Gtpase-Activating Proteins and Their Complexes Steven J Gamblin* and Stephen J Smerdon

195

GTPase-activating proteins and their complexes Steven J Gamblin* and Stephen J Smerdon

In the past year, crystallographic structures for four complexes Figure 1 of GTPase-activating proteins (GAPs) with their target G proteins have been described and substantially enhance our understanding of how these proteins function. GAPs specific for the Rho and Ras families of small G proteins insert an GMPPNP arginine residue into the active site of the G protein, stabilise its switch regions and share an underlying topological relationship. The complex of a heterotrimeric G protein with its activating protein shows that the latter protein does not participate directly in the hydrolytic reaction and has a different structure to RhoGAP and RasGAP. P-loop

Addresses Protein Structure Division, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK *e-mail: [email protected] I Correspondence: Steven J Gamblin

Current Opinion in Structural Biology 1998, 8:195–201 http://biomednet.com/elecref/0959440X00800195

Abbreviations II BH BCR homology EF elongation factor Ga G protein α subunit GAP GTPase-activating protein Hs Homo sapiens Current Opinion in Structural Biology PH pleckstrin homology PI3-kinase phosphatidyl inositol 3-kinase RGS regulator of G-protein signalling The structure of the archetypal small G protein, Ras, bound to the SH src homology nonhydrolysable nucleotide analogue GMPPNP [6]. The switch I, switch II and P-loop regions (in black) are labelled. The small G proteins Ras, RhoA and Cdc42 are structurally closely related. An Introduction extra helical segment (residues 117–137) is present in the Rho family members [16•] and is located C-terminal to the switch and P-loop The G-protein family is made up of a diverse range of mol- regions. Thus, the Ras and Cdc42Hs residue numbers are directly ecules that control a complex array of biological processes comparable. Unfortunately, RhoA possess two additional N-terminal but have in common a structurally homologous GTP-bind- amino acids, so residues are numbered two higher than their structural ing domain. They act as molecular switches that cycle equivalents in Ras or Cdc42Hs. In the main text, residues will be between the active, GTP-bound form and the inactive, numbered and subscripted according to the molecule to which they belong, whilst secondary structural elements will be named according GDP-bound form [1–3]. In the active form, they are com- to the original publication. These terms will also be used for referring to petent to interact with a broad range of effector molecules. the heterotrimeric G proteins and their complexes, although the The lifetime of this active state is determined by the com- numbering of its residues is unrelated to that of the small G proteins. bination of slow intrinsic GTPase activity and the action of The Gα switch III region, which responds indirectly to GTP hydrolysis, is specific to these proteins and has no counterpart in the small G GTPase-activating proteins (GAPs), which can accelerate protein relatives. GTP hydrolysis by as many as five orders of magnitude [4,5]. GTP hydrolysis causes conformational changes in the G protein that are localised to two distinct regions of the molecule, switch I (residues 30–40 in Ras, also known subfamily of small G proteins (Rac, Cdc42 and RhoA) as the effector loop) and switch II (residues 60–67 in Ras). [16•,17,18]. G proteins also contain a conserved region The locations of the switch regions with respect to the known as the P-loop (residues 10–17 in Ras), which nucleotide are shown in Figure 1. The structural respons- forms a structural cradle for the β- and γ-phosphate es of the switch regions to the loss of the γ-phosphate have groups of the nucleotide and additionally supplies a lig- been comprehensively described for the archetypal G pro- and (serine or threonine) to the octahedrally coordinated tein, Ras [6–8], Rap2A [9], elongation factor Tu (EF-Tu) magnesium ion [19•]. Mutations within this region of [10,11], the heterotrimeric G protein α subunit (Gα) Ras, result in the loss of GTPase activity and are [12–15], and more recently, members of the Rho oncogenic [20]. 196 Macromolecular assemblages

GAPs also form a structurally and functionally diverse fam- As mentioned before, the GAPs for Ras and Rho are unre- ily of molecules that appear to be specific for defined sub- lated at the protein sequence level but it is apparent that families of G proteins. At the time of writing, three crystal there is some similarity in terms of the arrangement of sec- structures of isolated GAP molecules [21–23] and four ondary structure elements in the G-protein binding sites of complexes of GAPs with their cognate G protein these GAPs. In RasGAP the G-protein binding site is gen- [24••–27••] have been solved. The purpose of this review erated from two adjacent helices and two associated loops is to assess the current state of our structural understand- [25••]. In the RhoGAP complexes, although there are ing of GAP-mediated G-protein regulation. some detailed differences in the interface between the ground and transition states, the binding surface again GAPs for small G proteins involves two adjacent helices and an adjoining loop GAP activity domains occur in many proteins, usually in [26••,27••]. combination with other signalling modules that may include src homology (SH) 2 and SH3 domains, and pleck- Comparison of the ground and transition state strin homology (PH) domains together with proline-rich G-protein complexes of RhoGAP regions [3,5]. Crystal structures have been solved for GAP The Cdc42Hs–RhoGAP complex contains the nonhy- domains from the p85α subunit of phosphatidyl inositol 3- drolysable GTP analogue GMPPNP, which can be regard- kinase (PI3-kinase) [21], p120RasGAP [22] and ed as representing the nucleotide substrate in its ground p50RhoGAP [23], which will be referred to hereafter as state complex. Crystal structures of nucleotide complexes BH (BCR homology domain), RasGAP and RhoGAP, of Ras have demonstrated that GMPPNP is a good struc- respectively. These structures reveal that all three GAP tural analogue of GTP [8]. The RhoA–RhoGAP complex α – domains are extensively -helical in composition. BH has contains GDP·AlF4 , which is thought to be an analogue of sequence and structural homology with RhoGAP and both the transition state of the phosphoryl transfer reaction. bind to members of the Rho family of small G proteins. Indeed adenylate cyclase was first identified 40 years ago BH does not enhance GTPase activity, however, [21,28,29] because aluminofluorides are potent activators of het- and this has led to the suggestion that Asn194 of RhoGAP erotrimeric G proteins [31–33]. These compounds – is important in GAP function since it is conserved in all (GMPPNP and GDP·AlF4 ) have subsequently been used RhoGAP domains that possess GAP activity, but not in BH to great effect in establishing the molecular basis of het- [23]. There is essentially no sequence homology between erotrimeric G-protein function [13,14]. Using these two RasGAPs and RhoGAPs and the occurrence of 15 helical compounds, structural snapshots of the RhoGAP–Rho pro- segments in RasGAP compared with nine in the smaller tein complex at two different points along the reaction RhoGAP fragment gives the two types of molecule quite pathway have been obtained. different appearances. Both RasGAP and RhoGAP, however, have a pair of conserved basic residues important for A comparison of the ground [26••] and transition-state their function (Arg789 and Arg903 in RasGAP [22], and complex [27••] structures reveals a substantial rearrange- Arg85 and Lys122 in RhoGAP [23]) and it is now apparent ment that can largely be described as a 20° rigid-body rota- that they share a common fold [30•] as shown in Figure 2. tion of the G protein with respect to RhoGAP about an axis that passes close to the phenolic sidechain of Tyr66RhoA. In Three small G protein–GAP complexes the transition state, but not the ground-state complex, Crystal structures have now been described for the follow- helix 3 of the G protein interacts with the C-terminal end •• ing complexes: Ras·GDP·AlF3–RasGAP [25 ], of the catalytic loop of RhoGAP (A–A1) and accounts for – •• 2 RhoA·GDP·AlF4 –RhoGAP [27 ] and Cdc42Hs·GMPP- an additional 460 Å of interaction surface largely consist- NP–RhoGAP [26••], which are shown in Figure 3a–c. ing of direct and solvent-mediated hydrogen bonds. Some Knowledge of these structures enables us to address a limited, local changes in the conformation of the A–A1 number of important issues. Firstly, since Cdc42Hs and loop of RhoGAP occur during its relocation such that RhoA both belong to the Rho family of small G proteins, Arg85 can now interact with the GTP and assist in cataly- what insights into the structural changes that take place sis. Both switch I and II are better ordered in the transi- during the GAP-assisted GTP hydrolysis reaction are evi- tion-state complex and this change probably constitutes an dent from comparing these two structures? Secondly, how important contribution to GAP activity. A significant do the two distinct Ras–RasGAP and Rho–RhoGAP fami- amount of the increased order of the switch I region is lies compare at the structural and functional level? These achieved through Tyr34RhoA. The mainchain carbonyl of questions will be addressed in the following two sections. this residue hydrogen bonds with the essentially conserved Asn194RhoGAP, whilst the aromatic ring of the sidechain Since all three small G proteins involved in these com- forms an electrostatic interaction with the repositioned plexes are closely related, it is not surprising that all three Arg85RhoGAP guanidinum group. This stabilisation of present the same structural components to the interface switch I probably enhances catalysis through its effects on with the GAP. All three G proteins make contacts with the nucleotide-associated magnesium ion. The interaction their GAP through parts of the protein intimately involved between Asp65RhoA and both Asn202RhoGAP (F helix) and in their GTPase activity: switch I, switch II and the P-loop. Arg126RhoGAP (B helix) is maintained from the ground state GTPase-activating proteins and their complexes Gamblin and Smerdon 197

Figure 2

(a)

(b) RhoGAP RasGAP

A1 R85 C R789

6ac

ABEFGDC 1c 2c 5c 7c 4c 3c C 8c 6bc

N A0 6ex 4 1 N ex ex 3ex

2ex

5ex

Current Opinion in Structural Biology

The topological relationship of the core structures of RhoGAP and RasGAP. (a) The core structures of the two molecules, with helices shown as cylinders [47] and labelled according to their original secondary structure description [22,23]. The two helices that constitute the major G-protein binding surface in each case are shown in black. Helical insertions within the RhoGAP core are shown as white cylinders. (b) Topological relationship between the helices within the GAP core structures. Additional, nonequivalent helices are in white, and the binding surface helices are again in black.

complex, although the formation of an additional interac- stabilise both the switch regions. With the recent observation between the sidechain of Gln63RhoA and the main- tion that RasGAP and RhoGAP are topologically related chain carbonyl of Arg85RhoGAP seems important in switch II [30], it becomes possible to compare their structures more stabilisation. As a consequence of this stabilisation, insightfully. With the two complexes oriented according to Gln63RhoA is locked into a position that enables it to pre- the common core structure of the GAPs, it is apparent that sent the hydrolytic water molecule in an optimal position. the two G proteins bind to their respective GAPs in different, but related ways. Rho binds to a shallow pocket on Comparison of Rho–RhoGAP with RhoGAP composed of helices B and F and the catalytic Ras–RasGAP loop (A–A1). Ras binds to a groove on RasGAP generated The mechanism of GTP hydrolysis and the involvement by helices α6c and α7c and the catalytic L1c and L6c of catalytic arginine residues in accelerating this reaction loops. The first three components broadly correspond to has already been widely discussed and we shall not be con- helices F and G and the catalytic A–A1 loop of RhoGAP, cerned with it in detail here [34,35•,36,37]. Superficially, it respectively. Thus, as noted before, the two GAPs each is apparent that both Rho and Ras GAPs carry out a similar use two helices as a major component of their binding sites α function in that they supply an arginine residue in trans for G proteins. The FRhoGAP and 6cRasGAP helices are into the active site of the G protein and, in addition, topologically equivalent, and RhoGAP utilises helix B 198 Macromolecular assemblages

Figure 3

(a) (b)

Current Opinion in Structural Biology

Four G protein–GAP complexes; all are aligned by superposition of their G-protein components, shown on the left of each panel in light grey, with the GAP molecules on the right in black. The Ras–RasGAP complex was modelled from available coordinates of the individual components (Brookhaven accession codes 5p21 and 1wer, respectively) and combined as described in [25••] (figure produced with MOLSCRIPT [48]). – – (a) Ras·GDP·AlF3–RasGAP. (b) RhoA·GDP·AlF4 –RhoGAP. (c) Cdc42Hs·GMPPNP–RhoGAP. (d) Giα·GDP·AlF4 –RGS4.

(equivalent to α2c) and RasGAP uses α7c (equivalent to helices B and F lie approximately in a plane while helix G helix G) to complete the binding site. This change in bind- is substantially rotated out of this plane. In contrast, in ing surface necessarily occurs because the helices of the RasGAP, helices α6c (helix F) and α7c (helix G) lie common core structure of the two GAPs pack against each approximately antiparallel in a plane while helix α2c (helix other somewhat differently. As mentioned before, there is B) does not. a broad topological equivalence within the common core structures of RasGAP and RhoGAP but there is no accu- There is a notable correspondence between structure and rate alignment of the secondary structure elements. In par- function in terms of the loop structures that carry the cat- ticular, there is an important difference in the way helices alytic arginines of the two GAPs. In both GAPs, this argi- B (α2c), F (α6c) and G (α7c) pack together. In RhoGAP, nine residue is preceded by two hydrophobic residues GTPase-activating proteins and their complexes Gamblin and Smerdon 199

(Leu-Phe in RasGAP and Ile-Phe in RhoGAP), which complex of Cdc42Hs·GMPPNP–RhoGAP, in which the anchor the catalytic loop to the core of the GAP domain. equivalent Arg85RhoGAP carbonyl oxygen is located some α •• Conserved residues in a helical bundle comprising helices 7 Å from the Gly12Cdc42 C [26 ]. A (α1c), B (α2c) and F (α6c) form a hydrophobic clamp (for the two hydrophobic loop residues) that acts to lock down Complex of a heterotrimeric G protein and its the catalytic loop. Again, the differences between the GAP packing of the core helices in RasGAP and RhoGAP are Heterotrimeric G proteins are a family of signalling mole- reflected in the positioning of the catalytic loops. In cules that respond to serpentine-receptor mediated activa- RasGAP, the L1c catalytic loop is closer to the surface tion [38,39]. In their inactive form, they consist of βγ made by helices α6c–α7c than its equivalent in RhoGAP. dimers associated with GDP-bound α subunits. Activation In both RasGAP and RhoGAP, a basic GAP residue inter- involves exchange of GDP for GTP, which leads to confor- acts with, and presumably stabilises, both the catalytic loop mational changes in the switch regions of the α subunit of the GAP and the switch II region of the G protein. The and its dissociation from the heterotrimeric complex. In importance of this interaction for these two key compo- this activated form, it can interact with a variety of down- nents of the GAP-activated GTPase active site is high- stream effectors. Heterotrimeric G-protein complexes lighted by the fact that both Lys122RhoGAP and its have been extensively characterised both biochemically •• equivalent, Arg903RasGAP, are conserved. In RasGAP, and structurally [12–15,24 ,40]. Crystallographic studies Arg903 is situated on α6c (equivalent of the F helix in have revealed that Gα subunits consist of two domains, one RhoGAP) and interacts with the mainchain carbonyl of structurally related to small G proteins and the other Arg789RasGAP and the sidechain of Glu63Ras. In RhoGAP, extensively helical and unique to G proteins of this class. Lys122 is located on the B helix (equivalent to α2c of Like small G proteins, Gα subunits possess intrinsic RasGAP) and contacts the mainchain carbonyl of Phe84 on GTPase activity that may also be stimulated through inter- the catalytic loop, and Glu65RhoA (equivalent Glu63Ras). action with GAP molecules of the regulator of G-protein This functional equivalence, together with the differences signalling (RGS) family, of which more than twenty mem- in the structure of the G-protein binding surface described bers have been identified [41,42]. The intrinsic GTPase above, dictate that this second basic residue (Arg903RasGAP activity of Gα is substantially greater (~100-fold) than that and Lys122RhoGAP) must be located on different helices in of small G proteins due to the presence of a catalytic argi- the two GAP structures. nine located on an extension of the switch I region that is, in turn, elaborated to form the extra helical domain inser- There are two further G protein–GAP interactions that tion. Indeed, biochemical studies have elegantly demon- appear to be common to both RasGAP and RhoGAP. For strated that the helical domain containing Arg201 of Gsα both complexes, there are important interactions between can act as a soluble GAP in trans when combined with suit- switch II of the G protein and the GAP. In particular ably deleted Gsα molecules [43]. Crystal structures of Gtα – Tyr66RhoA makes a polar interaction with the mainchain and Giα subunits complexed with GDP·AlF4 transition- carbonyl of Val197RhoGAP (F helix) whereas the equivalent state analogue [13,14] show that their respective catalytic Tyr64Ras likewise makes an interaction with the mainchain arginines interact with the transition state analogue in a α of Leu902RasGAP ( 6c). Finally, the mainchain carbonyl of similar way to that subsequently observed for the equiva- Ala88 on the catalytic loop of RhoGAP interacts with lent Ras–RasGAP and RhoA–RhoGAP complexes •• •• Asn94 on helix 3 of Rho, and there is a tentative assign- [25 ,27 ,37]. This occurs despite the fact that in Gtα and α ment of a mainchain interaction of Thr791 on the catalytic Giα, the C positions of the catalytic arginines are located loop of RasGAP with Lys88, which is also located on helix 14 Å from that observed for the equivalent arginines in the 3 (of Ras). Although the preceding discussion has only RasGAP and RhoGAP complexes. The GAP activity of dealt with a subset of the interactions within the two dif- RGS molecules is not, therefore, dependent on the supply ferent GAP complexes, it appears there is a significant of an arginine into the active site of the G protein. The degree of correspondence in the character of the molecular crystal structure of a complex between Giα and RGS4 interactions involved [25••,27••]. (shown in Figure 3d) [24••] indicates that the additional 40-fold activation by RGS molecules is most likely Since RasGAP and RhoGAP have apparently diverged achieved through binding to, and stabilisation of, the Giα from a common ancestor, it would seem plausible that the switch regions. RGS4, like Rho and Ras family GAP mol- Ras–RasGAP system also undergoes a substantial confor- ecules is extensively α-helical in composition. It consists of mational rearrangement on going from the ground to the a total of nine helical segments arranged to form a four- transition state. Wittinghofer and his colleagues [25••] helix bundle (α4–α7), with an additional excursion formed have already suggested that the catalytic arginine by the remaining helices, α1–α3 and α8–α9. Aside from (Arg789RasGAP) is remote from the vicinity of Gly12Ras in the helical bundle, RGS4 seems to share no other signifi- the ground state. This is because mutants at this position cant tertiary structural homology with RhoGAP or (that may be oncogenic) would clash with the Arg789 car- RasGAP. The G-protein binding surface of RGS4 is bonyl oxygen in the transition-state complex. This sugges- formed largely by residues derived from loops connecting tion is entirely in keeping with the known ground-state α3–α4, α5–α6 and α7–α8 at the base of the four-helix 200 Macromolecular assemblages

bundle. These residues interact with switch I, switch II 4. Gideon P, John J, Frech M, Lautwein A, Clark R, Scheffler JE, 2 Wittinghofer A: Mutational and kinetic analysis of the GTPase- and the P-loop of Giα and bury around 1100 Å of solvent activating protein (GAP)-p21 interaction: the C-terminal domain of accessible surface in the complex. Like the Rho and Ras GAP is not sufficient for full activity. Mol Cell Biol 1992, 12:2050- 2056. small G proteins, Gα subunits have a conserved glutamine residue derived from the switch II region that is essential 5. Lamarche N, Hall A: GAPs for rho-related GTPases. Trends Genet 1994, 10:436-440. for GTPase activity and also seems to be involved in the 6. Pai EF, Krengel U, Petsko GA, Goody RS, Kabsch W, Wittinghofer A: orientation and polarisation of the nucleophilic water mol- Refined crystal structure of the triphosphate conformation of H- ecule. As we have described, RhoGAP and RasGAP both ras p21 at 1.35 Å resolution: implications for the mechanism of appear to contribute to the orientation of the hydrolytic GTP hydrolysis. EMBO J 1990, 9:2351-2359. water molecule in the transition state through mainchain 7. Milburn MV, Tong L, deVos AM, Bruenger A, Yamaizumi Z, Nishimura S, Kim S-H: Molecular switch for signal transduction: structural carbonyl interactions with the sidechain of Gln63Rho differences between active and inactive forms of protooncogenic ras proteins. 247 /GLN61Ras. Although this situation does not occur in the Science 1990, :939-945. Giα–RGS complex, additional stabilisation of the equiva- 8. Schlichting I, Almo SC, Rapp G, Wilson K, Petratos, Lentfer A, Time- lent Gln204 of G α seems to be provided through a net- Wittinghofer, Kabsch W, Pai EF, Petsko GA, Goody RS: i resolved X-ray crystallographic study of the conformational work of hydrogen-bonding interactions with Asn128 from change in Ha-Ras p21 protein on GTP hydrolysis. Nature 1990, the RGS4 molecule. 345:309-315. 9. Cherfils J, Menetrey J, Le Bras G, Le Bras G, Janoueix-Lerosey I, de Grunzburg J, Garel J-R, Auzat I: Crystal structures of the small G Conclusions protein Rap2A in complex with its substrate GTP, with GDP and with GTPgS. 16 It is apparent that the activation of the intrinsic hydrolysis EMBO J 1997, :5582-5591. of G proteins by GAPs can be nominally divided into two 10. Kjeldgaard M, Nissen P, Thirup S, Nyborg J: The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP components. The insertion of a catalytic arginine from the conformation. Structure 1993, 1:35-50. GAP into the active site of their small G-protein partner 11. Bechtold H, Reshetnikova L, Reiser COA, Schirmer NK, Sprinzl M, represents a direct chemical contribution to catalysis. On Hilgenfeld R: Crystal structure of active elongation factor Tu the other hand, the interactions of all the GAPs described reveals major domain rearrangements. Nature 1993, 365:126-132. above appear to involve substantial stabilisation of the 12. Lambright DG, Noel JP, Hamm HE, Sigler PB: Structural determinants for activation of the a subunit of a heterotrimeric G switch regions associated with the intrinsic GTPase activi- protein. Nature 1994, 369:621-628. ty of the G proteins. These effects may be regarded as an 13. Coleman DE, Berghuis AM, Lee E, Linder ME, Gilman AG, Sprang allosteric contribution to catalysis. All of the complexes SR: Structures of active conformations of Gia1 and the described above involve the interaction of GAPs with the mechanism of GTP hydrolysis. Science 1994, 265:1405-1412. switch I and II regions of the G proteins. Of the two known 14. Sondek J, Lambright DG, Noel JP, Hamm HE, Sigler PB: GTPase mechanism of G-proteins from the 1.7 Å crystal structure of crystal structures of G proteins bound to their downstream – transducin a·GDP·AlF4 . Nature 1994, 372: 276-279. effectors, both involve interactions between the switch 15. Raw AS, Coleman DE, Gilman AG, Sprang SR: Structural and •• – – regions of the G protein and the effector [44,45 ]. These biochemical characterization of the GTPgS , GDP·Pi , and GDP- 42 structures underline the biochemical observation that the bound forms of a GTPase-deficient Gly -Val mutant of Gia1. Biochemistry 1997, 36:15660-15669. interactions of GAPs and effectors with G proteins are • 16. Hirshberg M, Stockley RW, Dodson G, Webb MR: The crystal mutually exclusive, for example [35 ]. This appears to be • structure of human rac1, a member of the rho-family complexed the case for RGS4 inhibition of the interaction of Gqα with with a GTP analogue. Nat Struct Biol 1997, 4:147-152. phospholipase-Cβ [46] and may represent an additional This paper reports the the first structure determination of a Rho subfamily small G protein, rac1A. mechanism for G protein down regulation by GAPs that is 17. Feltham JL, Doetsch V, Raza S, Manor D, Cerione RA, Sutcliffe MJ, independent of GTPase activation. Wagner G, Oswald RE: Definition of the switch surface in the solution structure of Cdc42Hs. Biochemistry 1997, 36:8755-8766. Acknowledgements 18. Wei Y, Zhang Y, Derewenda U, Liu X, Minor W, Nakamoto RK, Somlyo We are particularly grateful to our colleagues Philip Walker, John Eccleston, AV, Somlyo AP, Derewenda ZS: Crystal structure of RhoAGDP: Guy Dodson and Willy Taylor from the Structural Biology Group at the implications for function of GEFs and Clostridium toxins. Nat National Institute for Medical Research for stimulating discussions Struct Biol 1997, 4:699-703. regarding many of the aspects of the work presented here and also to Katrin The GTP binding motif: Rittinger and John Skehel for helpful suggestions regarding this manuscript. 19. Kjeldgaard M, Nyborg J, Clark BFC: • variations on a theme. FASEB J 1996, 10:1347-1368. An excellent review describing the widespread occurrence of the GTP- binding motif. References and recommended reading Papers of particular interest, published within the annual period of review, 20. Barbacid M: Ras genes. Annu Rev Biochem 1987, 56:779-828. have been highlighted as: 21. Musacchio A, Cantley LC, Harrison SC: Crystal structure of the breakpoint cluster region-homology domain from • of special interest a •• of outstanding interest phosphoinositide 3-kinase p85 subunit. Proc Natl Acad Sci USA 1996, 93:14373-14378. 1. 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