©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Wortmannin-sensitive Activation of p70 by Endogenous and Heterologously Expressed G-coupled Receptors (*)

(Received for publication, December 18, 1995; and in revised form, February 5, 1996)

Moira Wilson (1) (2) Andrew R. Burt (2) Graeme Milligan (2) Neil G. Anderson (1)(§)

From the  (1)Hannah Research Institute, Ayr KA6 5HL, Scotland, United Kingdom and the (2)Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In order to study the regulation of the ribosomal protein S6 kinase, p70, by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the alpha(2) adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human alpha receptor, with the alpha(2) agonist UK 14304 led to a transient increase in p70 activity. UK 14304 also activated p70 in a clone expressing the porcine alpha receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70 in alpha(2) receptor-transfected and in nontransfected cells. Activation of p70 by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70 by both agents. Activation of p70 by UK 14304 and by LPA, but not by platelet-derived growth factor (PDGF), was blocked by preincubation of cells with pertussis toxin. Wortmannin, a selective inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of p70 by UK 14304, LPA, and PDGF. These data indicate that p70 is regulatable by G(i)-coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70 by such agents appears to involve an isoform of PI 3-kinase.


INTRODUCTION

Mitogenic stimulation of cells results in increased phosphorylation of the ribosomal protein S6. Increased S6 phosphorylation correlates with elevated rates of translation and may be partly responsible for the increase in protein synthesis triggered by mitogens(5) . Phosphorylation of S6 occurs on five closely positioned sites and is catalyzed by a protein kinase known as p70(6, 7, 8) . Prevention of the activation of p70 represses cell growth and in some cell types blocks entry into S phase(9, 10, 11, 12) .

The mechanism of activation of p70 has not been fully characterized but appears to involve its phosphorylation on multiple sites by more than one protein kinase(13, 14) . Various lines of evidence indicate that p70 activation is independent of the p21/MAP (^1)kinase pathway(15, 16) . For example, p21 and p74 mutants block activation of MAP kinases but not p70 and certain PDGF receptor mutants, which activate p21 normally, fail to activate p70(16) . The immunosuppressive macrolide rapamycin completely blocks the activation of p70 but has no effect on MAP kinase activation (17, 18) and as such has become a useful tool for delineating p70-specific signaling events. Other studies have shown that the PI 3-kinase inhibitor wortmannin prevents activation of p70 by a range of agonists(19, 20, 21, 22, 23) , suggesting that PI 3-kinase lies upstream of p70.

Mitogen receptors can be subdivided into those which either are or couple to a tyrosine kinase (the tyrosine kinase class) and those which couple to heterotrimeric G proteins (the GPCRs). Although it was considered that these two classes signalled via distinct and mutually exclusive mechanisms, recent evidence has challenged this view. For example, the p21/MAP^1 kinase pathway, originally thought only to be activated by tyrosine kinase receptors, is now known to be activated by several GPCRs(24, 25, 26, 27, 28, 29) . These include the alpha(2) adrenergic, m(2) muscarinic, and LPA receptors which couple to the pertussis toxin-sensitive G(i) subfamily. The mechanisms linking G(i)-linked receptors with the MAP kinase and other mitogenic signaling pathways are still unclear but accumulating evidence from several laboratories indicates an involvement of G protein beta subunit complexes(30, 31, 32) .

As a means to study the mitogenic signaling of GPCRs, we have previously transfected Rat-1 fibroblasts with the human alpha(2)-C10 adrenergic receptor and shown that the receptor interacts directly with two pertussis toxin-sensitive G proteins, G and G, and thus induces both adenylyl cyclase inhibition and phospholipase D activation(33, 34) . Activation of the receptor also results in a mitogenic response characterized by a pertussis toxin-sensitive activation of the p21/MAP kinase pathway and DNA synthesis(26, 29) . Given the role of p70 in mitogenic signaling, we reasoned that this kinase should also be activated following stimulation of alpha(2) receptors in these cells. Our results show that this does occur and thus provide the first demonstration of p70 activation by a receptor acting solely through G(i). We further show that wortmannin attenuates activation of p70 suggesting that an isoform of PI 3-kinase is involved in the activation mechanism.


EXPERIMENTAL PROCEDURES

Materials

Cell culture media, calf serum, PDGF, hygromycin B, and geneticin were purchased from Life Technologies, Paisley, UK. Pertussis toxin was supplied by Speywood. Protein A-agarose was purchased from Pierce-Warriner, Chester, UK, and P(i) was from ICN Biomedicals. [-P]ATP, ECL reagents, and nitrocellulose were supplied by Amersham International, UK. All other reagents were from Sigma. Peptides were synthesized at the Hannah Research Institute core facility. The p70 antiserum was raised against a synthetic peptide representing amino acids 1-30 of rat p70(35) .

Cell Culture and Treatment

Rat-1 fibroblasts were transfected with the human alpha receptor (clone 1C cells) as described previously (33) or with an HA-tagged variant of the porcine alpha adrenoreceptor(36, 37) , equivalent to the human alpha receptor as follows. Cells were transfected in a 1:10 ratio with the plasmid pBABE hygro, which is able to direct expression of the hygromycin B resistance marker, and an HA-tagged porcine alpha adrenoreceptor in plasmid pCMV4 using Lipofectin reagent according to the manufacturer's instructions (Life Technologies, Inc.). Clones which demonstrated resistance to hygromycin B (200 µg/ml) were selected and expanded. The clone TAG WT3 displayed high affinity binding of [^3H]yohimbine (data not shown) and were used in this study. All cells were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum and either geneticin (500 µg/ml for clone 1C cells) or hygromycin B (50 µg/ml for TAG WT3 cells). Cells were grown to confluency in 100-mm cell culture dishes and serum-starved for 16-20 h prior to use. In some experiments, pertussis toxin was included in the serum starving medium at a final concentration of 25 ng/ml. Wortmannin was stored as a 10 mM solution in dimethyl sulfoxide at -20 °C and was diluted into water just prior to use. Rapamycin (a generous gift from Dr Sehgal, Wyeth-Ayerst, Princeton, NJ) was stored in ethanol at -20 °C and diluted with water just prior to use.

Immunoprecipitation of p70

After treatment, cells were washed twice in ice-cold phosphate-buffered saline and scraped into 1 ml of lysis buffer containing 50 mM TrisbulletHCl, pH 8.0 (4 °C), 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 5 mM EGTA, 10 mM sodium pyrophosphate, 30 mM 4-nitrophenyl phosphate, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each of leupeptin, aprotinin, and pepstatin A. After 20 min, lysates were centrifuged for 10 min at 14,000 times g. The supernatant was precleared by addition to 20 µl (packed volume) of protein A-agarose for 1 h. Precleared lysates (250 µg of protein) were immunoprecipitated with 2.5 µl of p70 antiserum for 2 h before addition to 20 µl of protein A-agarose for 1 h. Immunoprecipitates were washed twice with lysis buffer and twice with kinase assay buffer (25 mM MOPS, pH 7.2 (30 °C), 1 mM EDTA, 0.05% (v/v) Triton X-100, 1 mM DTT, and 20 mM 4-nitrophenyl phosphate). Immunoprecipitates were then resuspended in kinase assay buffer (50 µl) containing S6 substrate (a 32-amino acid peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) from the C-terminal sequence of S6; 20 µM) and 2 µM cyclic AMP-dependent protein kinase inhibitor. After a 2-min preincubation at 30 °C, the reaction was initiated by the addition of ATP/MgCl(2) (25 µM [-P]ATP (5000 cpm/pmol), 10 mM MgCl(2)). After 10 min, 30 µl of the reaction was spotted onto P81 paper which was then washed five times in 75 mM phosphoric acid. Dried papers were placed in 4 ml of scintillant and counted.

Metabolic Labeling of Cells

Serum-starved cells were incubated for 4 h in 4 ml of phosphate-free Dulbecco's modified Eagle's medium containing 1 mCi of P(i). Stimulants were then added following which cells were rinsed twice with phosphate-buffered saline, lysed, and immunoprecipitated with p70 antibodies as described above. Proteins eluted from the immune complex were electrophoresed on 9% (30:0.8 acrylamide:bisacrylamide) gels. The p70 bands were located by autoradiography, excised from the gel, and counted.

Immunoblotting of p70

Immunoprecipitates or cell lysate samples (25 µg) were electrophoresed on 9% (30:0.32; acrylamide:bisacrylamide) gels and transferred to nitrocellulose for 2 h at 400 mA. Following blocking in 3% bovine serum albumin, immunoblots were incubated with anti-p70 (1:1000 dilution) for 3 h followed by horseradish peroxidase-conjugated anti-rabbit IgG (1:5000 dilution) for 1 h. Immunoreactive bands were detected by enhanced chemiluminescence (ECL, Amersham) according to the manufacturer's instructions.

Phosphatase Treatment of p70

p70 was immunoprecipitated from cells as described above. The immunoprecipitates were washed twice with lysis buffer and twice with phosphatase buffer (20 mM PIPES, pH 6.8, 20 mM KCl, 1 mM dithiothreitol, 1 mM MgCl(2), 0.05% Brij 35). The complexes were then incubated for 30 min at 25 °C in 50 µl of phosphatase buffer containing 0.375 unit of potato acid phosphatase (Calbiochem-Novabiochem). Following incubation with phosphatase, an equal volume of stop buffer (20 mM potassium phosphate, pH 7.2, 0.1 mM sodium vanadate, 20 mM beta-glycerophosphate) was added, and the complexes were washed twice with Tris-buffered saline. Proteins were eluted from the complex by boiling in SDS-sample buffer, and the samples were then immunoblotted with p70 antibodies as described above.


RESULTS AND DISCUSSION

To examine possible coupling of GPCRs to p70, we used Rat-1 fibroblasts stably transfected with either the human alpha adrenergic receptor (clone 1C) or with an equivalent HA-tagged version of the porcine receptor (clone TAG WT3). Stimulation of 1C cells (which express around 3.5 pmol of receptor/mg of membrane protein(33) ) with the alpha(2) agonist UK 14304 led to a transient increase in p70 activity measured by an immunocomplex kinase assay (Table 1). Maximal activation (approximately 3-fold) was observed around 10-20 min after stimulation and although activity declined thereafter it remained above basal levels for at least 3 h (data not shown). In TAG WT3 cells (which express around 400 fmol of receptor/mg of membrane protein), UK 14304 activated p70 to a lesser extent than in 1C cells (Table 1). In parental Rat-1 cells, which do not express detectable levels of alpha(2) receptor(33) , UK 14304 did not significantly increase p70 activity. In addition to the effects of UK 14304, the mitogenic glycerophospholipid LPA, acting through endogenously expressed GPCRs, activated p70 in all three cell types (Table 1). Thus, activation of p70 by G protein-coupled agonists may be a generalized phenomenon in these cells which does not require heterologous high level expression of the appropriate receptor.



As expected, the tyrosine kinase receptor ligand PDGF also strongly coupled to p70 activation in both alpha(2) receptor-transfected and nontransfected cells (Table 1). The relative activation of p70 induced by PDGF, and indeed by LPA, was substantially higher in the untransfected cells, but this appears to reflect elevated basal p70 activities in both the TAG WT3 and 1C cells (Table 1). Attempts to down-regulate basal p70 activity in these cells by manipulating the conditions used for serum deprivation were unsuccessful (data not shown). Similarly, elevated MAP kinase basal activity in these cells has previously been reported(29) .

Activation of p70 correlates strongly with its phosphorylation on multiple sites(13, 14) . Therefore, we next examined whether the alpha(2) receptor-mediated activation of p70 was accompanied by enhanced phosphorylation of the protein. Phosphorylation of p70 results in a reduction in the relative mobility of the protein on SDS-polyacrylamide gel electrophoresis, and this is often used as an indicator of p70 activation status(17) . Fig. 1shows an immunoblot of p70 following treatment of cells with different agonists. In parental Rat-1 cells, PDGF and, to a lesser extent, LPA induced the appearance of slower migrating forms of p70, whereas UK 14304 had no such effect. Immunoblotting of clone 1C cells revealed that most of the p70 protein was detected as the slower migrating forms even in unstimulated cells, consistent with the elevated basal p70 activities in these cells (Table 1). Phosphatase treatment of p70 immunoprecipitated from LPA- or UK 14304-treated clone 1C cells resulted in the disappearance of the most slowly migrating forms of p70 and the coincident appearance of a single band with faster mobility (Fig. 1B). This effect was also evident in samples prepared from unstimulated clone 1C cells indicating that p70 exhibits increased basal phosphorylation as well as elevated basal activity in these cells.


Figure 1: G protein-coupled receptor activation results in increased phosphorylation of p70. A, Rat-1 cells (left panel) or clone 1C cells (right panel) were left untreated (C) or treated for 10 min with 10 ng/ml PDGF (P), 10 µM LPA (L), or 1 µM UK 14304 (U). Lysates were prepared, and 25 µg of lysate protein from each sample was immunoblotted with anti-p70. The fastest and slowest of the four immunoreactive bands representing various phosphorylation states of the enzyme are indicated by the arrowheads. B, p70 immunoprecipitated from control, LPA, or UK 14304-stimulated clone 1C cells were treated or not with potato acid phosphatase and then subjected to immunoblotting with anti-p70 antibodies as described under ``Experimental Procedures.'' C, clone 1C cells were metabolically labeled with P(i) and then left untreated (control) or treated with LPA or UK 14304. p70 was immunoprecipitated from the cells as described under ``Experimental Procedures'' and electrophoresed, and the dried gel was exposed to x-ray film for 24 h. The p70 bands were excised from the gel and counted for radioactivity. Duplicate immunoprecipitations from a single experiment, performed on one other occasion with similar results, are shown.



To confirm that activation of p70 by GPCRs results in increased phosphorylation of the protein, cells were metabolically labeled with P(i), and the levels of phosphate incorporated into p70 were measured. Fig. 1C shows that both UK 14304 and LPA induce an increase in the phosphorylation of p70. Counting of the P incorporated into the p70 bands revealed that LPA induced a 43.0 ± 4.0% (n = 4) increase and UK 14304 induced a 65.9 ± 4.3% (n = 4) increase in p70 phosphorylation. For comparison, PDGF, the most potent activator of p70 used in this study, increased the phosphorylation of p70 by around 2-fold in Rat-1 cells (data not shown). Taken together, these results show that activation of p70 by GPCRs correlates with enhanced phosphorylation of the protein. Whether G(i)-mediated signals result in phosphorylation of the same sites on p70 induced by other effectors remains to be determined.

The immunosuppressive drug rapamycin blocks the activation of p70 by preventing or reversing its phosphorylation at key sites necessary for activity(11, 12, 13, 14, 17, 18) . The mechanism underlying this effect has not been characterized completely, but recent reports have identified the protein kinase FRAP/RAFT (38, 39, 40) as a specific target for rapamycin in mammalian cells. The activation of p70 by UK 14304, LPA, and PDGF in the present study was completely blocked by pretreatment of cells with rapamycin (Fig. 2) indicating that there are commonalities in the mechanisms employed by both G protein-coupled agonists and other agents to elicit activation of p70. In contrast, only the activation of p70 by UK 14304 and LPA was blocked by pretreatment of cells with pertussis toxin (Fig. 3), consistent with their effects being mediated via the G(i) family of heterotrimeric G proteins. Although pertussis toxin pretreatment also led to a significant reduction in the p70 activity precipitated from PDGF-stimulated cells (Fig. 4), this is entirely accounted for by pertussis toxin-mediated inhibition of basal p70 activity. Thus, the relative increases in p70 activity induced by PDGF in control and pertussis toxin-treated cells were 42 ± 4% and 45 ± 3% (n = 3), respectively.


Figure 2: Rapamycin prevents activation of p70. Clone 1C cells were pretreated (open bars) or not (filled bars) with 100 nM rapamycin for 10 min prior to the addition of PDGF (10 ng/ml), LPA (10 µM), or UK 14304 (1 µM) for 10 min. Lysates were prepared, and p70 activity was measured on the immune complex as described under ``Experimental Procedures.'' *, significant inhibitory effect of rapamycin (p < 0.05, Student's t test)




Figure 3: Pertussis toxin prevents activation of p70 by G(i)-linked agonists. Clone 1C cells were pretreated (open bars) or not (filled bars) with 25 ng/ml pertussis toxin for 16 h prior to the addition of PDGF (10 ng/ml), LPA (10 µM), or UK 14304 (1 µM) for an additional 10 min. Lysates were prepared, and p70 activity was measured on the immune complex as described under ``Experimental Procedures.'' *, significant effect of pertussis toxin treatment (p < 0.05, Student's t test).




Figure 4: Wortmannin blocks activation of p70 by UK14304. Clone 1C cells were pretreated for 5 min with the indicated concentrations of wortmannin prior to the addition of 1 µM UK 14304 for 10 min. Lysates were prepared, and p70 activity was measured on the immune complex as described under ``Experimental Procedures.'' Data are expressed as a percentage of the maximal stimulatory effect of UK 14304 in the absence of wortmannin and are taken from a single experiment done on three other occasions with similar results.



Although the precise mechanisms of activation of p70 are unknown, a number of studies using the selective inhibitor wortmannin indicate that PI 3-kinase lies upstream of p70 in a signaling cascade induced by a number of tyrosine kinase receptors (19, 20, 21, 22, 23) . More recent work suggests that p70 contains a set of wortmannin-sensitive phosphorylation sites(13, 14, 41) . We therefore sought evidence for PI 3-kinase involvement in the activation of p70 by GPCRs. Pretreatment of clone 1C cells with wortmannin led to a dose-dependent attenuation of p70 activation by UK 14304 (Fig. 4). Approximately 50% inhibition occurred with concentrations of wortmannin around 30 nM with complete inhibition evident at 50-100 nM. These concentrations are similar to those reported to inhibit p70 activation by other growth factors(19, 20, 21, 22, 23) . Wortmannin also prevented the full activation of p70 by LPA (Table 2). We also tested the effects of LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), another PI 3-kinase inhibitor, structurally unrelated to wortmannin(20) . Preincubation of clone 1C cells with 50 µM LY 294002 completely blocked the activation of p70 induced by UK 14304 as well as by LPA and PDGF (data not shown). Together, these data suggest that PI 3-kinase is required for the activation of p70 by G(i)-linked agonists in Rat-1 cells. Whether this is the same PI 3-kinase isoform believed to regulate p70 in response to tyrosine kinase receptor ligands is not known. It is worth noting that specific G protein beta-activated forms of PI 3-kinase have been identified (1, 2, 3) and, very recently, a novel G protein-activated PI 3-kinase was isolated and cloned(4) . This protein, termed PI 3-kinase-, is regulated directly by both alpha and beta G protein subunits and is also inhibited by wortmannin. It will be of interest to determine whether this isoform of PI 3-kinase is stimulated by UK 14304 and other G protein-coupled ligands and whether it is required for the activation of p70.



In conclusion, we have shown that stimulation of both endogenous and heterologously expressed G(i)-coupled receptors results in a pertussis toxin-sensitive activation and coincident hyperphosphorylation of the S6 kinase p70. The activation is sensitive to both rapamycin and wortmannin and as such may be similar mechanistically to that stimulated by ligands signaling via tyrosine kinase receptors. Future work will be directed toward understanding the precise mechanisms underlying the effects of G(i)-coupled agonists on p70, particularly the potential role of beta signaling complexes.


FOOTNOTES

*
This work was supported by the Biotechnology and Biological Sciences Research Council. 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. Tel. 44-1292-476-013; Fax: 44-1292-671-052.

(^1)
The abbreviations used are: MAP kinase, mitogen-activated protein kinase; PI 3-kinase, phosphoinositide 3-OH kinase; GPCR, G protein-coupled receptor; PDGF, platelet-derived growth factor; LPA, lysophosphatidic acid; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Lee Limbird (Vanderbilt University, Nashville) for the HA-tagged alpha2A cDNA, Peter Downes (Dundee University) for LY 294002, and Sandra Woodburn for technical assistance.


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