Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors.

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 α adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human α receptor, with the α agonist UK 14304 led to a transient increase in p70 activity. UK 14304 also activated p70 in a clone expressing the porcine α receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70 in α 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-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.

In order to study the regulation of the ribosomal protein S6 kinase, p70 s6k , by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the ␣ 2 adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human ␣ 2C10 receptor, with the ␣ 2 agonist UK 14304 led to a transient increase in p70 s6k activity. UK 14304 also activated p70 s6k in a clone expressing the porcine ␣ 2A receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70 s6k in ␣ 2 receptortransfected and in nontransfected cells. Activation of p70 s6k by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70 s6k by both agents. Activation of p70 s6k 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 s6k by UK 14304, LPA, and PDGF. These data indicate that p70 s6k is regulatable by G i -coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70 s6k by such agents appears to involve an isoform of PI 3-kinase.
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 s6k (6 -8). Prevention of the activation of p70 s6k represses cell growth and in some cell types blocks entry into S phase (9 -12).
The mechanism of activation of p70 s6k 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 s6k activation is independent of the p21 ras /MAP 1 kinase pathway (15,16). For example, p21 ras and p74 raf mutants block activation of MAP kinases but not p70 s6k and certain PDGF receptor mutants, which activate p21 ras normally, fail to activate p70 s6k (16). The immunosuppressive macrolide rapamycin completely blocks the activation of p70 s6k but has no effect on MAP kinase activation (17,18) and as such has become a useful tool for delineating p70 s6kspecific signaling events. Other studies have shown that the PI 3-kinase inhibitor wortmannin prevents activation of p70 s6k by a range of agonists (19 -23), suggesting that PI 3-kinase lies upstream of p70 s6k .
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 ras /MAP 1 kinase pathway, originally thought only to be activated by tyrosine kinase receptors, is now known to be activated by several GPCRs (24 -29). These include the ␣ 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 ␤␥ subunit complexes (30 -32).
As a means to study the mitogenic signaling of GPCRs, we have previously transfected Rat-1 fibroblasts with the human ␣ 2 -C10 adrenergic receptor and shown that the receptor interacts directly with two pertussis toxin-sensitive G proteins, G i2 and G i3 , 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 ras /MAP kinase pathway and DNA synthesis (26,29). Given the role of p70 s6k in mitogenic signaling, we reasoned that this kinase should also be activated following stimulation of ␣ 2 receptors in these cells. Our results show that this does occur and thus provide the first demonstration of p70 s6k activation by a receptor acting solely through G i . We further show that wortmannin attenuates activation of p70 s6k suggesting that an isoform of PI 3-kinase is involved in the activation mechanism.
with an HA-tagged variant of the porcine ␣ 2A adrenoreceptor (36,37), equivalent to the human ␣ 2C10 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 HAtagged porcine ␣ 2A adrenoreceptor in plasmid pCMV4 using Lipofectin reagent according to the manufacturer's instructions (Life Technologies, Inc.). Clones which demonstrated resistance to hygromycin B (200 g/ml) were selected and expanded. The clone TAG WT3 displayed high affinity binding of [ 3 H]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 g/ml for clone 1C cells) or hygromycin B (50 g/ml for TAG WT3 cells). Cells were grown to confluency in 100-mm cell culture dishes and serumstarved 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 Ϫ20°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 Ϫ20°C and diluted with water just prior to use.
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 32 P i . Stimulants were then added following which cells were rinsed twice with phosphate-buffered saline, lysed, and immunoprecipitated with p70 s6k antibodies as described above. Proteins eluted from the immune complex were electrophoresed on 9% (30:0.8 acrylamide:bisacrylamide) gels. The p70 s6k bands were located by autoradiography, excised from the gel, and counted.
Phosphatase Treatment of p70 s6k -p70 s6k 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 l 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 ␤-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 s6k antibodies as described above.

RESULTS AND DISCUSSION
To examine possible coupling of GPCRs to p70 s6k , we used Rat-1 fibroblasts stably transfected with either the human ␣ 2C10 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 ␣ 2 agonist UK 14304 led to a transient increase in p70 s6k activity measured by an immunocomplex kinase assay (Table I). 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 s6k to a lesser extent than in 1C cells (Table I). In parental Rat-1 cells, which do not express detectable levels of ␣ 2 receptor (33), UK 14304 did not significantly increase p70 s6k activity. In addition to the effects of UK 14304, the mitogenic glycerophospholipid LPA, acting through endogenously expressed GPCRs, activated p70 s6k in all three cell types (Table I). Thus, activation of p70 s6k 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 s6k activation in both ␣ 2 receptor-transfected and nontransfected cells (Table I). The relative activation of p70 s6k induced by PDGF, and indeed by LPA, was substantially higher in the untransfected cells, but this appears to reflect elevated basal p70 s6k activities in both the TAG WT3 and 1C cells (Table I). Attempts to down-regulate basal p70 s6k 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 s6k correlates strongly with its phosphorylation on multiple sites (13,14). Therefore, we next examined whether the ␣ 2 receptor-mediated activation of p70 s6k was accompanied by enhanced phosphorylation of the protein. Phosphorylation of p70 s6k 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 s6k activation status (17). Fig. 1 shows an immunoblot of p70 s6k 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 s6k , whereas UK 14304 had no such effect. Immunoblotting of clone 1C cells revealed that most of the p70 s6k protein was detected as the slower migrating forms even in unstimulated cells, consistent with the elevated basal p70 s6k activities in these cells (Table I). Phosphatase treatment of p70 s6k immunoprecipitated from LPA-or UK 14304-treated clone 1C cells resulted in the disappearance of the most slowly migrating forms of p70 s6k 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 s6k exhibits increased basal phosphorylation as well as elevated basal ac- tivity in these cells.
To confirm that activation of p70 s6k by GPCRs results in increased phosphorylation of the protein, cells were metabolically labeled with 32 P i , and the levels of phosphate incorporated into p70 s6k were measured. Fig. 1C shows that both UK 14304 and LPA induce an increase in the phosphorylation of p70 s6k . Counting of the 32 P incorporated into the p70 s6k bands revealed that LPA induced a 43.0 Ϯ 4.0% (n ϭ 4) increase and UK 14304 induced a 65.9 Ϯ 4.3% (n ϭ 4) increase in p70 s6k phosphorylation. For comparison, PDGF, the most potent activator of p70 s6k used in this study, increased the phosphorylation of p70 s6k by around 2-fold in Rat-1 cells (data not shown). Taken together, these results show that activation of p70 s6k by GPCRs correlates with enhanced phosphorylation of the protein.
Whether G i -mediated signals result in phosphorylation of the same sites on p70 s6k induced by other effectors remains to be determined.
The immunosuppressive drug rapamycin blocks the activation of p70 s6k by preventing or reversing its phosphorylation at key sites necessary for activity (11-14, 17, 18). The mechanism underlying this effect has not been characterized completely, but recent reports have identified the protein kinase FRAP/ RAFT (38 -40) as a specific target for rapamycin in mammalian cells. The activation of p70 s6k 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 s6k . In contrast, only the activation of p70 s6k 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 s6k activity precipitated from PDGF-stimulated cells (Fig. 4), this is entirely accounted for by pertussis toxin-mediated inhibition of basal p70 s6k activity. Thus, the relative increases in p70 s6k activity induced by PDGF in control and pertussis toxintreated cells were 42 Ϯ 4% and 45 Ϯ 3% (n ϭ 3), respectively.
Although the precise mechanisms of activation of p70 s6k are unknown, a number of studies using the selective inhibitor wortmannin indicate that PI 3-kinase lies upstream of p70 s6k in a signaling cascade induced by a number of tyrosine kinase receptors (19 -23). More recent work suggests that p70 s6k 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 s6k by GPCRs. Pretreatment of clone 1C cells with wortmannin led to a dose-dependent attenuation of p70 s6k 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 s6k activation by other growth factors (19 -23). Wortmannin also prevented the full activation of p70 s6k by LPA (Table II). We also tested the effects of LY 294002 (2-(4-morpholinyl)-8phenyl-4H-1-benzopyran-4-one), another PI 3-kinase inhibitor, structurally unrelated to wortmannin (20). Preincubation of Lysates were prepared, and 25 g of lysate protein from each sample was immunoblotted with anti-p70 s6k . The fastest and slowest of the four immunoreactive bands representing various phosphorylation states of the enzyme are indicated by the arrowheads. B, p70 s6k 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 s6k antibodies as described under "Experimental Procedures." C, clone 1C cells were metabolically labeled with 32 P i and then left untreated (control) or treated with LPA or UK 14304. p70 s6k 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 s6k 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. clone 1C cells with 50 M LY 294002 completely blocked the activation of p70 s6k 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 s6k by G i -linked agonists in Rat-1 cells. Whether this is the same PI 3-kinase isoform believed to regulate p70 s6k in response to tyrosine kinase receptor ligands is not known. It is worth noting that specific G protein ␤␥-activated forms of PI 3-kinase have been identified (1-3) 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 ␣ and ␤␥ 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 s6k .
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 s6k . 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 underly-ing the effects of G i -coupled agonists on p70 s6k , particularly the potential role of ␤␥ signaling complexes.

TABLE II
Wortmannin attenuates activation of p70 s6k Clone 1C cells were preincubated with 100 nM wortmannin for 5 min prior to stimulation with the agonists as given in Table I. Lysates were prepared and p70 s6k activity was measured on the immune complex as described under "Experimental Procedures." Data are means Ϯ S.E. of triplicate assays and are representative of experiments carried out on three separate occasions.