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*

Sonja KrugmannDagger §, Phillip T. HawkinsDagger , Nancy Pryerparallel , and Sylvia Braselmannparallel

From Dagger  Signalling Programme, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom and parallel  Onyx Pharmaceuticals, Richmond, California 94806-5206

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recently, we have reported the purification and cloning of a novel G protein beta gamma subunit-activated phosphoinositide 3-kinase from pig neutrophils. The enzyme comprises a p110gamma catalytic subunit and a p101 regulatory subunit. Now we have cloned the human ortholog of p101 and generated panels of p101 and p110gamma truncations and deletions and used these in in vitro and in vivo assays to determine the protein domains responsible for subunit interaction and activation by beta gamma subunits. Our results suggest large areas of p101 including both N- and C-terminal portions interact with the N-terminal half of p110gamma . While modifications of the N terminus of p110gamma could modulate its intrinsic catalytic activity, binding to the N-terminal region of p101 was found to be indispensable for activation of heterodimers with Gbeta gamma .

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphoinositide 3-kinases (PI 3-kinases)1 are responsible for the phosphorylation of inositol phospholipids in the D-3 position of the inositol ring. Their lipid products (PtdIns3P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3) function as second messengers in eukaryotic cells. Indeed, PI 3-kinases appear to be involved in the control of a host of cellular responses ranging from intracellular transport to cell motility and the suppression of apoptosis (see Refs. 1-3, for reviews).

Three classes of PI 3-kinases are distinguished (4). Type I PI 3-kinases can be rapidly activated by cell-surface receptors and in vivo make predominantly PtdIns(3,4,5)P3 (5). They are heterodimeric enzymes comprising a 110-kDa catalytic subunit and a regulatory subunit. Type IA PI 3-kinases contain an alpha , beta , or delta  p110 catalytic subunit (6, 7) and a p50, p55, or p85 (alpha  or beta ) regulatory subunit (8-10). The regulatory subunits contain two SH2 domains which allow the enzyme to bind to, and be activated by key phosphotyrosine residues found in the cytoplasmic tails of growth factor receptors and various adapter proteins (11).

Type IB PI 3-kinase is made up of a p110gamma catalytic subunit and a p101 regulatory subunit (12). This PI 3-kinase seems to be specifically stimulated by receptors capable of activating heterotrimeric G proteins (12, 13). It appears, that this effect is mediated by G protein beta gamma subunits which can directly activate p101/p110gamma PI 3-kinase (12). Although several reports show that both the biological effects of p110gamma and its intrinsic sensitivity to Gbeta gamma are substantially amplified by the presence of p101, some data suggest that p110gamma alone can be substantially catalytically activated by Gbeta gamma and have clear biological effects (14, 15). We have begun to address the issue of the role of p101, if any, in these events by analyzing the regions of p101 involved in interactions with p110gamma and furthermore, for those constructs that bind, how they affect the activation of the complex by Gbeta gamma . We analyzed also the part played by p110gamma in the process of Gbeta gamma activation and p101 binding.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Site-directed Mutagenesis of p101 and p110gamma -- All point mutations, novel N or C termini, and internal deletions within porcine p101 cDNA were done using polymerase chain reaction-based strategies with mutagenic primers and Taq polymerase (Promega). Polymerase chain reaction-generated fragments were ligated back into pCMV3 with an N-terminal (EE)- or Myc-tag for expression in mammalian cells and into pAcOG3 with an N-terminal (EE)-tag for constructing baculovirus transfer vectors. All polymerase chain reaction-generated inserts were sequenced.

N- and C-terminal deletions of the human p110gamma were either done by digesting with the appropriate restriction enzymes at designated sites. The isolated DNA fragments were religated into pcDNA3 containing N-terminal Myc-tag linkers. The Ras-binding domain deletions were done by fusing the internal fragments in-frame to the N-terminal 169 amino acids, creating a three amino acid linker. Full-length human p101 was cloned into an N-terminal EE-tag containing pcDNA3 vector.

Cell Culture-- Sf9 cells were grown in TNM-FH medium with 11% fetal bovine serum in spinner flasks at 27 °C at 0.5-2 × 106 cells/ml. COS-7 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum.

Sf9 Transfections and Production of Recombinant Protein-- Sf9 cells were lipofected using Insectin (Invitrogen) liposomes with baculogold (Pharmingen) linearized baculoviral DNA and a baculovirus transfer vector. Viral particles were plaque purified prior to amplification. Infection times were 2.3 days for p101 and 1.8 days for p110gamma . Harvested cells were pelleted, washed in 7 mM NaH2PO4 (pH 6.2), 20 mM MgCl2, 0.7% KCl, 2.66% sucrose, snap frozen in liquid N2 and stored at -80 °C.

Purification of Proteins from Sf9 Cells-- 6H-tagged porcine p110gamma and EE-tagged porcine p101 were purified as described previously (12) with metal ion chelate columns or immunoprecipitation, respectively. The p110gamma purification was modified as follows. Buffer C was 50 mM sodium phosphate (pH 7.1, 4 °C), 1% betaine, 0.05% Tween 20, 0.1 M NaCl; buffer D was 1% betaine, 30 mM Tris-Cl (pH 7.5, 4 °C), 0.15 M NaCl, 0.02% Tween 20, 10% ethylene glycol; buffer E was as C supplemented with 7 mM imidazole (pH 7.5, 4 °C); elution was in buffer F which was as C supplemented with 100 mM imidazole (pH 7.5, 4 °C). Eluted p110gamma in buffer F was supplemented with 1 mM dithiothreitol and 1 mM EGTA (F') or, if it was to be bound to p101, passed through a PD10 column (Pharmacia) equilibrated in buffer H (10 mM Tris-Cl (pH 7.5, 4 °C), 0.15 M NaCl, 1% betaine, 0.02% Tween 20, 10% ethylene glycol, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 1% Triton X-100). In some cases, the p110gamma (6H)-tag was cleaved with thrombin (Sigma) at 12 units/ml at 4 °C for 12 h.

The p101 purification was modified as follows, cells were sonicated into 0.15 M NaCl, 25 mM Hepes (pH 7.2, 4 °C), 2 mM EGTA, 1 mM MgCl2 plus antiproteases; cytoplasmic fractions were not pre-cleared with anti-Myc beads; washes after the anti-EE beads (Onyx Pharmaceuticals) were five times in 0.4 M NaCl, 20 mM Hepes (pH 7.4, 4 °C), 1 mM EGTA, 1% Triton X-100, 0.4% cholate, and three further times in buffer H. To bind the subunits, p101 bound to packed anti-EE beads was mixed end on end for 2.5 h with a 25-fold molar excess of free p110gamma in a small volume of buffer H. Heterodimer on anti-EE beads was washed in buffer J (1% Triton X-100, 0.15 M NaCl, 20 mM Hepes (pH 7.4, 4 °C), 1 mM EGTA) and in buffer F'. p101-p110gamma heterodimer was eluted in buffer F' supplemented with 125 µg/ml EY peptide (EYMPTD). Typical yields were 2 mg of p110gamma and 25 µg of p101/500 ml of Sf9 culture.

GST-p110gamma (the relevant recombinant baculovirus was a gift from R. Wetzker and encoded the human form of the protein) was purified from cytosolic fractions or Triton X-100 lysates of Sf9 cells as described by Leopoldt et al. (Ref. 15 and references therein). This GST construct could not be cleaved by thrombin to yield full-length untagged p110gamma .

Transient Transfections in COS-7-- Exponentially growing COS-7 cells were trypsinized, washed twice in phosphate-buffered saline, and resuspended in 30 mM NaCl, 0.12 M KCl, 8.1 mM Na2HPO4, 1.46 mM KH2PO4, 5 mM MgCl2 at 1 × 107 cells/450 µl. They were mixed with 30 µg of plasmid DNA (15 µg of pCMV3(EE)101 constructs and 15 µg of pCMV3(myc)110gamma or 15-30 µg of irrelevant plasmid DNA) per 107 cells aliquot. Aliquots were placed into 0.4-cm gap electroporation cuvettes (Bio-Rad) and electroporated in a single pulse (250 V, 980 microfarads). Cells from each electroporation were seeded into 175-ml tissue culture flasks in full growth medium. After 28 h, cells were harvested by trypsinization, washed once in phosphate-buffered saline, and cell pellets lysed in 1 × phosphate-buffered saline, 1 mM EGTA, 1% Triton X-100. Cytoplasmic fractions were immunoprecipitated with anti-EE beads followed by washes in 2 × phosphate-buffered saline, 1 mM EGTA, 1% Triton X-100. Samples for PI 3-kinase assays were washed further in 25 mM Hepes (pH 7.4, 4 °C), 1 mM EGTA. Remaining samples were resolved by SDS-PAGE. Gels were either Coomassie stained or wet-blotted onto polyvinylidene fluoride membranes (Millipore) and immunoblotted according to their tags with anti-Myc antibody (obtained from Onyx Pharmaceuticals) and anti-EE ascites fluid (Babco) or with an anti-p101 antiserum (Microchemical Facilities, Babraham Institute).

PI 3-Kinase Assays-- Free, purified protein from Sf9 cells or COS-7 derived protein on anti-EE beads was diluted in sample dilution buffer (2 mg/ml fatty acid free bovine serum albumin, 0.1 M KCl, 20 mM Hepes (pH 7.4, 4 °C), 1 mM dithiothreitol). Lipid mixtures containing phosphatidylethanolamine and PtdIns(4,5)P2 were dried down and sonicated into 0.1 M NaCl, 25 mM Hepes (pH 7.4, 4 °C), 1 mM EGTA, 0.1% cholate for final concentrations of 50 µM phosphatidylethanolamine (Sigma) and 5 µM PtdIns(4,5)P2 (13). They were supplemented with G protein beta gamma subunits (prepared as in Ref. 16) or its vehicle (1% cholate, 0.15 M NaCl, 1 mM dithiothreitol, 5 mM EGTA, 0.2 mM EDTA) to a final concentration of 0.3 µM Gbeta gamma . [gamma -32P]ATP was diluted to a concentration of 5 µCi/10 µl into 0.1 M NaCl, 25 mM Hepes (pH 7.4, 4 °C), 1 mM EGTA. For the assay, 5 µl of diluted protein were kept on ice until 20 µl of lipid mixture with or without Gbeta gamma was added and the mixture transferred to a 30 °C waterbath. 4 min later, 50 µl more sample dilution buffer supplemented with MgCl2 to give a final concentration of 3.5 mM was added, another 4 min later, 10 µl of diluted ATP was added. Assays were stopped after 12 min incorporation time by addition of 160 µl of 1.25 M HCl. Assays were extracted with 800 µl of CHCl3:MeOH (2:1) and then with CHCl3, MeOH, 1 M HCl (3:48:47). Lipids were deacylated and resolved on PEI TLC plates as described before (13). Alternatively, assays were conducted precisely as described by Leopoldt et al. (15) with PtdIns acting as the substrate.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

First, we simply reproduced the results of previous work showing purified p110gamma could bind p101 both in vitro and in vivo and that this association substantially increased the scale of activation of the PI 3-kinase with Gbeta gamma from 1-2-fold to 50-150-fold, but now using modified procedures and porcine and human versions of the proteins. Human p101 (GenBank accession number AF128881) was 88% identical to porcine p101 (c.f. human p110gamma is 94% idential to porcine p110gamma ). We found the species orthologs behaved interchangeably in these assays and gave results identical to those of the earlier work (Ref. 12, data not shown).

Analysis of p101 Structure/Function in Vitro-- In order to understand the interactions between p101 and p110gamma and the ability of the heterodimer to respond to Gbeta gamma subunits, we made panels of p101 and p110gamma constructs. Analysis of p101 structures involved in binding to p110gamma was first approached with the following assay. NH2-terminal (EE)-tagged p101 constructs were purified from Sf9 cells using anti-(EE)-beads. After washing, an excess of purified, NH2-terminal (6H)-tagged full-length p110gamma from Sf9 cells was mixed with the immobilized p101 constructs. The beads were washed again and eluted with an epitope (EY) peptide. The released proteins were analyzed by SDS-PAGE and PI 3-kinase assays with and without Gbeta gamma subunits. This analysis revealed that all of the p101 constructs tested (Fig. 1A) had reduced, but still significant capacity to specifically bind p110gamma relative to full-length. Thus both the large N-and C-terminal truncations of p101 (Delta 1-163 and 1-733) resulted in a 50% reduction in the recovery of p110gamma , while Delta 283-581 reduced recovery by about 25% (Fig. 1B). Some p110gamma specifically bound to N-terminal (p101 1-163) and C-terminal fragments of p101 (p101Delta 1-574) (about 10 and 30% of wild-type, respectively) (Fig. 1C).


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Fig. 1.   In vitro study of p101 association with p110gamma and activation of complexes by Gbeta gamma . A, panel of p101 constructs used for expression and purification from Sf9 cells. All p101 proteins were N-terminal (EE)-tagged (open circle ). B and C, in vitro association of the (EE)-p101 proteins to independently purified, N-terminal (6H)-tagged p110gamma as detailed under "Experimental Procedures." Fractions of eluted heterodimer were visualized by SDS-PAGE and Coomassie staining. p101b was prepared from an equivalent extract of Sf9 cells infected with wild-type baculovirus and thus represents a control for nonspecific binding of p120 to the anti-(EE) beads. All data shown is representative of at least four independent experiments. D, PI 3-kinase assays of fractions of the heterodimers shown in B and C using PtdIns(4,5)P2 as substrate, with and without addition of Gbeta gamma as indicated. The data are mean ± range (n = 2-5) drawn from independent experiments. The activity in the absence of Gbeta gamma was normalized to the amount of p110gamma protein in each assay (estimated from the equivalent SDS-PAGE gels).

All p101-p110gamma heterodimers were also assayed for PI 3-kinase activity in the presence or absence of Gbeta gamma (Fig. 1D). All of the heterodimers created containing p101 truncations and deletions had reduced activation by G protein beta gamma subunits. It is striking, however, that the N-terminal truncation (Delta 1-163) completely abrogated activation by Gbeta gamma , while the remaining truncations resulted merely in decreased ability of the respective heterodimers to be activated by Gbeta gamma . Neither N- nor C-terminal p101 peptides bound to p110gamma yielded considerable activation when assayed in the presence of Gbeta gamma subunits. Overall this suggests a number of regions in p101 are involved in its ability to interact with p110gamma , particularly the N and C termini. Similarly, multiple areas in p101 are required for heterodimers to give maximal activation by Gbeta gamma subunits, however, the N terminus is absolutely required for this process.

Analysis of p101 Structure/Function in Vivo-- To address issues such as (a) the possibility that purification and our handling of the p101 constructs had resulted in varying levels of denaturation and that this effect generated the differential binding we observed, and (b) that some binding only occurs in vitro in the absence of competing proteins found in the cell, we examined the ability of p101 derivatives to bind to p110gamma when the proteins are transiently co-expressed in COS-7 cells. In addition, to allow for more detailed mapping of the impact of different areas of p101 on both binding to p110gamma and activation of the heterodimer with Gbeta gamma , a number of further constructs were introduced (Fig. 2A).


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Fig. 2.   In vivo association of p101 constructs with p110gamma and activation of resulting heterodimers by Gbeta gamma . A, further p101 constructs for use in COS-7 transient transfection assays included further N- and C-terminal peptides and larger deletions. Furthermore, an unusual acidic region was replaced by a stretch of serines, threonines, and prolines (DE328-341STP); a region bearing a vague similarity to PH domains of known signaling proteins as well as potential key residues therein were mutated (Delta 161-263, Y193C, W252A); a possible spliced variant of p101 is also included (p84, this sequence diverges from p101 at residue 733 resulting in a truncated version of the protein; P. Hawkins and A. Eguinoa, unpublished data). Again, all p101 constructs included N-terminal (EE)-tags (open circle ). B, COS-7 cells were transiently transfected with mammalian expression vectors encoding a (EE)-p101 construct and a (Myc)-p110gamma (for controls, the total amount of DNA was made up to with irrelevant DNA). Cytoplasmic fractions of transfected cells were subjected to immunoprecipitations with (EE)-beads. The amount of co-immunoprecipitated (Myc)-p110gamma was visualized on Coomassie-stained protein gels. Binding ratios were estimated by eye with a minimum of three independent transfections being taken into account for each p101 construct. C, graphical illustration of all binding data obtained from the COS-7 transient transfection assays. D, aliquots of the COS-7-derived p101-p110gamma heterodimers (on the beads) were assayed for PI 3-kinase activity in the presence or absence of Gbeta gamma . Fold activation obtained with Gbeta gamma for the different constructs is detailed comparatively in this graph. The legend on the y axis indicates with which construct p110gamma had been expressed.

All p101 constructs were (EE)-tagged and co-expressed with (Myc)-tagged p110gamma in COS-7 cells in transient transfection assays. Cytosolic fractions of harvested cells were subjected to anti-(EE) immunoprecipitations, half of which were used to estimate binding stoichiometries on gels or blots (see Fig. 2B, for an example) while the other half was assayed for PI 3-kinase activity in the presence or absence of G protein beta gamma subunits.

The data describing binding of p101 derivatives to p110gamma in this assay is summarized in Fig. 2C. Binding to p110gamma was affected in all p101 constructs except for the point mutations and DE328-341STP constructs, supporting the conclusion from the in vitro studies indicating that more than one area of p101 contributes to binding p110gamma . It appears, however, that in the COS-7 cell assays compared with the in vitro assays, the N-terminal deletions lead to a larger decrease in p110gamma binding ability than C-terminal deletions (25 versus 70% binding) and N-terminal peptides can rescue more binding than C-terminal peptides (50 versus 12% binding).

We also assayed all constructs immunoprecipitated from COS-7 cells for PI 3-kinase activity. The resulting data (Fig. 2D) underlines the data already obtained from the in vitro assays from Sf9-produced protein. Again, we found that both C and N termini of p101 contribute to full activation of the complex by G protein beta gamma subunits. Any deletion within p101 interfered with the ability of the heterodimer to be completely activated. Interestingly, deletion of the C-terminal 150 amino acids (1-733 and p84) and deletion of much larger C-terminal portions (1-314, 1-265, and 1-163) lead to a similar decrease in activation by Gbeta gamma to less than 25% of that of full-length p101, stressing the role for the very C-terminal part. The most dramatic result, however, is induced by deletion of the very N-terminal portion of p101 (constructs Delta 1-153 and Delta 1-263) which lead to complete loss of activation, confirming the conclusions from the in vitro assays that this region of p101 is essential for activation of the complex by G protein beta gamma subunits.

Analysis of p110gamma Structure/Function-- To define the sites in p110gamma involved in both interaction with p101 and activation by Gbeta gamma , we prepared a set of constructs shown in Fig. 3 for expression in COS-7 and Sf9 cells. These constructs were used in assays aimed at defining the regions that interacted with p101 that were relevant to the Gbeta gamma activation of p101-p110gamma heterodimers and were required for basal PI 3-kinase activity.


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Fig. 3.   p110gamma domain structure; all truncation and deletion constructs. p110gamma contains two regions of high homology with p110alpha /beta . These include the N-terminal Ras-binding domain (RBD) and the C-terminal kinase domain. All constructs used in our study are drawn to scale; they were N-terminal tagged (Myc or 6H) except where stated otherwise in the main text.

PI 3-Kinase Activity of p110gamma Constructs-- Increasing N-terminal truncation of p110gamma systematically reduced its basal catalytic activity (Fig. 4A). Deletions beyond residue 369 had no detectable PI 3-kinase activity. N-terminal tags seemed to increase the basal catalytic activity of p110gamma . The activity of the N-terminal (6H)-tagged p110gamma (6H-p110gamma ) was reduced 2-fold by removal of the tag with thrombin and the activity of defined p110gamma constructs was reduced by half by switching from an N-terminal to a C-terminal (6H)-tag. Furthermore, N-terminal (GST)-tagged p110gamma (GST-p110gamma ) possessed an approximately 10-15-fold higher specific activity than wild-type p110gamma . In contrast, and as previously reported (12), binding of p101 reduced the basal catalytic activity of p110gamma by 5-fold.


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Fig. 4.   The intrinsic activity of p110gamma and its activation by Gbeta gamma are affected by deletions of the very N-terminal portion of the molecule. A, comparison of p110gamma constructs basal catalytic activities. All proteins were derived from Sf9 cells, and purified according to their tags. The basal catalytic activity was compared with that of N-terminal (6H)-tagged p110gamma for each individual construct. Note that thrombin cleavage of the (6H)-tag results in loss of 26 N-terminal amino acids due to an internal thrombin cleavage site (R. Williams, personal communication). GST-p110gamma could not be successfully cleaved by thrombin. B, porcine p101, porcine (6H)-p110gamma , porcine p101/(6H)-p110gamma , and human GST-p110gamma were purified from Sf9 cells as described under "Experimental Procedures" and aliquots were assayed for PI 3-kinase activity in the presence or absence of Gbeta gamma according to the protocol of Leopoldt et al. (15). Samples were normalized for their basal activity (this is not equivalent to amount of p110gamma in the assay, see A for this comparison).

Reports that GST-p110gamma can be substantially activated by Gbeta gamma in vitro (15) lead us to test the effect of manipulating p110gamma structure on its responsiveness to Gbeta gamma , despite the fact that we have never detected significant effects of Gbeta gamma on porcine p110gamma activity in the absence of p101. Testing a range of catalytically active porcine and human p110gamma constructs with or without N-terminal or C-terminal (6H) tags in the presence and absence of Gbeta gamma showed up to 2-fold activations of human (but not porcine) proteins with either our standard assay procedure or that of Leopoldt et al. (15; see below). However, with human GST-p110gamma we found that although our assay procedure (using PtdIns(4,5)P2 and 3.5 mM MgCl2) failed to reveal any activation by Gbeta gamma , using PtdIns and 10 mM MgCl2 in the assay (15), we could reproducibly detect 6-7-fold activations by Gbeta gamma (Fig. 4B); this was unaffected if the constructs were purified from membrane or cytosolic fractions (data not shown).

Interaction of p110gamma Constructs with p101 and Activation of Heterodimers by Gbeta gamma -- Experiments performed with proteins expressed in either COS-7 or Sf9 cells indicated that the N terminus of p110gamma was critical for binding to p101. p110gamma deletions through the series Delta 1-122, Delta 1-133, and Delta 1-144 (Fig. 5A, for examples) showed decreasing ability to bind p101, a Delta 1-169 construct showed no detectable binding (Fig. 5B). However, other regions were clearly involved in binding p101 (see Fig. 5C for an overview). Hence, N-terminal peptides (e.g. 1-169) had low p101 binding potential and further deletion of either the Ras-binding domain (178-330) or the central regions (330-775) of p110gamma also significantly reduced p101 binding. It is significant that those p110gamma constructs capable of binding p101, once bound, were all very similarly activated by Gbeta gamma (Fig. 5D). Catalytically active p110gamma constructs incapable of binding p101 remained insensitive to Gbeta gamma when assayed in the presence of p101 (Fig. 5C).


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Fig. 5.   Interaction of p110gamma constructs with p101. A, in vitro interaction assays to assay binding ability of free (6H)-p110gamma constructs to immobilized (EE)-p101 as described under "Experimental Procedures" are shown here with full-length p110gamma and two N-terminal truncations in comparison. Left-hand lanes show p110gamma derivatives after two different purification steps and right-hand lanes show the p110gamma derivatives obtained via binding to the (EE)-p101 beads. Binding of Delta 1-144 could not be detected on the gel, since the proteins co-migrated, however, activity assays showed that a small degree of binding did take place (see below). B, Myc-tagged human p110gamma and EE-tagged human p101 were transiently co-transfected into COS-7 cells as indicated and the protein complexes were co-immunoprecipitated and immunoblotted. I, p101 expression: anti-p101 Western blot of an anti-EE immunoprecipitation; II, p101 binding to p110gamma : anti-p101 Western blot of an anti-Myc immunoprecipitation; III, p110gamma expression: anti-Myc Western blot of the cell lysates. C, overview of the ability of all p110gamma constructs to bind p101 as estimated by eye from Coomassie-stained gels or Western blottings derived from in vitro interaction assays and COS-7 transient transfection assays. It is indicated also, which constructs are catalytically active and where complexes are activated by Gbeta gamma . D, extent of fold-activation of catalytic activity by Gbeta gamma for aliquots of the three p101-p110gamma heterodimers prepared in vitro from Sf9-derived proteins and shown in Fig. 2A.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is clear from the above results that multiple regions of p101 are involved in either direct (i.e. physical contact) or indirect interactions with p110gamma . Interestingly, the Gbeta gamma sensitivity of the p110gamma heterodimers formed by using various p101 constructs clearly correlated with how tightly they formed heterodimers, but absolutely depended on the presence of the N terminus of p101. We have previously published some binding data showing Gbeta gamma can apparently bind about five times more effectively to p101 than p110gamma (12), but we cannot discount important binding of Gbeta gamma to p110gamma , as others have identified clear effects of Gbeta gamma on GST-p110gamma (13, 15; see also Fig. 4B). There are, therefore, two types of explanation for this data. First, that a lot of the contacts between p101 and p110gamma are required for Gbeta gamma to have their full effects on p110gamma catalytic activity (whether the Gbeta gamma bind to p101, p110gamma , or both). Second, that the primary sites of Gbeta gamma binding that influence the activity of the heterodimer are located on p101 and are "co-incidentally" disrupted by changes which impinge on p101-p110gamma interaction. This apparently highly interwoven structure-function relationship between p101, Gbeta gamma , and p110gamma is not surprising if it is accepted that binding of p101 and Gbeta gamma can have such a profound effect on the catalytic site in p110gamma . The binding site for p101 in p110gamma , although primarily involving the N terminus of the protein, is a far larger region extending deeper into the protein. Considering the fact that several parts of p101 are involved in binding to p110gamma , it would seem reasonable that a substantial piece of p110gamma is involved in binding p101 (note that in p110alpha a relatively short region of the N terminus is thought to bind to a short inter-SH2 domain segment of p85 (17, 18)). In contrast to the situation with p101, where deletions affecting the total contact area and strength of binding to p110gamma also affected the sensitivity of the heterodimer to Gbeta gamma , deletions through the N terminus of p110gamma , that reduced the efficiency of binding to p101, had no effect on the activation of heterodimeric enzyme that could be recovered. Clearly, this analysis cannot be complete because deletions removing more than 200 N-terminal residues begin to strongly reduce the intrinsic catalytic activity of p110gamma , such that deletions beyond residue 369 activity is completely lost (see below), yet these deeper regions are clearly involved in binding to p101. However, the implication of this data is that some of the contacts between p101 and p110gamma , involved in their binding, can be eliminated by disruptions on the p110gamma side without effect on Gbeta gamma activation of the heterodimer, but that contacts broken by p101 disruptions reduce the ability of Gbeta gamma to activate the enzyme. The simplest explanation for these observations is that p101 disruptions are also interfering with Gbeta gamma -binding sites that are most important for activation of the heterodimer; i.e. that the latter of our proposed models (see above) is most likely to be correct.

In the process of engineering and assaying a range of p110gamma constructs it became clear that the N terminus of the protein had significant potential to influence catalytic activity. Hence the addition of N-terminal GST and (6H)-tags apparently increased the activity of the enzyme. In contrast, binding of p101, which appears to interact most strongly with the N terminus of p110gamma lead to a 5-fold inhibition of basal catalytic activity. This effect of p101 appears to have some parallels with observations that p85 binding to p110alpha suppresses its PI 3-kinase catalytic activity (19). Clearly the scale of activation of the p101/p110gamma heterodimer by Gbeta gamma in vitro indicates this "simple model" cannot apply here, however, this phenomenon lead us to test whether N-terminal tagging also influenced the sensitivity of p110gamma alone to Gbeta gamma subunits.

We found that of a large range of constructs GST-p110gamma alone was clearly activated by Gbeta gamma , but only under the conditions previously reported to show this effect (15). The very fact that Gbeta gamma can have some significant effect on GST-p110gamma in the absence of p101 may be taken to suggest one primary point of interactions of Gbeta gamma with p101/p110gamma is through direct binding to the p110gamma subunit. This gains some support from work indicating Gbeta gamma can bind to p110gamma alone. We have previously found that although five times more Gbeta gamma could be rescued from in vitro binding assays associated with p101 than with p110gamma we could detect above background binding to p110gamma (12). Furthermore, Leopoldt et al. (15) have shown Gbeta gamma can be recovered with p110gamma immunoprecipitated from Sf9 cells, and they went on to map this binding to two distinct regions within p110gamma . However, there are major problems with these approaches (15). First, neither directly demonstrated that the Gbeta gamma -binding sites were relevant to the activation of the PI 3-kinases and second as both analyses depended on an "immunoprecipitation wash" protocol they are subject to selective recovery of interactions with appropriate affinities and on/off rates; meaning much more physiologically important binding sites could be missed. As Gbeta gamma are notoriously prone to nonspecific hydrophobic interaction and for binding to effectors with low affinity but high on/off rates this means these assays are of very limited value in defining the regions that bind Gbeta gamma that lead to activation of the enzyme.

As a consequence of these considerations and in the light of (i) lack of effect of Gbeta gamma on other, non-GST-tagged p110gamma constructs, (ii) the unphysiological nature of the substrate and ionic conditions required to see Gbeta gamma activation of GST-p110gamma , (iii) the inevitable problem that in using p110gamma alone rather than in a complex with p101 further hydrophobic regions of protein interaction may be exposed, and (iv) that the presence of the GST fusion will drive homodimerization of p110gamma , we consider the evidence that the effects of Gbeta gamma on p101/p110gamma PI 3-kinase are only via direct interaction with p110gamma to be very weak.

Overall, our data suggests that p101 and p110gamma interact primarily through large areas covering the N and C termini of p101 and the N-terminal half of p110gamma and that the areas which bind Gbeta gamma giving the major effect on PI 3-kinase activity are probably located on p101. Given the substantial difficulties encountered in studying specific, low affinity interactions of Gbeta gamma subunits with various effectors in vitro (e.g. studying Gbeta gamma activation of PLCbeta s can be seen as an analogous problem), it is likely that a combination of further technologies, including detailed structural information, will be required to yield further insight into the mechanism of action of Gbeta gamma on p101/p110gamma PI 3-kinase.

    ACKNOWLEDGEMENTS

We thank Olga Perisic, Ed Walker, Christian Reid, and Roger Williams for providing Sf9 cells expressing some of the p110gamma constructs. We also thank the Protein Production Unit at Onyx Pharmaceuticals for the production of all the other p101 and p110gamma constructs in Sf9 cells.

    FOOTNOTES

* 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.

Fellow of the Biotechnology and Biological Sciences Research Council.

§ Supported by a scholarship from the Boehringer Ingelheim Foundation. To whom correspondence should be addressed. Tel.: 44-1223-496594; Fax: 44-1223-496043; E-mail: sonja.krugmann{at}bbsrc.ac.uk.

    ABBREVIATIONS

The abbreviations used are: PI 3-kinase, phosphoinositide 3-kinase; G protein, guanosine nucleotide-binding protein; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate; (EE)-tag, gluthamine-tag; (6H)-tag, hexa-histidine-tag; TNM-FH, insect cell medium; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PEI, polyethyleneimine cellulose; Sf9 cells, Spodoptera frugiperda cells.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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