HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Leopoldt, D. Articles by Nürnberg, B. Search for Related Content PubMed PubMed Citation Articles by Leopoldt, D. Articles by Nürnberg, B. Social Bookmarking                      What's this?

J Biol Chem, Vol. 273, Issue 12, 7024-7029, March 20, 1998

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

Daniela Leopoldt, Theodor Hanck^§¶, Torsten Exner, Udo Maier, Reinhard Wetzker^§, and Bernd Nürnberg^**

From the  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

Top 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 PI3K, -, and - isoforms coupled to receptor tyrosine kinases and a PI3K isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3K catalytic subunit (p110) and G complexes. When phosphatidylinositol was used as a substrate, G stimulated p110 lipid kinase activity more than 60-fold (EC₅₀, ~20 nM). Stimulation was inhibited by G_o-GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110 did not affect G-mediated enzymatic stimulation, whereas incubation of G with a synthetic peptide resembling a predicted G effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110. G complexes bound to N- as well as C-terminal deletion mutants of p110. Correspondingly, these enzymatically inactive N- and C-terminal mutants inhibited G-mediated activation of wild type p110. Our data suggest that (i) p110 directly interacts with G, (ii) the pleckstrin homology domain is not the only region important for G-mediated activation of the lipid kinase, and (iii) G binds to at least two contact sites of p110, one of which is close to or within the catalytic core of the enzyme.

	INTRODUCTION

Top 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 I_A)- or of G protein-coupled receptor (class I_B)-signaling pathways, although very recently one member (p110) has been suggested to respond synergistically to both G 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 I_A members p110, , and  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 I_B member p110 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 p110 in the absence of p101 both in vitro and in vivo (20-23). G are thought to be the dominant physiological stimulus, while G subunits of the G_i but not G_q or G₁₂ subfamilies only moderately activate p110.

Whereas a precise picture of the molecular mechanisms for receptor-induced activation of class I_A PI3Ks has been drawn, little is known about how G activates PI3K 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 I_A enzymes have an N-terminal stretch assumed to interact with its p85 subunits, whereas only p110 exhibits a Ras-GAP homology region, which may fold to form a pleckstrin homology (PH) domain (6, 20, 26). This region of p110 has been speculated to be involved in G-mediated activation of PI3K, since PH domains of G-regulated proteins such as -adrenergic receptor kinase or phosducin have previously been identified to bind G (27, 28). However, other enzymes with an inherent PH domain such as phospholipase C are insensitive to modulation by G, while some effectors lacking PH domains, e.g. adenylyl cyclases and potassium or calcium channels, are regulated by G complexes (29-32).

Therefore, the aim of the present study was to examine whether the putative PH domain of the catalytic subunit of PI3K is critical for interaction of p110 with G. Binding of a monoclonal antibody (mAb) to p110 that blocked the PH domain did not reduce G-mediated stimulation. Furthermore, results obtained with deletion mutants of p110 indicated that G 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 G-mediated activation of wild type p110 by sequestration of G. We conclude that the PH domain of p110 is not the only region interacting with G and hypothesize that N- and C-terminal stretches of p110 contribute to form a common G effector region.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Construction and Purification of PI3K-GST Fusion Proteins-- Construction of recombinant baculoviruses for expression of human GST-p110 fusion proteins and mutants thereof and of porcine p101 and GST-p101 were described previously (9, 19, 20, 22). Recombinant baculoviruses for G₁ and G₂ subunits and for GST-p110 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 G₁₂ 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 G.

Preparation of G Proteins-- For isolation of bovine retinal transducin as well as G_o subunits and G 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 G_o and G 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 G 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  subunits were determined by binding of [³⁵S]GTPS (35), amounts of G protein  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 p110 and antiserum AS 398 against G subunits were detailed elsewhere (22, 39). For detection of p110 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 -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 G 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 [-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 G 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

Top Abstract Introduction Procedures Results Discussion References

Our initial experiments with the purified recombinant p110 yielded a 5-6-fold stimulation with 1 µM G and an EC₅₀ of 200 nM for transducin (20). As this effect of G is moderate compared with effects on other G protein-regulated cellular targets, we optimized the conditions for p110 activation. Human recombinant p110 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 p110 (Fig. 1A) and was much more sensitive to G than the enzyme isolated from Sf9 membrane extracts (not shown). To investigate the stimulatory effect of G complexes we used a mixture of highly concentrated purified bovine brain G (see Fig. 1A) instead of using the less potent transducin . Under these experimental conditions with phosphatidylinositol (PI) as substrate, G complexes stimulated purified cytosolic p110 up to 60-fold with an EC₅₀ of ~20 nM in a bimodal manner (see Fig. 1, B and C). As expected, p110 was not stimulated by G (not shown). G increased the V_max of the recombinant p110 for ATP, corresponding to the data obtained with the native purified PI3K (23). Some variation in the efficiency of G on p110 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 p110. The specific covalent inhibitor wortmannin decreased G-stimulated enzymatic activity half-maximally at 15 nM and completely at 100 nM (Fig. 2A). An excess of GDP-bound G_o also inhibited G-mediated stimulation of p110 activity, most likely by sequestration of free G (Fig. 2B).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Stimulation of p110 activity by G subunits. A, recombinant p110 was expressed as a GST fusion protein in Sf9 cells and purified from cytosol, whereas G 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 p110 PI3K activity from basal (+buffer) is seen in the presence of 200 nM G. Note that addition of buffer alone results in a slight decrease in PI3K activity. C, representative concentration response curve of purified recombinant p110 PI3K activity by G purified from bovine brain. Enzyme activity was determined by measuring formation of radiolabeled PI-3P from PI and [-32P]ATP using a phosphorimaging system. The inset shows the corresponding autoradiogram of 32P incorporation into PI. Basal PI3K activity in the absence of G corresponds to the left most PI-3P spot and was about 1-3 nmol/min/mg of protein.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   A, inhibition of G-stimulated p110 PI3K activity by wortmannin. p110 was maximally stimulated with 200 nM G and incubated with wortmannin at increasing concentrations. Shown are mean values (±S.D.) of three independent experiments. B, inhibition of G-stimulated p110 PI3K activity by GDP-bound Go. p110 was incubated without (basal) or with half-maximally stimulating amounts of G (25 nM) in the absence or presence of a 2- or 6-fold molar excess of Go. Shown is one representative experiment.

To examine the involvement of the putative PH domain of p110 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 p110 were incubated with different volumes of antibody solution to ensure binding of saturating amounts of the mAb to the enzyme. The p110-antibody complex was purified on glutathione-Sepharose beads (Fig. 4, upper panels) and subsequently stimulated with G. As seen in Fig. 4 (lower panel), blocking the p110 PH domain by the mAb did not prevent G-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 p110 for the G complex (41, 42). Increasing concentrations of this peptide completely reversed G-mediated stimulation of p110 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 G-insensitive adenylyl cyclase 3 (AC3) did not compete for the G-stimulated activity (see Fig. 5).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Characterization of a monoclonal antibody (mAb) to p110. A, schematics of GST and GST fusion proteins of wild type p110 and mutants thereof, which were purified from baculovirus-infected Sf9 cells. B, immunoblot analysis of p110 and deletion mutants. Recombinant proteins were analyzed by immunoblotting after SDS-PAGE with a mAb to p110 as detailed under "Materials and Methods."

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effect of the p110 mAb on G-stimulated p110 PI3K activity. Immobilized p110 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 G (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).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effect of different concentrations of peptides derived from adenylyl cyclase type 2 (AC2) and type 3 (AC3) on basal and G-stimulated p110 PI3K activity. Peptides were preincubated without or with G (25 nM) before addition of lipid vesicles and reaction mix containing p110. The lipid kinase assay was performed as described under "Materials and Methods."

To encircle regions of p110 required for G-elicited activation of PI3K, we studied the copurification of G₁₂ with various GST-p110 constructs following coexpression of recombinant proteins in Sf9 cells. First, to test the specificity of this experimental approach we coexpressed G₁₂ with GST-p110 and GST-p110 (Fig. 6, center and upper panels). Since G does not activate p110 it should not copurify with p110, whereas it should copurify with the G-activated p110. p110 isoforms were isolated from cell homogenates on a glutathione affinity matrix. Subsequently proteins were subjected to SDS gels, and copurified G complexes were detected by immunoblotting with a G-specific antibody (see Fig. 6, lower panel). This assay revealed that G₁₂ indeed copurified with p110, whereas no G immunoreactivity was detected after purification of p110 or GST. Further experiments showed that G copurified only with membrane-bound p110 but not with cytosolic p110, as could be explained by the membrane association of G complexes (not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Copurification of recombinant G12 and GST-tagged PI3K isoforms after coexpression in Sf9 cells. G12 was coexpressed with constructs encoding GST alone or GST fused with p110 or p110 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 G-specific antibody AS 398 (center and bottom).

Next, we coexpressed wild type p110 or different p110 mutants as GST fusion proteins together with G₁₂ and studied copurification. As shown in Fig. 7, G copurified with different mutants expressing the N-terminal PH domain (lower panel 1-97, lane 5; 335-1068, lane 7; 741-1068, lane 9), but also with a mutant lacking the PH domain (75-398, lane 6) and with a C-terminal mutant (1-739, lane 8) bearing only the catalytic core of the enzyme. These results indicate that G specifically interacts with at least two binding sites of p110. One binding site is localized N-terminally, whereas the other one is found at the C terminus.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Copurification of recombinant G12 and GST-tagged p110-mutants after coexpression in Sf9 cells. G12 was expressed alone or coexpressed with constructs encoding GST or GST fused with full-length or mutant p110. Lysates of Sf9 cells were identified for expression of G with G-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 G in whole cell lysates (center panel, lane 1).

As has been shown before, all p110 deletion mutants lacking more than the first 97 amino acids were enzymatically inactive as well as a p110 construct containing a Lys  Arg mutation at position 799 (K799R), which abolishes wortmannin binding (9). To support the assumption that G interacts with two regions of p110, we tested whether the inactive N- and C-terminal mutants would compete with wild type p110 for G. This was done by coinfecting Sf9 cells with a constant amount of recombinant baculovirus encoding enzymatically active wild type p110-GST fusion protein and increasing amounts of viruses encoding the N terminus (741-1068) or the C terminus (1-739) of p110 as GST fusion proteins. The coexpressed proteins were affinity-purified from Sf9 cytosol on glutathione-Sepharose beads and stimulated by addition of bovine brain G complexes, and the fold activation of p110 was calculated. The C-terminal fragment bearing only the catalytic domain of p110 (1-739) as well as the N terminus of p110 (741-1068) inhibited G-mediated stimulation of wild type p110 in a concentration-dependent manner (Fig. 8). GST alone did not affect G-mediated stimulation of p110. The enzymatically inactive K799R p110 point mutant capable of binding to G (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 p110 exhibits two domains interacting with G. Therefore, we suppose that both the N and C terminus bearing the catalytic domain of p110 are important for direct interaction with G.

View larger version (34K): [in this window] [in a new window]   Fig. 8.   G-stimulated PI3K activity of coexpressed wild type and mutant p110 in Sf9 cells. Constant amounts of wild type p110 were coexpressed with increasing amounts of constructs encoding GST, GST fused with a catalytically inactive point mutant (K799R), or inactive N- (1-739) or C-terminally (741-1068) truncated forms of p110. The ratios of virus encoding mutant to wild type p110 are indicated. PI3K activity of GSH-affinity purified cytosolic proteins was determined in the absence and presence of G (25 nM) and the -fold stimulation calculated. Shown are data from one representative experiment.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

This study examines the direct interaction between p110 and G. We show that the catalytic subunit of PI3K is greatly stimulated by G 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 p110 by G as recently hypothesized (19). Furthermore, preliminary results employing a purified recombinant heterodimer consisting of p110 and p101 showed no further increase in PI-3P formation in response to G compared with p110 alone. At present there is no conclusive evidence for an essential role of p101 as an adaptor protein linking G protein-coupled receptors and p110 in a similar way as p85 mediates interaction of p110 with receptor tyrosine kinases (43). In addition, the recent observation that G can bind directly to the related p110 supports an interaction with p110 as well (10). Accordingly, in reconstituted neutrophils we recently observed stimulation of p110 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 p110 degrades rapidly in the absence of p101, this subunit could stabilize the enzymatic activity of p110. In addition, p101 could also affect the extent of autophosphorylation of p110 which may influence G-mediated activation of PI3K. Furthermore, the possibility that a small fraction of the recombinant human p110 associated with a putative insect cell-derived p101-like protein mediated all the G-sensitive activity is unlikely, since the basal specific activities of recombinant p110 recorded in this study was roughly 1 and 2 orders of magnitude larger than for recombinant p110 and p110/p101, respectively, as estimated from published results (19).

In our study we noticed that geranyl-geranylated G complexes from bovine brain were 10 times more potent in stimulating p110 than previously used farnesylated bovine transducin (20). This difference in potency was also seen in studies with other G-regulated effectors (31). We found that half-maximal stimulation of the PI3K catalytic subunit required only low nanomolar concentrations (~20 nM) of bovine brain G. These concentrations are similar to those required for regulation of other effectors such as phospholipase C-, 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 p110 was significantly stimulated by addition of exogenous G. The marginal responsiveness of the membrane-derived enzyme corresponds to the observation that recombinant G₁₂ copurified with membrane-extracted but not cytosolic p110. G complexes may therefore function as a membrane anchor for p110. This in turn could facilitate the access of the enzyme to its lipid substrates thereby enhancing p110 activity. Interestingly, for the purified native PI3K Tang and Downes (23) proposed cooperative kinetics for lipid substrates in the presence of G.

Direct interaction of p110 with G raises the question which region of p110 is critical for interaction with G. Since p110 significantly differs from the receptor tyrosine kinase-regulated enzymes by an inherent PH domain it was speculated that this stretch may function as a G-binding site (6, 20, 26). Blocking the PH domain of enzymatically active p110 by an specific antibody reacting with this p110 domain did not reduce the stimulatory activity of G on p110. Although the N-terminal half of p110 (741-1068) did bind G and inhibited G-mediated activation of the fully processed p110, we present evidence that interaction of G with the putative p110 PH domain is not exclusively responsible for stimulation of PI3K activity. In particular, p110 deletion mutants lacking the PH domain and adjacent stretches still bound G. Furthermore, coexpression of p110 with an enzymatically inactive C-terminal deletion mutant (1-739) significantly inhibited activation of fully processed p110 by G. Similar results were obtained after mixing of separately purified p110 with increasing concentrations of mutants (not shown). Our results suggest that the catalytic core is important for G-mediated activation. This is not unlikely since the amino acid identity with p110 and - in this region is only 45 and 46.5%, respectively. Recently, the motif QXXER has been proposed as a consensus binding site for G (41). p110 but not p110, -, or - 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 G-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 G domain, that for its part does not interact with PH-like structures, blocked G-induced activation of p110.

In summary, this study shows that the N as well as the C terminus of p110 interacts with G. 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 p110 one may suggest that N- and C-terminal portions of p110 form a common G-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 G-mediated p110 activation. This finding supports the emerging concept in molecular and cell biology of PH structures being not sufficient do define molecules as G-regulated effectors.

	ACKNOWLEDGEMENTS

We thank Antje Tomschegg for excellent technical assistance. We are grateful to Drs. Michael Waterfield for providing a p110-encoding virus, Martin Lohse for G- and G-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.

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 p110; p85, regulatory subunit of receptor tyrosine kinase activated PI3K; PH, pleckstrin homology; HR, homology region; G_o, -subunit of the major G protein in mammalian brain; G, -subunit from bovine brain; PIK, lipid kinase domain; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; GTPS, guanosine 5'-O-(thiotriphosphate).

	REFERENCES

Top Abstract Introduction Procedures Results Discussion References

1. Traynor-Kaplan, A. E., Harris, A. L., Thompson, B. L., Taylor, P., Sklar, L. A. (1988) Nature 334, 353-356[CrossRef][Medline] [Order article via Infotrieve]
2. Parker, P. J., and Waterfield, M. D. (1992) Cell Growth Differ. 3, 747-752[Medline] [Order article via Infotrieve]
3. Stephens, L. R., Jackson, T. R., and Hawkins, P. T. (1993) Biochim. Biophys. Acta 1179, 27-75[Medline] [Order article via Infotrieve]
4. Divecha, N., and Irvine, R. F. (1995) Cell 80, 269-278[CrossRef][Medline] [Order article via Infotrieve]
5. Toker, A., and Cantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve]
6. Zvelebil, M. J., Macdougall, L., Leevers, S., Volina, S., Vanhaesebroeck, B., Gout, I., Panayotou, G., Domin, J., Stein, R., Pages, F., Koga, H., Salim, K., Linacre, J., Das, P., Panaretou, C., Wetzker, R., and Waterfield, M. (1996) Phil. Trans. R. Soc. Lond. Ser. B Biol. 351, 217-223[Medline] [Order article via Infotrieve]
7. Carpenter, C. L., Auger, K. R., Duckworth, B. C., Hou, W. M., Schaffhausen, B. S., Cantley, L. C. (1993) Mol. Cell. Biol. 13, 1657-1665[Abstract/Free Full Text]
8. Dhand, R., Hara, K., Hiles, I., Bax, B., Gout, I., Panayotou, G., Fry, M. J., Yonezawa, K., Kasuga, M., Waterfield, M. D. (1994) EMBO J. 13, 4261-4272
9. Stoyanova, S., Bulgarelli-Leva, G., Kirsch, C., Hanck, T., Klinger, R., Wetzker, R., and Wymann, M. P. (1997) Biochem. J. 324, 489-495
10. Kurosu, H., Maehama, T., Okada, T., Yamamoto, T., Hoshino, S., Fukui, Y., Ui, M., Hazeki, O., and Katada, T. (1997) J. Biol. Chem. 272, 24252-24256[Abstract/Free Full Text]
11. Chang, H. W., Aoki, M., Fruman, D., Auger, K. R., Bellacosa, A., Tschlis, P. N., Cantley, L. C., Roberts, T. M., Vogt, P. K. (1997) Science 276, 1848-1850[Abstract/Free Full Text]
12. Carpenter, C. L., Duckworth, B. C., Auger, K. R., Cohen, B., Schaffhausen, B. S., Cantley, L. C. (1990) J. Biol. Chem. 265, 19704-19711[Abstract/Free Full Text]
13. Morgan, S. J., Smith, A. D., and Parker, P. J. (1990) Eur. J. Biochem. 191, 761-767[Medline] [Order article via Infotrieve]
14. Stephens, L. R., Smrcka, A., Cooke, F. T., Jackson, T. R., Sternweis, P. C., Hawkins, P. T. (1994) Cell 77, 83-89[CrossRef][Medline] [Order article via Infotrieve]
15. Thelen, M., Wymann, M. P., and Langen, H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4960-4964[Abstract/Free Full Text]
16. Thomason, P. A., James, S. R., Casey, P. J., Downes, C. P. (1994) J. Biol. Chem. 269, 16525-16528[Abstract/Free Full Text]
17. Domin, J., and Waterfield, M. D. (1997) FEBS Lett. 410, 91-95[CrossRef][Medline] [Order article via Infotrieve]
18. Vanhaesebroeck, B., Welham, M. J., Kotani, K., Stein, R., Warne, P. H., Zvelebil, M. J., Higashi, K., Volina, S., Downward, J., Waterfield, M. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4330-4335[Abstract/Free Full Text]
19. Stephens, L. R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F., Coadwell, J., Smrcka, A. S., Thelen, M., Cadwallader, K., Tempst, P., Hawkins, P. T. (1997) Cell 89, 105-114[CrossRef][Medline] [Order article via Infotrieve]
20. Stoyanov, B., Volinia, S., Hanck, T., Rubio, I., Loubtchenkov, M., Malek, D., Stoyanova, S., Vanhaesebroeck, B., Dhand, R., Nürnberg, B., Gierschik, P., Seedorf, K., Hsuan, J. J., Waterfield, M. D., Wetzker, R. (1995) Science 269, 690-693[Abstract/Free Full Text]
21. Kular, G., Loubtchenkov, M., Swigart, P., Whatmore, J., Ball, A., Cockcroft, S., and Wetzker, R. (1997) Biochem. J. 325, 299-301
22. Lopez-Ilasaca, M., Crespo, P., Pellici, P. G., Gutkind, J. S., Wetzker, R. (1997) Science 275, 394-397[Abstract/Free Full Text]
23. Tang, X., and Downes, C. P. (1997) J. Biol. Chem. 272, 14193-14199[Abstract/Free Full Text]
24. Vanhaesebroeck, B., Stein, R. C., and Waterfield, M. D. (1996) Cancer Surv. 27, 249-270[Medline] [Order article via Infotrieve]
25. Rodriguez-Viciana, P., Warne, P. H., Vanhaesebroeck, B., Waterfield, M. D., Downward, J. (1996) EMBO J. 15, 2442-2451[Medline] [Order article via Infotrieve]
26. Srinivasan, N., Waterfield, M. D., and Blundell, T. L. (1996) Biochem. Biophys. Res. Commun. 220, 697-702[CrossRef][Medline] [Order article via Infotrieve]
27. Touhara, K., Inglese, J., Pitcher, J. A., Shaw, G., Lefkowitz, R. J. (1994) J. Biol. Chem. 269, 10217-10220[Abstract/Free Full Text]
28. Gaudet, R., Bohm, A., and Sigler, P. B. (1996) Cell 87, 577-588[CrossRef][Medline] [Order article via Infotrieve]
29. Parker, P. J., Hemmings, B. A., and Gierschik, P. (1994) Trends Biol. Sci. 19, 54-55
30. Sunahara, R. K., Dessauer, C. W., and Gilman, A. G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 461-480[CrossRef][Medline] [Order article via Infotrieve]
31. Clapham, D. E., and Neer, E. J. (1997) Annu. Rev. Pharmacol. Toxicol. 37, 167-203[CrossRef][Medline] [Order article via Infotrieve]
32. Qin, N., Platano, D., Olcese, R., Stefani, E., and Birnbaumer, L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8866-8871[Abstract/Free Full Text]
33. Wieland, T., Ulibarri, I., Gierschik, P., and Jakobs, K. H. (1991) Eur. J. Biochem. 196, 707-716[Medline] [Order article via Infotrieve]
34. Degtiar, V., Harhammer, R., and Nürnberg, B. (1997) J. Physiol. 502, 321-333 [Abstract/Free Full Text]
35. Nürnberg, B., Spicher, K., Harhammer, R., Bosserhoff, A., Frank, R., Hilz, H., and Schultz, G. (1994) Biochem. J. 300, 387-394
36. Nürnberg, B. (1997) in Bacterial Toxins, Tools in Cell Biology (Aktories, K., ed), pp. 47-60, Chapman & Hall, Weinheim
37. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
38. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
39. Leopoldt, D., Harteneck, C., and Nürnberg, B. (1997) Naunyn Schmiedebergs Arch. Pharmacol. 356, 216-224[CrossRef][Medline] [Order article via Infotrieve]
40. Morris, A. J., Rudge, S. A., Mahlum, C. E., Jenco, J. M. (1995) Mol. Pharmacol. 48, 532-539[Abstract]
41. Chen, J., DeVivo, M., Dingus, J., Harry, A., Li, J., Sui, J., Carty, D. J., Blank, J. L., Lefkowitz, R. J., Logothetis, D. E., Hildebrandt, J. D., Iyengar, R. (1995) Science 268, 1166-1169[Abstract/Free Full Text]
42. Weng, G., Li, J., Dingus, J., Hildebrandt, J. D., Weinstein, H., Iyengar, R. (1996) J. Biol. Chem. 271, 26445-26448[Abstract/Free Full Text]
43. Pawson, T., and Scott, J. D. (1997) Science 278, 2075-2080[Abstract/Free Full Text]
44. Parish, C. A., Smrcka, A. V., and Rando, R. R. (1995) Biochemistry 34, 7722-7727[CrossRef][Medline] [Order article via Infotrieve]
45. Exton, J. H. (1997) Eur. J. Biochem. 243, 10-20[Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				T. Bohnacker, R. Marone, E. Collmann, R. Calvez, E. Hirsch, and M. P. Wymann PI3K{gamma} Adaptor Subunits Define Coupling to Degranulation and Cell Motility by Distinct PtdIns(3,4,5)P3 Pools in Mast Cells Sci. Signal., June 9, 2009; 2(74): ra27 - ra27. [Abstract] [Full Text] [PDF]

				C. Billottet, L. Banerjee, B. Vanhaesebroeck, and A. Khwaja Inhibition of Class I Phosphoinositide 3-Kinase Activity Impairs Proliferation and Triggers Apoptosis in Acute Promyelocytic Leukemia without Affecting Atra-Induced Differentiation Cancer Res., February 1, 2009; 69(3): 1027 - 1036. [Abstract] [Full Text] [PDF]

				E. Zebedin, O. Simma, C. Schuster, E. M. Putz, S. Fajmann, W. Warsch, E. Eckelhart, D. Stoiber, E. Weisz, J. A. Schmid, et al. Leukemic challenge unmasks a requirement for PI3K{delta} in NK cell-mediated tumor surveillance Blood, December 1, 2008; 112(12): 4655 - 4664. [Abstract] [Full Text] [PDF]

				T. Pisitkun, V. Jacob, S. M. Schleicher, C.-L. Chou, M.-J. Yu, and M. A. Knepper Akt and ERK1/2 pathways are components of the vasopressin signaling network in rat native IMCD Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1030 - F1043. [Abstract] [Full Text] [PDF]

				R. Heller, Q. Chang, G. Ehrlich, S. N. Hsieh, S. M. Schoenwaelder, P. J. Kuhlencordt, K. T. Preissner, E. Hirsch, and R. Wetzker Overlapping and distinct roles for PI3K{beta} and {gamma} isoforms in S1P-induced migration of human and mouse endothelial cells Cardiovasc Res, October 1, 2008; 80(1): 96 - 105. [Abstract] [Full Text] [PDF]

				I. Alcazar, M. Marques, A. Kumar, E. Hirsch, M. Wymann, A. C. Carrera, and D. F. Barber Phosphoinositide 3 kinase {gamma} participates in T cell receptor induced T cell activation J. Exp. Med., November 26, 2007; 204(12): 2977 - 2987. [Abstract] [Full Text] [PDF]

				D. Park, I. Park, D. Lee, Y. B. Choi, H. Lee, and Y. Yun The adaptor protein Lad associates with the G protein {beta} subunit and mediates chemokine-dependent T-cell migration Blood, June 15, 2007; 109(12): 5122 - 5128. [Abstract] [Full Text] [PDF]

				P. Voigt, M. B. Dorner, and M. Schaefer Characterization of p87PIKAP, a Novel Regulatory Subunit of Phosphoinositide 3-Kinase {gamma} That Is Highly Expressed in Heart and Interacts with PDE3B J. Biol. Chem., April 14, 2006; 281(15): 9977 - 9986. [Abstract] [Full Text] [PDF]

				T Balla Phosphoinositide-derived messengers in endocrine signaling J. Endocrinol., February 1, 2006; 188(2): 135 - 153. [Abstract] [Full Text] [PDF]

				S. Kang, A. Denley, B. Vanhaesebroeck, and P. K. Vogt Oncogenic transformation induced by the p110beta, -{gamma}, and -{delta} isoforms of class I phosphoinositide 3-kinase PNAS, January 31, 2006; 103(5): 1289 - 1294. [Abstract] [Full Text] [PDF]

				L. H. Mayeenuddin, W. E. McIntire, and J. C. Garrison Differential Sensitivity of P-Rex1 to Isoforms of G Protein beta{gamma} Dimers J. Biol. Chem., January 27, 2006; 281(4): 1913 - 1920. [Abstract] [Full Text] [PDF]

				Y. I. Cha, S.-H. Kim, D. Sepich, F. G. Buchanan, L. Solnica-Krezel, and R. N. DuBois Cyclooxygenase-1-derived PGE2 promotes cell motility via the G-protein-coupled EP4 receptor during vertebrate gastrulation Genes & Dev., January 1, 2006; 20(1): 77 - 86. [Abstract] [Full Text] [PDF]

				P. Voigt, C. Brock, B. Nurnberg, and M. Schaefer Assigning Functional Domains within the p101 Regulatory Subunit of Phosphoinositide 3-Kinase {gamma} J. Biol. Chem., February 11, 2005; 280(6): 5121 - 5127. [Abstract] [Full Text] [PDF]

				D. S. Hutchinson and T. Bengtsson {alpha}1A-Adrenoceptors Activate Glucose Uptake in L6 Muscle Cells through a Phospholipase C-, Phosphatidylinositol-3 Kinase-, and Atypical Protein Kinase C-Dependent Pathway Endocrinology, February 1, 2005; 146(2): 901 - 912. [Abstract] [Full Text] [PDF]

				V. Leblais, S.-H. Jo, K. Chakir, V. Maltsev, M. Zheng, M. T. Crow, W. Wang, E. G. Lakatta, and R.-P. Xiao Phosphatidylinositol 3-Kinase Offsets cAMP-Mediated Positive Inotropic Effect via Inhibiting Ca2+ Influx in Cardiomyocytes Circ. Res., December 10, 2004; 95(12): 1183 - 1190. [Abstract] [Full Text] [PDF]

				K. R. Kerchner, R. L. Clay, G. McCleery, N. Watson, W. E. McIntire, C.-S. Myung, and J. C. Garrison Differential Sensitivity of Phosphatidylinositol 3-Kinase p110{gamma} to Isoforms of G Protein {beta}{gamma} Dimers J. Biol. Chem., October 22, 2004; 279(43): 44554 - 44562. [Abstract] [Full Text] [PDF]

				M. B. Jones, D. P. Siderovski, and S. B. Hooks The G{beta}{gamma} DIMER as a NOVEL SOURCE of SELECTIVITY in G-Protein Signaling: GGL-ing AT CONVENTION Mol. Interv., August 1, 2004; 4(4): 200 - 214. [Abstract] [Full Text] [PDF]

				C. Ibarra, M. Estrada, L. Carrasco, M. Chiong, J. L. Liberona, C. Cardenas, G. Diaz-Araya, E. Jaimovich, and S. Lavandero Insulin-like Growth Factor-1 Induces an Inositol 1,4,5-Trisphosphate-dependent Increase in Nuclear and Cytosolic Calcium in Cultured Rat Cardiac Myocytes J. Biol. Chem., February 27, 2004; 279(9): 7554 - 7565. [Abstract] [Full Text] [PDF]

				J. Leemhuis, S. Boutillier, H. Barth, T. J. Feuerstein, C. Brock, B. Nurnberg, K. Aktories, and D. K. Meyer Rho GTPases and Phosphoinositide 3-Kinase Organize Formation of Branched Dendrites J. Biol. Chem., January 2, 2004; 279(1): 585 - 596. [Abstract] [Full Text] [PDF]

				T. M. Cabrera-Vera, J. Vanhauwe, T. O. Thomas, M. Medkova, A. Preininger, M. R. Mazzoni, and H. E. Hamm Insights into G Protein Structure, Function, and Regulation Endocr. Rev., December 1, 2003; 24(6): 765 - 781. [Abstract] [Full Text] [PDF]

				S. B. Kondapaka, S. S. Singh, G. P. Dasmahapatra, E. A. Sausville, and K. K. Roy Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation Mol. Cancer Ther., November 1, 2003; 2(11): 1093 - 1103. [Abstract] [Full Text] [PDF]

				N. Kaplan-Albuquerque, C. Garat, C. Desseva, P. L. Jones, and R. A. Nemenoff Platelet-derived Growth Factor-BB-mediated Activation of Akt Suppresses Smooth Muscle-specific Gene Expression through Inhibition of Mitogen-activated Protein Kinase and Redistribution of Serum Response Factor J. Biol. Chem., October 10, 2003; 278(41): 39830 - 39838. [Abstract] [Full Text] [PDF]

				D. Chodniewicz and D. V. Zhelev Novel pathways of F-actin polymerization in the human neutrophil Blood, September 15, 2003; 102(6): 2251 - 2258. [Abstract] [Full Text] [PDF]

				J. S. Popova and M. M. Rasenick G{beta}{gamma} Mediates the Interplay between Tubulin Dimers and Microtubules in the Modulation of Gq Signaling J. Biol. Chem., September 5, 2003; 278(36): 34299 - 34308. [Abstract] [Full Text] [PDF]

				M.-W. Chien, C.-S. Chien, L.-D. Hsiao, C.-H. Lin, and C.-M. Yang OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells J. Lipid Res., September 1, 2003; 44(9): 1667 - 1675. [Abstract] [Full Text] [PDF]

				S. Kraus, O. Benard, Z. Naor, and R. Seger c-Src Is Activated by the Epidermal Growth Factor Receptor in a Pathway That Mediates JNK and ERK Activation by Gonadotropin-releasing Hormone in COS7 Cells J. Biol. Chem., August 29, 2003; 278(35): 32618 - 32630. [Abstract] [Full Text] [PDF]

				C. Czupalla, M. Culo, E.-C. Muller, C. Brock, H. P. Reusch, K. Spicher, E. Krause, and B. Nurnberg Identification and Characterization of the Autophosphorylation Sites of Phosphoinositide 3-Kinase Isoforms beta and gamma J. Biol. Chem., March 21, 2003; 278(13): 11536 - 11545. [Abstract] [Full Text] [PDF]

				N. Djouder, E. Aneiros, A. Cavalie, and K. Aktories Effects of Large Clostridial Cytotoxins on Activation of RBL 2H3-hm1 Mast Cells Indicate Common and Different Roles of Rac in Fcepsilon RI and M1-Receptor Signaling J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1243 - 1250. [Abstract] [Full Text] [PDF]

				T. Minami, Md. R. Abid, J. Zhang, G. King, T. Kodama, and W. C. Aird Thrombin Stimulation of Vascular Adhesion Molecule-1 in Endothelial Cells Is Mediated by Protein Kinase C (PKC)-delta -NF-kappa B and PKC-zeta -GATA Signaling Pathways J. Biol. Chem., February 21, 2003; 278(9): 6976 - 6984. [Abstract] [Full Text] [PDF]

				C. Brock, M. Schaefer, H. P. Reusch, C. Czupalla, M. Michalke, K. Spicher, G. Schultz, and B. Nurnberg Roles of G{beta}{gamma} in membrane recruitment and activation of p110{gamma}/p101 phosphoinositide 3-kinase {gamma} J. Cell Biol., January 2, 2003; 160(1): 89 - 99. [Abstract] [Full Text] [PDF]

				A. Bell, A. Gagnon, P. Dods, D. Papineau, M. Tiberi, and A. Sorisky TSH signaling and cell survival in 3T3-L1 preadipocytes Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1056 - C1064. [Abstract] [Full Text] [PDF]

				A. Rahman, A. L. True, K. N. Anwar, R. D. Ye, T. A. Voyno-Yasenetskaya, and A. B. Malik G{alpha}q and G{beta}{gamma} Regulate PAR-1 Signaling of Thrombin-Induced NF-{kappa}B Activation and ICAM-1 Transcription in Endothelial Cells Circ. Res., September 6, 2002; 91(5): 398 - 405. [Abstract] [Full Text] [PDF]

				S. M. Miggin and B. T. Kinsella Regulation of Extracellular Signal-Regulated Kinase Cascades by alpha - and beta -Isoforms of the Human Thromboxane A2 Receptor Mol. Pharmacol., April 1, 2002; 61(4): 817 - 831. [Abstract] [Full Text] [PDF]

				X. Ferry, V. Eichwald, L. Daeffler, and Y. Landry Activation of {beta}{gamma} Subunits of Gi2 and Gi3 Proteins by Basic Secretagogues Induces Exocytosis Through Phospholipase C{beta} and Arachidonate Release Through Phospholipase C{gamma} in Mast Cells J. Immunol., November 1, 2001; 167(9): 4805 - 4813. [Abstract] [Full Text] [PDF]

				P. Friedl, S. Borgmann, and E.-B. Brocker Amoeboid leukocyte crawling through extracellular matrix: lessons from the Dictyostelium paradigm of cell movement J. Leukoc. Biol., October 1, 2001; 70(4): 491 - 509. [Abstract] [Full Text] [PDF]

				K. Christopherson II and R. Hromas Chemokine Regulation of Normal and Pathologic Immune Responses Stem Cells, September 1, 2001; 19(5): 388 - 396. [Abstract] [Full Text] [PDF]

				V. P. Krymskaya, A. J. Ammit, R. K. Hoffman, A. J. Eszterhas, and R. A. Panettieri Jr. Activation of class IA PI3K stimulates DNA synthesis in human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L1009 - L1018. [Abstract] [Full Text] [PDF]

				C. Bony, S. Roche, U. Shuichi, T. Sasaki, M. A. Crackower, J. Penninger, H. Mano, and M. Puceat A Specific Role of Phosphatidylinositol 3-Kinase {gamma}: A Regulation of Autonomic Ca2+ Oscillations in Cardiac Cells J. Cell Biol., February 20, 2001; 152(4): 717 - 728. [Abstract] [Full Text] [PDF]

				I. J. Gonzalez-Robayna, A. E. Falender, S. Ochsner, G. L. Firestone, and J. S. Richards Follicle-Stimulating Hormone (FSH) Stimulates Phosphorylation and Activation of Protein Kinase B (PKB/Akt) and Serum and Glucocorticoid-Induced Kinase (Sgk): Evidence for A Kinase-Independent Signaling by FSH in Granulosa Cells Mol. Endocrinol., August 1, 2000; 14(8): 1283 - 1300. [Abstract] [Full Text]

				I. A. Yamboliev, K. M. Wiesmann, C. A. Singer, J. C. Hedges, and W. T. Gerthoffer Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle Am J Physiol Cell Physiol, August 1, 2000; 279(2): C352 - C360. [Abstract] [Full Text] [PDF]

				J. Klein, M. Fasshauer, M. Benito, and C. R. Kahn Insulin and the {beta}3-Adrenoceptor Differentially Regulate Uncoupling Protein-1 Expression Mol. Endocrinol., June 1, 2000; 14(6): 764 - 773. [Abstract] [Full Text]

				U. Maier, A. Babich, N. Macrez, D. Leopoldt, P. Gierschik, D. Illenberger, and B. Nurnberg Gbeta 5gamma 2 Is a Highly Selective Activator of Phospholipid-dependent Enzymes J. Biol. Chem., April 28, 2000; 275(18): 13746 - 13754. [Abstract] [Full Text] [PDF]

				C. Murga, S. Fukuhara, and J. S. Gutkind A Novel Role for Phosphatidylinositol 3-Kinase beta in Signaling from G Protein-coupled Receptors to Akt J. Biol. Chem., April 14, 2000; 275(16): 12069 - 12073. [Abstract] [Full Text] [PDF]

				S. V. Naga Prasad, G. Esposito, L. Mao, W. J. Koch, and H. A. Rockman Gbeta gamma -dependent Phosphoinositide 3-Kinase Activation in Hearts with in Vivo Pressure Overload Hypertrophy J. Biol. Chem., February 18, 2000; 275(7): 4693 - 4698. [Abstract] [Full Text] [PDF]

				K. Wenzel-Seifert, J. M. Arthur, H.-Y. Liu, and R. Seifert Quantitative Analysis of Formyl Peptide Receptor Coupling to Gialpha 1, Gialpha 2, and Gialpha 3 J. Biol. Chem., November 19, 1999; 274(47): 33259 - 33266. [Abstract] [Full Text] [PDF]

				M. Laffargue, P. Raynal, A. Yart, C. Peres, R. Wetzker, S. Roche, B. Payrastre, and H. Chap An Epidermal Growth Factor Receptor/Gab1 Signaling Pathway Is Required for Activation of Phosphoinositide 3-Kinase by Lysophosphatidic Acid J. Biol. Chem., November 12, 1999; 274(46): 32835 - 32841. [Abstract] [Full Text] [PDF]

				U. Maier, A. Babich, and B. Nurnberg Roles of Non-catalytic Subunits in Gbeta gamma -induced Activation of Class I Phosphoinositide 3-Kinase Isoforms beta and gamma J. Biol. Chem., October 8, 1999; 274(41): 29311 - 29317. [Abstract] [Full Text] [PDF]

				A. Adomeit, A. Graness, S. Gross, K. Seedorf, R. Wetzker, and C. Liebmann Bradykinin B2 Receptor-Mediated Mitogen-Activated Protein Kinase Activation in COS-7 Cells Requires Dual Signaling via Both Protein Kinase C Pathway and Epidermal Growth Factor Receptor Transactivation Mol. Cell. Biol., August 1, 1999; 19(8): 5289 - 5297. [Abstract] [Full Text] [PDF]

				S. Krugmann, P. T. Hawkins, N. Pryer, and S. Braselmann Characterizing the Interactions between the Two Subunits of the p101/p110gamma Phosphoinositide 3-Kinase and Their Role in the Activation of This Enzyme by Gbeta gamma Subunits J. Biol. Chem., June 11, 1999; 274(24): 17152 - 17158. [Abstract] [Full Text] [PDF]

				R. Baier, T. Bondeva, R. Klinger, A. Bondev, and R. Wetzker Retinoic Acid Induces Selective Expression of Phosphoinositide 3-Kinase {{gamma}} in Myelomonocytic U937 Cells Cell Growth Differ., June 1, 1999; 10(6): 447 - 456. [Abstract] [Full Text]

				Y.-X. Wang, P. D. K. Dhulipala, L. Li, J. L. Benovic, and M. I. Kotlikoff Coupling of M2 Muscarinic Receptors to Membrane Ion Channels via Phosphoinositide 3-Kinase gamma  and Atypical Protein Kinase C J. Biol. Chem., May 14, 1999; 274(20): 13859 - 13864. [Abstract] [Full Text] [PDF]

				P. VIARD, T. EXNER, U. MAIER, J. MIRONNEAU, B. NÜRNBERG, and N. MACREZ Gß{gamma} dimers stimulate vascular L-type Ca2+ channels via phosphoinositide 3-kinase FASEB J, April 1, 1999; 13(6): 685 - 694. [Abstract] [Full Text]

				X. Su, P. Wang, A. Ibitayo, and K. N. Bitar Differential activation of phosphoinositide 3-kinase by endothelin and ceramide in colonic smooth muscle cells Am J Physiol Gastrointest Liver Physiol, April 1, 1999; 276(4): G853 - G861. [Abstract] [Full Text] [PDF]

				H. Yang and M. K. Raizada Role of Phosphatidylinositol 3-Kinase in Angiotensin II Regulation of Norepinephrine Neuromodulation in Brain Neurons of the Spontaneously Hypertensive Rat J. Neurosci., April 1, 1999; 19(7): 2413 - 2423. [Abstract] [Full Text] [PDF]

				A. Al-Aoukaty, B. Rolstad, and A. A. Maghazachi Recruitment of Pleckstrin and Phosphoinositide 3-Kinase {gamma} into the Cell Membranes, and Their Association with G{beta}{gamma} After Activation of NK Cells with Chemokines J. Immunol., March 15, 1999; 162(6): 3249 - 3255. [Abstract] [Full Text] [PDF]

				T. Exner, O. N. Jensen, M. Mann, C. Kleuss, and B. Nurnberg Posttranslational modification of Galpha o1 generates Galpha o3, an abundant G protein in brain PNAS, February 16, 1999; 96(4): 1327 - 1332. [Abstract] [Full Text] [PDF]

				A. Graness, A. Adomeit, R. Heinze, R. Wetzker, and C. Liebmann A Novel Mitogenic Signaling Pathway of Bradykinin in the Human Colon Carcinoma Cell Line SW-480 Involves Sequential Activation of a Gq/11 Protein, Phosphatidylinositol 3-Kinase beta , and Protein Kinase Cepsilon J. Biol. Chem., November 27, 1998; 273(48): 32016 - 32022. [Abstract] [Full Text] [PDF]

				S. J. Turner, J. Domin, M. D. Waterfield, S. G. Ward, and J. Westwick The CC Chemokine Monocyte Chemotactic Peptide-1 Activates both the Class I p85/p110 Phosphatidylinositol 3-Kinase and the Class II PI3K-C2alpha J. Biol. Chem., October 2, 1998; 273(40): 25987 - 25995. [Abstract] [Full Text] [PDF]

				K. Cieslik, C. S. Abrams, and K. K. Wu Up-regulation of Endothelial Nitric-oxide Synthase Promoter by the Phosphatidylinositol 3-Kinase gamma /Janus Kinase 2/MEK-1-dependent Pathway J. Biol. Chem., January 5, 2001; 276(2): 1211 - 1219. [Abstract] [Full Text] [PDF]

				R. K. Bommakanti, S. Vinayak, and W. F. Simonds Dual Regulation of Akt/Protein Kinase B by Heterotrimeric G Protein Subunits J. Biol. Chem., December 1, 2000; 275(49): 38870 - 38876. [Abstract] [Full Text] [PDF]

				D. Bacqueville, P. Deleris, C. Mendre, M.-T. Pieraggi, H. Chap, G. Guillon, B. Perret, and M. Breton-Douillon Characterization of a G Protein-activated Phosphoinositide 3-Kinase in Vascular Smooth Muscle Cell Nuclei J. Biol. Chem., June 15, 2001; 276(25): 22170 - 22176. [Abstract] [Full Text] [PDF]

				H. P. Reusch, S. Zimmermann, M. Schaefer, M. Paul, and K. Moelling Regulation of Raf by Akt Controls Growth and Differentiation in Vascular Smooth Muscle Cells J. Biol. Chem., August 31, 2001; 276(36): 33630 - 33637. [Abstract] [Full Text] [PDF]

				E. S. Park, C. O. Echetebu, S. Soloff, and M. S. Soloff Oxytocin stimulation of RGS2 mRNA expression in cultured human myometrial cells Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E580 - E584. [Abstract] [Full Text] [PDF]

				N. Macrez, C. Mironneau, V. Carricaburu, J.-F. Quignard, A. Babich, C. Czupalla, B. Nurnberg, and J. Mironneau Phosphoinositide 3-Kinase Isoforms Selectively Couple Receptors to Vascular L-Type Ca2+ Channels Circ. Res., October 12, 2001; 89(8): 692 - 699. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Leopoldt, D. Articles by Nürnberg, B. Search for Related Content PubMed PubMed Citation Articles by Leopoldt, D. Articles by Nürnberg, B. Social Bookmarking                      What's this?