography on a Mono Q HR10/10 column using a gradient of NaCl (0-200 mM). Membranes from Sf9 cells expressing type V adenylyl cyclase were generously provided by Dr. Christiane Kleuss (this laboratory), while the recombinant baculovirus encoding PKCalpha was a gift from Dr. Shigeo Ohno (Yokohama City University).


RESULTS

Phosphorylation of alpha and alpha(z) by PKC

The results of Dhanasekaran et al.(10) , described above, prompted examination of possible phosphorylation of alpha by PKC. Of the nine G protein alpha subunits tested, only alpha and alpha(z) were phosphorylated in vitro by PKC (Fig. 1A); the effect on alpha(z) was anticipated based on the work of Lounsbury et al.(17) . The related alpha subunit, alpha, is not a substrate for PKC, also consistent with Dhanasekaran et al.(10) . Phosphorylation of alpha was dependent on Ca and phosphatidylserine, characteristic of PKC (Fig. 1B). Of the several types of PKC tested (PKCalpha, -, -, and -), all showed the same pattern, phosphorylating only alpha and alpha(z) (data not shown).


Figure 1: Phosphorylation of G protein alpha subunits by PKC. A, G subunits (2.5 pmol) were incubated with 2.5 milliunits of PKC for 20 min. The products were separated by SDS-PAGE, stained with silver, and subjected to autoradiography. Upper panel, silver staining of alpha subunits; lower panel, autoradiography. alpha, alpha, alpha, alpha, alpha, alpha(z), and alpha(q) were purified from Sf9 cells as described under ``Experimental Procedures.'' alpha(o) and alpha(s) were purified from bovine brain and E. coli, respectively. B, alpha (2.5 pmol) was incubated with 2.5 milliunits of recombinant PKCalpha for 20 min in the presence or absence of 5 µM PMA, 10 µg/ml phosphatidylserine, or 125 µM CaCl(2) as indicated. Proteins were resolved by SDS-PAGE and subjected to autoradiography.



We examined NIH 3T3 cells that had been stably transfected with an expression plasmid encoding alpha to test phosphorylation of the protein in vivo. Immunoblotting of membranes from these cells (NIH 3T3-G12) demonstrates significant expression of alpha (Fig. 2A); we could not detect the protein in these cells prior to transfection (using antiserum J169). This antiserum could be used to immunoprecipitate alpha from a membrane extract of [S]methionine-labeled NIH 3T3-G12 cells (Fig. 2B), and phosphorylated alpha was immunoprecipitated from cells labeled with [P]P(i) after exposure to PMA (Fig. 2C). Thus, alpha appears to be phosphorylated in vivo after PKC is activated by phorbol esters.


Figure 2: Western blotting and immunoprecipitation of NIH 3T3-G12 cells. A, membranes from NIH 3T3 cells (lane 1) or NIH 3T3-G12 cells (lane 2) (10 µg of each) were subjected to SDS-PAGE and immunoblotted with antibody J169. B, [S]methionine-labeled NIH 3T3-G12 cell lysate was immunoprecipitated with control rabbit IgG (lane 1) or with J169 IgG (lane 2). The precipitates were resolved by SDS-PAGE and subjected to autoradiography. C, NIH 3T3-G12 cells were labeled with [P]P(i) and treated with vehicle (lane 1) or 5 µM PMA (lane 2) for 20 min. The cell lysates were immunoprecipitated with J169 IgG. The precipitates were resolved by SDS-PAGE and subjected to autoradiography. The arrows in A, B, and C indicate the position of alpha.



The time course and stoichiometry of phosphorylation of alphain vitro are shown in Fig. 3A. Since the substrate is over 90% pure (based on silver staining; Fig. 1A) and other phosphorylated proteins do not appear in the reaction mixtures (Fig. 1B), we estimated stoichiometry by filtration. When 7 pmol of alpha was included in the assay, the maximal incorporation of phosphate was about 3 pmol. Since the stoichiometry of binding of GTPS to the alpha used here was about 50% (based on the protein assay), we believe that 1 mol of phosphate is incorporated per mol of alpha. (alpha is not phosphorylated when denatured; data not shown.) Of interest, alpha is phosphorylated very poorly after incubation with a 2-fold excess of beta(1)(2) (Fig. 3A); the reaction is almost completely suppressed when alpha and beta(1)(2) are present at equimolar concentrations (Fig. 3B). Similar results were obtained with alpha(z) (Fig. 3C). Nonprenylated beta subunit complexes have reduced affinity for at least certain G subunits(22) ; appropriately, the beta complex comprised of beta(1) and the nonprenylated Cys Ser (2) mutant was a less potent inhibitor of alpha phosphorylation (Fig. 3B). Since beta(1)(2) did not inhibit the activity of PKC when a specific substrate peptide from myelin basic protein (MBP) was utilized (data not shown), we conclude that alpha and alpha(z) are not substrates for PKC when associated with beta in the G protein heterotrimer.


Figure 3: Inhibition of phosphorylation of alpha and alpha(z) by beta. A, alpha (70 nM) was incubated on ice for 10 min with or without beta(1)(2) (140 nM) and then phosphorylated with PKC. Aliquots (100 µl) were withdrawn at the indicated times, filtered, and counted as described under ``Experimental Procedures.'' B, alpha (50 nM) was incubated with the indicated concentration of beta(1)(2) or beta(1)(2)C68S on ice and then phosphorylated with PKC at 30 °C for 20 min. Aliquots were then filtered and counted. C, alpha or alpha(z) (50 nM) was incubated with the indicated concentration of beta(1)(2) on ice for 10 min and then phosphorylated with PKC for 20 min at 30 °C. Data are expressed as percent phosphorylation relative to that observed in the absence of beta(1)(2). In A, B, and C, data are the average of duplicate determinations from a single experiment that is representative of three such experiments.



Both the GDP-bound and the GTPS-bound forms of alpha and alpha(z) are phosphorylated almost equally well by PKC (Fig. 4A); there was no significant difference in the time course of phosphorylation of both forms of both proteins (data not shown). Lounsbury et al.(17) reported that the GDP-bound form of alpha(z) was phosphorylated more efficiently than the GTPS-activated species. The discrepancy may be explained by the fact that the alpha subunits used in this work were purified from Sf9 cells and thus myristoylated at their amino termini; the protein used by Lounsbury et al.(17) was synthesized in Escherichia coli and was not so modified. Myristoylation of the amino terminus may alter the conformation of this domain, which is the site of phosphorylation (see below).


Figure 4: Phosphorylation of the GDP- or GTPS-bound forms of alpha(z) or alpha. A, GDP- or GTPS-bound alpha(z) or alpha (2.5 pmol of each) was phosphorylated with PKC for 20 min at 30 °C. The products were resolved by SDS-PAGE and subjected to autoradiography. To prepare the GTPS-bound alpha subunits, alpha was incubated with 100 µM GTPS in the presence of 10 mM MgSO(4) at 30 °C for 120 min; alpha(z) was incubated with 100 µM GTPS in the presence of 5 mM EDTA and 3 mM MgCl(2) at 30 °C for 90 min. Proteins were then gel-filtered into 50 mM NaHepes (pH 8.0), 100 mM NaCl, 3 mM MgCl(2), 1 mM EDTA, 2 mM DTT, and 0.7% CHAPS. The amount of protein was estimated by staining with Amido Black. B, alpha(z)-GDP () or alpha(z)-GTPS (bullet) (0.5 nM) was incubated with indicated concentrations of beta(1)(2) and then phosphorylated with PKC and 1 µM [-P]ATP (70 cpm/fmol) at 30 °C for 30 min in a total volume of 100 µl. Aliquots were filtered and counted as described under ``Experimental Procedures.'' Data shown are the average of duplicate determinations from a single experiment that is representative of three such experiments.



Since beta inhibits the phosphorylation of alpha and alpha(z), we assessed the dependence of this effect on beta concentration (using both GDP- and GTPS-bound forms of alpha(z) at the lowest possible concentrations (0.5 nM)) in an attempt to estimate the affinity of beta for the protein (Fig. 4B). Efforts to measure these affinities have been thwarted in the past by the very high affinity of alpha-GDP for beta and resultant difficulty in detection of an effect of beta on alpha at appropriately low concentrations. However, phosphorylation of alpha by PKC offers a very sensitive signal. The concentrations of beta(1)(2) required to inhibit (by 50%) phosphorylation of alpha(z)-GDP and alpha(z)-GTPS were 0.5 and 50 nM, respectively. Since the effect of beta(1)(2) on alpha(z)-GDP was still close to stoichiometric, there exists at least a 100-fold difference in apparent affinity of beta(1)(2) for alpha(z)-GDP and alpha(z)-GTPS.

The Site of Phosphorylation of alpha

Phosphorylated alpha was digested with trypsin either in the presence of GDP or GDP + AMF. Activation of alpha by AMF protects the bulk of the protein from digestion, and a 40-kDa fragment accumulates (Fig. 5). This fragment is recognized by antiserum J169, which was generated using a peptide corresponding to the carboxyl terminus of alpha (Fig. 5, lane 2). However, this fragment is no longer phosphorylated (Fig. 5, lane 5). Thus, phosphorylated alpha can still be activated by AMF, and phosphorylation by PKC occurs near the amino terminus. Similar results were obtained with alpha(z), in which Ser and Ser both appear to be phosphorylated by PKC(17) .


Figure 5: Tryptic digestion of alpha. alpha (2.5 pmol) was phosphorylated with PKC and [-P]ATP as described under ``Experimental Procedures'' and then incubated with 100 µM GDP, 10 mM NaF, 30 µM AlCl(3), and 20 mM MgSO(4) (lanes 2 and 5) or 100 µM GDP alone (lanes 3 and 6) on ice for 10 min. TPCK-treated trypsin (20% of the alpha mass) was then added and incubation was continued at 30 °C for 20 min. The products were resolved by SDS-PAGE, followed by immunoblotting with antiserum J169 (lanes 1-3) or autoradiography (lanes 4-6). Lanes 1 and 4 show the sample before digestion with trypsin.



Characterization of Phosphorylated alpha and alpha(z)

alpha and alpha(z) were phosphorylated and repurified as described under ``Experimental Procedures.'' The stoichiometry of phosphorylation of alpha was approximately 0.5 based on total protein concentration (presumed stoichiometry approximately 1), while that for alpha(z) was 1-1.5; it is possible that alpha(z) is phosphorylated at more than one site(17) . Phosphorylation did not change the time course of GTPS binding (and thus of GDP dissociation) for either alpha or alpha(z) (Fig. 6).


Figure 6: Time course of GTPS binding to phosphorylated alpha or alpha(z). Nonphosphorylated (bullet) or phosphorylated () alpha (A) or alpha(z) (B) (100 nM each) was incubated at 30 °C with 5 µM [S]GTPS and 10 mM MgSO(4) (alpha) or 0.3 mM MgSO(4) (alpha(z)) in the presence of 1 mM EDTA. Aliquots (50 µl) were withdrawn at the indicated times, filtered, and counted. Data shown are the average of duplicate determinations from a single experiment that is representative of two such experiments.



The rate of binding of GTPS to nonphosphorylated alpha or alpha(z) is inhibited by beta (Fig. 7). This reflects the well-known capacity of beta to stabilize the GDP-bound form of G protein alpha subunits. However, the rate of GTPS binding to phosphorylated alpha (Fig. 7A) or phosphorylated alpha(z) (Fig. 7B) is not inhibited substantially by a 10-fold molar excess of beta(1)(2), and the modest effects seen could reflect the presence of small amounts of nonphosphorylated protein in the preparations. The effect of beta on GTPS binding to mock-treated proteins (PKC in the absence of ATP) was the same as that on the nonphosphorylated proteins (data not shown).


Figure 7: Inhibition by beta(1)(2) of the rate of GTPS binding to alpha or alpha(z). Phosphorylated () or nonphosphorylated (bullet) alpha (A) or alpha(z) (B) (140 nM) was mixed with the indicated concentration of beta(1)(2) and then incubated at 30 °C for 60 min with 5 µM [S]GTPS and 10 mM MgSO(4) (alpha) or 0.3 mM MgSO(4) (alpha(z)) in the presence of 1 mM EDTA. After 60 min, aliquots (50 µl) were withdrawn, filtered, and counted. The amount of GTPS bound is shown as the percentage bound relative to that observed in the absence of beta(1)(2). For nonphosphorylated or phosphorylated alpha, 100% was 1.0 and 0.8 pmol, respectively. For nonphosphorylated or phosphorylated alpha(z), 100% was 1.4 and 1.3 pmol, respectively. Data shown in A and B are the average of duplicate determinations from a single experiment that is representative of three such experiments.



We also examined the interaction of alpha and beta by gel filtration. The peaks of both phosphorylated and nonphosphorylated alpha were in fractions 38-40 (Fig. 8), corresponding to molecular weights of about 45,000. Addition of beta(1)(2) to nonphosphorylated alpha shifted the peak of alpha about three fractions (35-37), while the migration of phosphorylated alpha was unchanged after incubation with beta(1)(2). The results shown in Fig. 7and Fig. 8indicate that phosphorylation of alpha and alpha(z) interfere with their capacity to form oligomers with the G protein beta subunit complex.


Figure 8: Superdex 200 gel filtration of alpha and beta(1)(2). Nonphosphorylated alpha (A) or phosphorylated alpha (B) (250 pmol) was incubated with or without 750 pmol of beta(1)(2) prior to application to a Superdex 200 gel filtration column. Fractions (20 µl) were subjected to SDS-PAGE and stained with silver. Positions of molecular weight standards are: void volume (fraction 25), -globulin (fraction 31), ovalbumin (fraction 39), myoglobin (fraction 43), and vitamin B (fraction 52).



Finally, we examined the effect of phosphorylation of alpha(z) on its ability to inhibit the activity of type V adenylyl cyclase(5) , since this is the only assay available for interaction of alpha(z) or alpha with an effector (Fig. 9). Okadaic acid (1 µM) was included in the assay to inhibit phosphatases that might be present in the Sf9 cell membranes utilized as the source of adenylyl cyclase. Phosphorylation of alpha(z) had little or no effect on its inhibitory interactions with adenylyl cyclase.


Figure 9: Inhibition of type V adenylyl cyclase by phosphorylated alpha(z). The indicated concentrations of alpha(z) were mixed with 20 µg of membranes from Sf9 cells expressing type V adenylyl cyclase in the presence of 50 nM GTPS-alpha(s). Adenylyl cyclase activity was assayed as described under ``Experimental Procedures.'' alpha subunits were nonphosphorylated alpha(z)-GDP (), nonphosphorylated alpha(z)-GTPS (bullet), phosphorylated alpha(z)-GDP (), phosphorylated alpha(z)-GTPS (). The concentrations of GTPS-activated alpha subunits were estimated from [S]GTPS binding. Data shown are the average of duplicate determinations from a single experiment that is representative of three such experiments.




DISCUSSION

We have demonstrated that alpha is phosphorylated by PKC both in vitro and in vivo; the homologous subfamily member alpha is not a substrate. Among the large number of G protein alpha subunits tested, the only other efficient substrate for phosphorylation by various isoforms of PKC was alpha(z). The stoichiometry of phosphorylation of alpha was equal to that for GTPS binding and is thus assumed to be 1.

Phosphorylation of alpha occurs within the amino-terminal domain that is removed by trypsin selectively from activated G protein alpha subunits (Table 1). Examination of corresponding sites of proteolysis in other G subunits indicates that trypsin probably removes the first 49 or 50 residues from alpha. There are three serine residues (2, 9, and 38) and one threonine (7) within the relevant sequence. Although Ser^9 and Ser are both candidates for phosphorylation by PKC(30) , Ser is surrounded by basic residues (RRRSR) and corresponds to one of the phosphorylated serine residues in alpha(z) (Ser; RRSRR). There is no equivalent of alpha residues Ser^2, Thr^7, or Ser^9 in alpha(z), and there is no equivalent of alpha(z) residue Ser (the other phosphorylation site) in alpha. Although these arguments appear to implicate Ser in alpha as the site of phosphorylation, Ser^9 cannot be ruled out. Of interest, both Ser^9 and Ser have homologs in alpha, which is not phosphorylated.



Phosphorylation of alpha and alpha(z) does not appear to change their basic guanine nucleotide binding properties, nor the interactions of alpha(z) with type V adenylyl cyclase. However, the affinity of both alpha subunits for beta is clearly reduced by phosphorylation, and, reciprocally, their phosphorylation is inhibited by prior interaction with beta. Similar results with alpha(z) were just reported by Fields and Casey(21) . This effect suggests that phosphorylation of these proteins could play a role in desensitization of the relevant signaling pathways if PKC was stimulated simultaneously. Activation of the G protein causes dissociation of alpha from beta, and PKC-mediated phosphorylation would thus be favored. Subsequent inhibition of oligomerization as a result of phosphorylation of alpha would presumably attenuate signaling because of the requirement for beta for receptor-mediated activation of alpha.

Although the signaling pathway that is regulated by alpha is unknown, expression of constitutively activated alpha activates Na/H exchange in a PKC-dependent manner(10) . Perhaps alpha activates certain isoforms of PKC either directly or indirectly to stimulate Na/H exchange, while PKC attenuates the activity of alpha in a classic feedback loop.

The crystal structure of alpha has been determined in its GTPS-, free GDP-, and GDP/beta-bound forms(31, 32, 33) . The conformation of the amino terminus of the alpha subunit is a particularly dynamic aspect of the nucleotide- and beta-induced structural changes that have been observed. The amino terminus is disordered when alpha is activated by GTPS; it forms a compact subdomain with the carboxyl terminus of alpha in the free GDP-bound form; it is extended in a long alpha helix that forms extensive contacts with the beta subunit in the heterotrimer. The serine residue in alpha (Ser) that is analogous to Ser in alpha and Ser in alpha(z) is part of this interface and is hydrogen-bonded to Lys in beta(1), consistent with the effect of phosphorylation at this site on interactions of alpha with beta.

The compact subdomain formed by the amino and carboxyl termini of alpha in the free GDP-bound state is also of interest. This domain appears to be stabilized by interactions between arginine residues at positions 15, 21, and 32 and a sulfate ion contributed by the crystallization solution. Sulfate ions are capable of binding at sites that normally interact with phosphate or phosphoserine(34) , and the arginine residues involved are close to the sites of phosphorylation of alpha and alpha(z). It will be interesting to determine if phosphorylation alters the structure of this microdomain. The specificity of phosphorylation of G protein alpha subunits by PKC seems problematic, particularly if phosphorylation regulates a property as fundamental as alpha subunit oligomerization. Although alpha is apparently phosphorylated following activation of PKC in hepatocytes or the promyelocytic cell line U937 (19, 20) and phosphorylation in vitro of a mixture of isoforms of alpha(i) by PKC was also described(18) , we were not able to demonstrate phosphorylation of specific isoforms of alpha(i) with the preparations of PKC used in this study. Perhaps phosphorylation of G protein alpha subunits is a more general phenomenon than suspected and the appropriate kinases have not yet been identified.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM34497, American Cancer Society Grant BE30-O, the Lucille P. Markey Charitable Trust, and the Raymond and Ellen Willie Chair of Molecular Neuropharmacology (to A. G. G.). 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.

§
To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235.

(^1)
The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; PKC, protein kinase C; GTPS, guanosine 5`-3-O-(thio)triphosphate; CE, polyoxyethylene 10-lauryl ether; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; PMA, phorbol 12-myristate 13-acetate; TPCK, tosylphenylalanyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Jeffrey Laidlaw for excellent technical assistance, Shigeo Ohno for a recombinant baculovirus encoding PKCalpha, Bruce Posner for beta(1)(2)C68S, and Patrick Casey for communicating results prior to publication.


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