Gbeta gamma Stimulates Phosphoinositide 3-Kinase-gamma by Direct Interaction with Two Domains of the Catalytic p110 Subunit*

Daniela LeopoldtDagger , Theodor Hanck§, Torsten ExnerDagger , Udo MaierDagger par , Reinhard Wetzker§, and Bernd NürnbergDagger **

From the Dagger  Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, D-14195 Berlin (Dahlem), Germany and § Max-Planck-Arbeitsgruppe "Molekulare Zellbiologie," Friedrich-Schiller-Universität, Jena, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3Kalpha , -beta , and -delta isoforms coupled to receptor tyrosine kinases and a PI3Kgamma isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3Kgamma catalytic subunit (p110gamma ) and Gbeta gamma complexes. When phosphatidylinositol was used as a substrate, Gbeta gamma stimulated p110gamma lipid kinase activity more than 60-fold (EC50, ~20 nM). Stimulation was inhibited by Galpha o-GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110gamma did not affect Gbeta gamma -mediated enzymatic stimulation, whereas incubation of Gbeta gamma with a synthetic peptide resembling a predicted Gbeta gamma effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110gamma . Gbeta gamma complexes bound to N- as well as C-terminal deletion mutants of p110gamma . Correspondingly, these enzymatically inactive N- and C-terminal mutants inhibited Gbeta gamma -mediated activation of wild type p110gamma . Our data suggest that (i) p110gamma directly interacts with Gbeta gamma , (ii) the pleckstrin homology domain is not the only region important for Gbeta gamma -mediated activation of the lipid kinase, and (iii) Gbeta gamma binds to at least two contact sites of p110gamma , one of which is close to or within the catalytic core of the enzyme.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Phosphatidylinositol 3,4,5-trisphosphate (PI-(3,4,5)P3)1 is absent in quiescent cells but is rapidly produced upon exposure to various stimuli (1-4). Hence, PI-(3,4,5)P3 has been suggested to act as a second messenger eliciting a wide array of cellular responses (5). PI-(3,4,5)P3-dependent functions include regulation of cell growth, protein sorting, exocytosis, and cytoskeletal rearrangements. The enzymes responsible for the formation of PI-(3,4,5)P3 are phosphoinositide 3-kinases (PI3K; EC 2.7.1.137), which catalyze phosphorylation of phosphoinositides at the 3 position of the inositol ring (6). Depending on substrate specificity and enzyme structures, three classes of PI3Ks have been distinguished. Class I enzymes are involved in receptor-induced hormonal responses. They utilize PI, PI-4P, and PI-(4,5)P2 in vitro though within the cell they exhibit a preference for PI-(4,5)P2. In addition, they show a moderate serine/threonine protein kinase activity (7-9). Further discrimination of the class I members is based on their association with receptor tyrosine kinase (class IA)- or of G protein-coupled receptor (class IB)-signaling pathways, although very recently one member (p110beta ) has been suggested to respond synergistically to both Gbeta gamma and tyrosine-phosphorylated peptides (10). Additionally a related retrovirus-encoded PI3K causing hemangiosarcomas was found (11).

Class I enzymes purify as heterodimers with a molecular mass of about 200 kDa containing a catalytic subunit of 110 kDa (p110) and a regulatory subunit (12-16). Several mechanisms for regulating their enzymatic activity in response to extracellular stimuli have been elucidated. Among them the class IA members p110alpha , beta , and delta  are stimulated by tyrosine-phosphorylated proteins through interaction with regulatory PI3K subunits such as p85 or p55 (17, 18). They in turn bind to the N terminus of the catalytic p110 subunit, thereby inducing PI3K activity. In contrast, the only known class IB member p110gamma does not bind to p85 adaptors, but instead associates with a noncatalytic p101 subunit (19). However, several lines of evidence indicate that G proteins stimulate p110gamma in the absence of p101 both in vitro and in vivo (20-23). Gbeta gamma are thought to be the dominant physiological stimulus, while Galpha subunits of the Gi but not Gq or G12 subfamilies only moderately activate p110gamma .

Whereas a precise picture of the molecular mechanisms for receptor-induced activation of class IA PI3Ks has been drawn, little is known about how Gbeta gamma activates PI3Kgamma and which structures of the enzyme are involved. Comparison of the deduced amino acid sequences of the catalytic subunits of class I members revealed several highly conserved regions of homology (HR) but also parts which are quite diverse (6). All enzymes have a C-terminally located catalytic domain (HR1). Interestingly, this domain requires N-terminal regions for enzymatic activity (9). Therefore a horseshoe-like folding of the p110, enabling interaction between the N- and C-terminal half of the enzyme, has been assumed (24). HR2 represents a PIK domain found in all PI3- and PI4-kinases. Other regions of homology are specific for PI3Ks (HR3) and class I PI3Ks (HR4). All class I PI3Ks also contain a Ras-binding motif (25). In contrast, only class IA enzymes have an N-terminal stretch assumed to interact with its p85 subunits, whereas only p110gamma exhibits a Ras-GAP homology region, which may fold to form a pleckstrin homology (PH) domain (6, 20, 26). This region of p110gamma has been speculated to be involved in Gbeta gamma -mediated activation of PI3Kgamma , since PH domains of Gbeta gamma -regulated proteins such as beta -adrenergic receptor kinase or phosducin have previously been identified to bind Gbeta gamma (27, 28). However, other enzymes with an inherent PH domain such as phospholipase Cgamma are insensitive to modulation by Gbeta gamma , while some effectors lacking PH domains, e.g. adenylyl cyclases and potassium or calcium channels, are regulated by Gbeta gamma complexes (29-32).

Therefore, the aim of the present study was to examine whether the putative PH domain of the catalytic subunit of PI3Kgamma is critical for interaction of p110gamma with Gbeta gamma . Binding of a monoclonal antibody (mAb) to p110gamma that blocked the PH domain did not reduce Gbeta gamma -mediated stimulation. Furthermore, results obtained with deletion mutants of p110gamma indicated that Gbeta gamma binds to an N-terminal region as well as to a region near or within the C-terminally located catalytic core. Correspondingly, inactive N- and C-terminal mutants inhibited Gbeta gamma -mediated activation of wild type p110gamma by sequestration of Gbeta gamma . We conclude that the PH domain of p110gamma is not the only region interacting with Gbeta gamma and hypothesize that N- and C-terminal stretches of p110gamma contribute to form a common Gbeta gamma effector region.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction and Purification of PI3K-GST Fusion Proteins-- Construction of recombinant baculoviruses for expression of human GST-p110gamma fusion proteins and mutants thereof and of porcine p101 and GST-p101 were described previously (9, 19, 20, 22). Recombinant baculoviruses for Gbeta 1 and Ggamma 2 subunits and for GST-p110alpha were generous gifts from Drs. M. Lohse (Würzburg) and M. D. Waterfield (London). For protein expression cells were infected at a multiplicity of infection of 1 virus per cell. After 48-60 h of infection cells were pelleted by centrifugation (1,000 × g), washed with phosphate-buffered saline twice, and resuspended in ice-cold buffer A containing 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 50 mM Tris/HCl, pH 8.0, 10 mM dithiothreitol, 10 µg/ml each of aprotinin, benzamidin, leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride. Cells were disrupted by N2 cavitation (30 min at 4 °C, 25 bar) or by forcing the cell suspension through a 22-gauge needle (20 times) and subsequently through a 26-gauge needle (10 times). Nuclei and debris were discarded. The cytosolic and membranous fraction were recovered by centrifugation at 100,000 × g for 50 min. Membrane extract (0.5% Lubrol PX in buffer A) and cytosol were incubated overnight with glutathione-Sepharose 4B beads (Pharmacia Biotech Inc.) prewashed with buffer A. The Sepharose-bound GST fusion proteins could be stored at -20 °C in buffer B containing 50% glycerol, 1 mM EDTA, 40 mM Tris/HCl, pH 8.0, 1 mM dithiothreitol, and 1.57 mg/ml benzamidin. For enzymatic assays GST fusion proteins were freshly eluted with buffer C consisting of buffer A with 10 mM glutathione for 1 h at 4 °C. Purified proteins were quantified by Coomassie Blue staining following SDS-PAGE with bovine serum albumin as the standard.

For coexpression experiments with recombinant Gbeta 1gamma 2 equal multiplicity of infection numbers for all recombinant baculoviruses were used. After 58-64 h of infection, cells were harvested by centrifugation (1,000 × g, 10 min) and resuspended in 3 ml of ice-cold lysis buffer containing 0.5% Lubrol PX in buffer A. Lysates were incubated with glutathione-Sepharose 4B, and eluted GST fusion proteins were analyzed for binding of Gbeta gamma .

Preparation of G Proteins-- For isolation of bovine retinal transducin beta gamma as well as Galpha o subunits and Gbeta gamma complexes from bovine brain, we employed standard techniques with modifications (33, 34). Bovine brain G protein subunits were purified to apparent homogeneity in the presence of aluminum fluoride. Isolation and final purification of Galpha o and Gbeta gamma was achieved using a Mono Q (Pharmacia) fast protein liquid chromatography column (35). G protein subunits were identified by their immunoreactivity. Contamination by other pertussis toxin-sensitive Galpha subunits was excluded by analysis of autoradiographic signals after pertussis toxin-mediated [32P]ADP-ribosylation with a BAS 1500 Fuji-Imager (Raytest, Straubenhardt, Germany) (36). Concentrations of G protein heterotrimers and their alpha  subunits were determined by binding of [35S]GTPgamma S (35), amounts of G protein beta  subunits were determined by the method of Lowry et al. (37) and by Coomassie Blue staining following SDS-PAGE with bovine serum albumin as the standard (38). Purified proteins were stored at -70 °C until use.

Gelelectrophoresis, Immunoblotting, and Antibodies-- Generation of the monoclonal antibody against p110gamma and antiserum AS 398 against Gbeta subunits were detailed elsewhere (22, 39). For detection of p110gamma or the G protein subunit, preparations were fractionated by SDS-PAGE transferred to nitrocellulose or polyvinylidene difluoride membranes (Millipore, Eschborn, Germany). Visualization of specific antisera was performed using the ECL chemiluminescence system (Amersham, Braunschweig, Germany) or the CDP-Star chemiluminescence reagent (Tropix, Bedford, MA) according to the manufacturers' instructions.

Lipid Kinase Assay-- The assays were conducted in a final volume of 50 µl containing 0.1% bovine serum albumin, 2 mM EGTA, 0.2 mM EDTA, 10 mM MgCl2, 120 mM NaCl, 40 mM HEPES, pH 7.4, 1 mM dithiothreitol, 1 mM beta -glycerophosphate as described previously (20) with some modifications. Briefly, 30 µl of lipid vesicles (320 µM phosphatidylethanolamine, 300 µM phosphatidylethanolserine, 140 µM phosphatidylethanolcholine, 30 µM sphingomyelin, and 320 µM PI) were mixed with either Gbeta gamma complexes or their vehicle and incubated on ice for 8 min. Thereafter the enzyme fraction (1-10 ng) was added and the mixture was incubated for further 10 min at 4 °C in a final volume of 40 µl. The assay was then started by adding 40 µM ATP (1 µCi of [gamma -32P]ATP) in 10 µl of the above assay buffer (30 °C). Water-dissolved peptides (Eurogentec, Brussels, Belgium) used in this study were incubated with Gbeta gamma complexes before adding lipid vesicles. Wortmannin was stored in dimethyl sulfoxide (20 mM) in the dark at -20 °C and added to the kinase immediately before the experiment. After 15 min the reaction was stopped with ice-cold 150 µl of 1 N HCl and placing the tubes on ice. The lipids were extracted by vortexing samples with 450 µl of chloroform/methanol (1:1). After centrifugation and removing of the aqueous phase, the organic phase was washed twice with 200 µl of 1 N HCl. Subsequently, 40 µl of the organic phase were resolved on potassium oxalate-pretreated TLC plates (Whatman, Cliffton, NJ) with 35 ml of 2 N acetic acid and 65 ml of 1-propanol as the mobile phase. Dried TLC plates were exposed to Fuji-Imaging plates, and autoradiographic signals were quantitated with a BAS 1500 Fuji-Imager.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Our initial experiments with the purified recombinant p110gamma yielded a 5-6-fold stimulation with 1 µM Gbeta gamma and an EC50 of 200 nM for transducin beta gamma (20). As this effect of Gbeta gamma is moderate compared with effects on other G protein-regulated cellular targets, we optimized the conditions for p110gamma activation. Human recombinant p110gamma was expressed as GST fusion protein in Sf9 cells. Purified cytosolic enzyme exhibited a basal specific activity of 1-3 nmol of PI-3P/min/mg of p110gamma (Fig. 1A) and was much more sensitive to Gbeta gamma than the enzyme isolated from Sf9 membrane extracts (not shown). To investigate the stimulatory effect of Gbeta gamma complexes we used a mixture of highly concentrated purified bovine brain Gbeta gamma (see Fig. 1A) instead of using the less potent transducin beta gamma . Under these experimental conditions with phosphatidylinositol (PI) as substrate, Gbeta gamma complexes stimulated purified cytosolic p110gamma up to 60-fold with an EC50 of ~20 nM in a bimodal manner (see Fig. 1, B and C). As expected, p110alpha was not stimulated by Gbeta gamma (not shown). Gbeta gamma increased the Vmax of the recombinant p110gamma for ATP, corresponding to the data obtained with the native purified PI3Kgamma (23). Some variation in the efficiency of Gbeta gamma on p110gamma activity was probably due to either a well known variability in the quality of PI-liposome preparations used as substrates (40) or to the degree of autophosphorylation of p110gamma . The specific covalent inhibitor wortmannin decreased Gbeta gamma -stimulated enzymatic activity half-maximally at 15 nM and completely at 100 nM (Fig. 2A). An excess of GDP-bound Galpha o also inhibited Gbeta gamma -mediated stimulation of p110gamma activity, most likely by sequestration of free Gbeta gamma (Fig. 2B).


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Fig. 1.   Stimulation of p110gamma activity by Gbeta gamma subunits. A, recombinant p110gamma was expressed as a GST fusion protein in Sf9 cells and purified from cytosol, whereas Gbeta gamma was isolated from bovine brain membranes as detailed under "Materials and Methods." Proteins were separated by SDS-PAGE and stained by Coomassie Blue. Apparent molecular masses of marker proteins are indicated. DF indicates the dye front of the gel. B, a 60-fold stimulation of p110gamma PI3K activity from basal (+buffer) is seen in the presence of 200 nM Gbeta gamma . Note that addition of buffer alone results in a slight decrease in PI3K activity. C, representative concentration response curve of purified recombinant p110gamma PI3K activity by Gbeta gamma purified from bovine brain. Enzyme activity was determined by measuring formation of radiolabeled PI-3P from PI and [gamma -32P]ATP using a phosphorimaging system. The inset shows the corresponding autoradiogram of 32P incorporation into PI. Basal PI3K activity in the absence of Gbeta gamma corresponds to the left most PI-3P spot and was about 1-3 nmol/min/mg of protein.


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Fig. 2.   A, inhibition of Gbeta gamma -stimulated p110gamma PI3K activity by wortmannin. p110gamma was maximally stimulated with 200 nM Gbeta gamma and incubated with wortmannin at increasing concentrations. Shown are mean values (±S.D.) of three independent experiments. B, inhibition of Gbeta gamma -stimulated p110gamma PI3K activity by GDP-bound Galpha o. p110gamma was incubated without (basal) or with half-maximally stimulating amounts of Gbeta gamma (25 nM) in the absence or presence of a 2- or 6-fold molar excess of Galpha o. Shown is one representative experiment.

To examine the involvement of the putative PH domain of p110gamma we took advantage of a mAb raised against the purified enzyme (22). The mAb binds specifically to an amino acid stretch of the enzyme corresponding to the PH domain postulated between amino acids 87 and 302 (6, 26). Fig. 3 shows that the mAb did not recognize constructs lacking amino acids 75-398 (see Fig. 3B, lane 8) or amino acids 1-739 (lane 4), whereas it bound to constructs lacking amino acids 1-97 (lane 7), 335-1068 (lane 6), or 741-1068 (lane 5). Preparations containing active p110gamma were incubated with different volumes of antibody solution to ensure binding of saturating amounts of the mAb to the enzyme. The p110gamma -antibody complex was purified on glutathione-Sepharose beads (Fig. 4, upper panels) and subsequently stimulated with Gbeta gamma . As seen in Fig. 4 (lower panel), blocking the p110gamma PH domain by the mAb did not prevent Gbeta gamma -mediated stimulation. Conversely, a peptide derived from the adenylyl cyclase 2 (AC2) (amino acids 956-982; Fig. 5, upper part) not containing a PH domain did compete with p110gamma for the Gbeta gamma complex (41, 42). Increasing concentrations of this peptide completely reversed Gbeta gamma -mediated stimulation of p110gamma with an IC50 of 50 µM (see Fig. 5, lower part). The adenylyl cyclase 2 peptide did not change basal activity, and a control peptide derived from the corresponding region of the Gbeta gamma -insensitive adenylyl cyclase 3 (AC3) did not compete for the Gbeta gamma -stimulated activity (see Fig. 5).


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Fig. 3.   Characterization of a monoclonal antibody (mAb) to p110gamma . A, schematics of GST and GST fusion proteins of wild type p110gamma and mutants thereof, which were purified from baculovirus-infected Sf9 cells. B, immunoblot analysis of p110gamma and deletion mutants. Recombinant proteins were analyzed by immunoblotting after SDS-PAGE with a mAb to p110gamma as detailed under "Materials and Methods."


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Fig. 4.   Effect of the p110gamma mAb on Gbeta gamma -stimulated p110gamma PI3K activity. Immobilized p110gamma was preincubated in the absence or presence of two different volumes of mAb-containing solution (0.5 and 5 ml), eluted and stimulated by addition of half-maximally stimulating amounts of Gbeta gamma (25 nM) or vehicle only. PI3K activity was determined as described, and proteins were detected by immunoblot analysis using specific antisera (upper panels). Note that anti-IgG antibody recognized the heavy chain of the mAb (upper band).


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Fig. 5.   Effect of different concentrations of peptides derived from adenylyl cyclase type 2 (AC2) and type 3 (AC3) on basal and Gbeta gamma -stimulated p110gamma PI3K activity. Peptides were preincubated without or with Gbeta gamma (25 nM) before addition of lipid vesicles and reaction mix containing p110gamma . The lipid kinase assay was performed as described under "Materials and Methods."

To encircle regions of p110gamma required for Gbeta gamma -elicited activation of PI3K, we studied the copurification of Gbeta 1gamma 2 with various GST-p110 constructs following coexpression of recombinant proteins in Sf9 cells. First, to test the specificity of this experimental approach we coexpressed Gbeta 1gamma 2 with GST-p110alpha and GST-p110gamma (Fig. 6, center and upper panels). Since Gbeta gamma does not activate p110alpha it should not copurify with p110alpha , whereas it should copurify with the Gbeta gamma -activated p110gamma . p110 isoforms were isolated from cell homogenates on a glutathione affinity matrix. Subsequently proteins were subjected to SDS gels, and copurified Gbeta gamma complexes were detected by immunoblotting with a Gbeta -specific antibody (see Fig. 6, lower panel). This assay revealed that Gbeta 1gamma 2 indeed copurified with p110gamma , whereas no Gbeta immunoreactivity was detected after purification of p110alpha or GST. Further experiments showed that Gbeta gamma copurified only with membrane-bound p110gamma but not with cytosolic p110gamma , as could be explained by the membrane association of Gbeta gamma complexes (not shown).


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Fig. 6.   Copurification of recombinant Gbeta 1gamma 2 and GST-tagged PI3K isoforms after coexpression in Sf9 cells. Gbeta 1gamma 2 was coexpressed with constructs encoding GST alone or GST fused with p110alpha or p110gamma as described under "Materials and Methods." Lysates of Sf9 cells (center) were purified on glutathione-Sepharose beads (top and bottom), separated by SDS-PAGE, and analyzed by Coomassie Blue staining (top) and immunoblotting with a Gbeta -specific antibody AS 398 (center and bottom).

Next, we coexpressed wild type p110gamma or different p110gamma mutants as GST fusion proteins together with Gbeta 1gamma 2 and studied copurification. As shown in Fig. 7, Gbeta gamma copurified with different mutants expressing the N-terminal PH domain (lower panel Delta 1-97, lane 5; Delta 335-1068, lane 7; Delta 741-1068, lane 9), but also with a mutant lacking the PH domain (Delta 75-398, lane 6) and with a C-terminal mutant (Delta 1-739, lane 8) bearing only the catalytic core of the enzyme. These results indicate that Gbeta gamma specifically interacts with at least two binding sites of p110gamma . One binding site is localized N-terminally, whereas the other one is found at the C terminus.


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Fig. 7.   Copurification of recombinant Gbeta 1gamma 2 and GST-tagged p110gamma -mutants after coexpression in Sf9 cells. Gbeta 1gamma 2 was expressed alone or coexpressed with constructs encoding GST or GST fused with full-length or mutant p110gamma . Lysates of Sf9 cells were identified for expression of Gbeta gamma with Gbeta -specific antibody AS 398 (center) followed by purification on glutathione-Sepharose beads. Subsequently, proteins were separated by SDS-PAGE and analyzed by immunoblotting with a GST-specific antibody (top) and AS 398 (bottom) as described under "Materials and Methods." Note detection of endogenous Gbeta in whole cell lysates (center panel, lane 1).

As has been shown before, all p110gamma deletion mutants lacking more than the first 97 amino acids were enzymatically inactive as well as a p110gamma construct containing a Lys right-arrow Arg mutation at position 799 (K799R), which abolishes wortmannin binding (9). To support the assumption that Gbeta gamma interacts with two regions of p110gamma , we tested whether the inactive N- and C-terminal mutants would compete with wild type p110gamma for Gbeta gamma . This was done by coinfecting Sf9 cells with a constant amount of recombinant baculovirus encoding enzymatically active wild type p110gamma -GST fusion protein and increasing amounts of viruses encoding the N terminus (Delta 741-1068) or the C terminus (Delta 1-739) of p110gamma as GST fusion proteins. The coexpressed proteins were affinity-purified from Sf9 cytosol on glutathione-Sepharose beads and stimulated by addition of bovine brain Gbeta gamma complexes, and the fold activation of p110gamma was calculated. The C-terminal fragment bearing only the catalytic domain of p110gamma (Delta 1-739) as well as the N terminus of p110gamma (Delta 741-1068) inhibited Gbeta gamma -mediated stimulation of wild type p110gamma in a concentration-dependent manner (Fig. 8). GST alone did not affect Gbeta gamma -mediated stimulation of p110gamma . The enzymatically inactive K799R p110gamma point mutant capable of binding to Gbeta gamma (see Fig. 7, lower panel, lane 10) inhibited enzymatic activity to the same extent as the deletion mutants (see Fig. 8). This observation is in accordance with results from the copurification experiments (see above) and underlines the hypothesis that p110gamma exhibits two domains interacting with Gbeta gamma . Therefore, we suppose that both the N and C terminus bearing the catalytic domain of p110gamma are important for direct interaction with Gbeta gamma .


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Fig. 8.   Gbeta gamma -stimulated PI3K activity of coexpressed wild type and mutant p110gamma in Sf9 cells. Constant amounts of wild type p110gamma were coexpressed with increasing amounts of constructs encoding GST, GST fused with a catalytically inactive point mutant (K799R), or inactive N- (Delta 1-739) or C-terminally (Delta 741-1068) truncated forms of p110gamma . The ratios of virus encoding mutant to wild type p110gamma are indicated. PI3K activity of GSH-affinity purified cytosolic proteins was determined in the absence and presence of Gbeta gamma (25 nM) and the -fold stimulation calculated. Shown are data from one representative experiment.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

This study examines the direct interaction between p110gamma and Gbeta gamma . We show that the catalytic subunit of PI3Kgamma is greatly stimulated by Gbeta gamma in the absence of the recently reported p101 subunit. It extents our previous findings (20-22) supported by Tang and Downes (23) and points against an indispensable function of p101 for the stimulation of p110gamma by Gbeta gamma as recently hypothesized (19). Furthermore, preliminary results employing a purified recombinant heterodimer consisting of p110gamma and p101 showed no further increase in PI-3P formation in response to Gbeta gamma compared with p110gamma alone. At present there is no conclusive evidence for an essential role of p101 as an adaptor protein linking G protein-coupled receptors and p110gamma in a similar way as p85 mediates interaction of p110alpha with receptor tyrosine kinases (43). In addition, the recent observation that Gbeta gamma can bind directly to the related p110beta supports an interaction with p110gamma as well (10). Accordingly, in reconstituted neutrophils we recently observed stimulation of p110gamma lipid kinase activity by the agonist of the G protein-coupled fMet-Leu-Phe receptor in the absence of p101 (21). A possible explanation for this apparent discrepancy is the observation that purified recombinant human p110gamma degrades rapidly in the absence of p101, this subunit could stabilize the enzymatic activity of p110gamma . In addition, p101 could also affect the extent of autophosphorylation of p110gamma which may influence Gbeta gamma -mediated activation of PI3Kgamma . Furthermore, the possibility that a small fraction of the recombinant human p110gamma associated with a putative insect cell-derived p101-like protein mediated all the Gbeta gamma -sensitive activity is unlikely, since the basal specific activities of recombinant p110gamma recorded in this study was roughly 1 and 2 orders of magnitude larger than for recombinant p110gamma and p110gamma /p101, respectively, as estimated from published results (19).

In our study we noticed that geranyl-geranylated Gbeta gamma complexes from bovine brain were 10 times more potent in stimulating p110gamma than previously used farnesylated bovine transducin beta gamma (20). This difference in potency was also seen in studies with other Gbeta gamma -regulated effectors (31). We found that half-maximal stimulation of the PI3Kgamma catalytic subunit required only low nanomolar concentrations (~20 nM) of bovine brain Gbeta gamma . These concentrations are similar to those required for regulation of other effectors such as phospholipase C-beta , potassium, or calcium channels, but are much lower than those reported previously by other groups studying native or recombinant heterodimeric PI3K (14, 16, 19, 23, 31, 40, 44, 45).

Only cytosolic but not membrane-extracted purified p110gamma was significantly stimulated by addition of exogenous Gbeta gamma . The marginal responsiveness of the membrane-derived enzyme corresponds to the observation that recombinant Gbeta 1gamma 2 copurified with membrane-extracted but not cytosolic p110gamma . Gbeta gamma complexes may therefore function as a membrane anchor for p110gamma . This in turn could facilitate the access of the enzyme to its lipid substrates thereby enhancing p110gamma activity. Interestingly, for the purified native PI3Kgamma Tang and Downes (23) proposed cooperative kinetics for lipid substrates in the presence of Gbeta gamma .

Direct interaction of p110gamma with Gbeta gamma raises the question which region of p110gamma is critical for interaction with Gbeta gamma . Since p110gamma significantly differs from the receptor tyrosine kinase-regulated enzymes by an inherent PH domain it was speculated that this stretch may function as a Gbeta gamma -binding site (6, 20, 26). Blocking the PH domain of enzymatically active p110gamma by an specific antibody reacting with this p110gamma domain did not reduce the stimulatory activity of Gbeta gamma on p110gamma . Although the N-terminal half of p110gamma (Delta 741-1068) did bind Gbeta gamma and inhibited Gbeta gamma -mediated activation of the fully processed p110gamma , we present evidence that interaction of Gbeta gamma with the putative p110gamma PH domain is not exclusively responsible for stimulation of PI3Kgamma activity. In particular, p110gamma deletion mutants lacking the PH domain and adjacent stretches still bound Gbeta gamma . Furthermore, coexpression of p110gamma with an enzymatically inactive C-terminal deletion mutant (Delta 1-739) significantly inhibited activation of fully processed p110gamma by Gbeta gamma . Similar results were obtained after mixing of separately purified p110gamma with increasing concentrations of mutants (not shown). Our results suggest that the catalytic core is important for Gbeta gamma -mediated activation. This is not unlikely since the amino acid identity with p110alpha and -beta in this region is only 45 and 46.5%, respectively. Recently, the motif QXXER has been proposed as a consensus binding site for Gbeta gamma (41). p110gamma but not p110alpha , -beta , or -delta contains these amino acids within the catalytic core (amino acids 888-893), although they lack the correct spacing of this consensus motif. Unfortunately, peptides derived from corresponding regions of p110 isozymes strongly inhibited basal enzymatic activity preventing further analysis. In this context it should be noticed that a recent study on G protein-regulated calcium channels identified two Gbeta gamma -interacting domains that do not contain a QXXER motif (32). Nevertheless, a peptide derived from the adenylyl cyclase 2, which is thought to interact with a Gbeta domain, that for its part does not interact with PH-like structures, blocked Gbeta gamma -induced activation of p110gamma .

In summary, this study shows that the N as well as the C terminus of p110gamma interacts with Gbeta gamma . This could be explained by a folding of the enzyme which results in proximity of N and C terminus. Indeed, several lines of evidence point to a horseshoe-like structure of p110 (24) since N-terminal regions outside the C-terminal catalytic core are mandatory for enzymatic activity and wortmannin binding (9). Based on the proposed horseshoe-like structure of p110gamma one may suggest that N- and C-terminal portions of p110gamma form a common Gbeta gamma -effector region for regulation of enzymatic activity. The putative PH domain may be a part of this effector region but additional structures are required for Gbeta gamma -mediated p110gamma activation. This finding supports the emerging concept in molecular and cell biology of PH structures being not sufficient do define molecules as Gbeta gamma -regulated effectors.

    ACKNOWLEDGEMENTS

We thank Antje Tomschegg for excellent technical assistance. We are grateful to Drs. Michael Waterfield for providing a p110alpha -encoding virus, Martin Lohse for Gbeta - and Ggamma -encoding viruses, as well as Len Stephens and Phil Hawkins for providing us with the pcDNA3-p101 construct. Valuable discussions with Drs. Doris Koesling and Alan V. Smrcka are appreciated. We are indebted to Günter Schultz for critical reading of the manuscript and for his support.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Institut für Neurobiologie, Otto von Guericke Universität Magdeburg, Magdeburg, Germany.

par Recipient of the Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie.

** To whom correspondence should be addressed. Tel.: 49-30 838 4210; Fax: 49-30 831 5954; E-mail: bnue{at}zedat.fu-berlin.de.

1 The abbreviation used are: PI, phosphatidylinositol (locants of other phosphates on the inositol ring shown in parentheses); PI3K, phosphoinositide 3-kinase; GST, glutathione S-transferase; p110, catalytic subunit of PI3K; p101, subunit associated with p110gamma ; p85, regulatory subunit of receptor tyrosine kinase activated PI3K; PH, pleckstrin homology; HR, homology region; Galpha o, alpha -subunit of the major G protein in mammalian brain; Gbeta gamma , beta gamma -subunit from bovine brain; PIK, lipid kinase domain; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; GTPgamma S, guanosine 5'-O-(thiotriphosphate).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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