Ras is an indispensable coregulator of the class IBphosphoinositide 3-kinase p87p110Barbara Kurigab Aliaksei Shymanetsab Thomas Bohnackerc Prajwalab Carsten Brockd1 Mohammad Reza AhmadianbMichael Schaeferde Antje Gohlab2 Christian Hartenecka Matthias P Wymannc Elisabeth Jeanclosb2and Bernd Nurnbergabd3

aDepartment of Pharmacology and Experimental Therapy Institute of Experimental and Clinical Pharmacology and Toxicology Eberhard Karls UniversityHospitals and Clinics and Interfaculty Center of Pharmacogenomics and Pharmaceutical Research University of Tubingen 72074 Tubingen GermanybInstitute of Biochemistry and Molecular Biology II Heinrich Heine University Hospitals and Clinics 40225 Dusseldorf Germany cInstitute of Biochemistryand Genetics Deparment of Biomedicine University of Basel CH-4051 Basel Switzerland dInstitute of Pharmacology Free University of Berlin 14195 BerlinGermany and eRudolf Boehm Institute of Pharmacology and Toxicology University of Leipzig 04109 Leipzig Germany

Edited by Peter K Vogt The Scripps Research Institute La Jolla CA and approved October 6 2009 (received for review May 19 2009)

Class IB phosphoinositide 3-kinase (PI3K) elicits various immu-nologic and cardiovascular responses however the molecularbasis for this signal heterogeneity is unclear PI3K consists of acatalytic p110 and a regulatory p87PIKAP (p87 also p84) or p101subunit Hitherto p87 and p101 are generally assumed to exhibitredundant functions in receptor-induced and G protein (G)-mediated PI3K regulation Here we investigated the molecularmechanism for receptor-dependent p87p110 activation By an-alyzing GFP-tagged proteins expressed in HEK293 cells PI3K-complemented bone marrowndashderived mast cells (BMMCs) fromp110-- mice and purified recombinant proteins reconstituted tolipid vesicles we elucidated a novel pathway of p87-dependent Gproteinndashcoupled receptor (GPCR)-induced PI3K activation Al-though p101 strongly interacted with G thereby mediatingPI3K membrane recruitment and stimulation p87 exhibited onlya weak interaction resulting in modest kinase activation and lackof membrane recruitment Surprisingly Ras-GTP substituted themissing G-dependent membrane recruitment of p87p110 bydirect interaction with p110 suggesting the indispensability ofRas for activation of p87p110 Consequently interference withRas signaling indeed selectively blocked p87p110 but not p101p110 kinase activity in HEK293 and BMMC cells revealing animportant crosstalk between monomeric and trimeric G proteins forp87p110 activation Our data display distinct signaling require-ments of p87 and p101 conferring signaling specificity to PI3K thatcould open up new possibilities for therapeutic intervention

confocal life cell imaging G protein receptor signaling mast cells

Chemotaxis the release of antimicrobial molecules fromgranules or production of reactive oxygen species (ROS)

displays some of the most potent defense mechanisms of theimmune system These immune responses are under spatial andtemporal control of diverse stimuli including chemokines che-motactic peptides or nucleosides which typically signal throughthe G proteinndashcoupled receptor (GPCR)-dependent phospho-inositide 3-kinase (PI3K) (1) Thus gene-targeted micelacking the catalytic subunit of PI3K show severe defects inimmune response and are completely protected against systemicanaphylaxis (2ndash5) Moreover in models of rheumatoid arthritissystemic lupus erythematosus and atherosclerosis loss of PI3Kactivity resulted in partial or even complete protection againstdisease progression (6ndash8) Thus PI3K is considered a prom-ising drug target for the treatment of chronic inflammation andallergy (9 10) The vital role of PI3K is not restricted to theimmune system however it exhibits additional functions in thecardiovascular system (11ndash14) Although this reflects the abilityof PI3K to regulate multiple effects elucidating how signal-specificity is achieved may facilitate the design of strategies forselective pharmacologic interventions

To some degree specificity within PI3K-dependent signalingis accomplished by the existence of 4 class I PI3K isoforms all

of which are regulated by transmembrane receptors andor activeRas Class I PI3Ks are heterodimers composed of a catalyticp110 and a regulatory subunit and are further subdivided intoclass IA and class IB (15 16) Within the class IA PI3Ks 3 distinctcatalytic p110 isoforms contribute to heterogeneity and signal-specificity of PI3Ks and (17ndash21) Although class IA PI3Kis under the control of GPCRs and receptor tyrosine kinasesPI3K is considered to be exclusively GPCR-regulated and thusthe only member of class IB (11 19ndash28) PI3K is composed ofp110 and the regulatory p101 or the novel p87PIKAP (p87 alsocalled p84) subunit (29ndash32) The discovery of p87 opened thepossibility of conferring PI3K signal-specificity on the 2 regu-latory subunits but p87 is considered functionally identical top101 Very recently we demonstrated that p87p110 andp101p110 differ in their capability to regulate distinct ade-nosine-induced cellular functions (33) Although we correlateddistinct cell responses elicited by the 2 dimers with the formationof PtdIns (345)P3 pools at different membrane compartmentsthe regulatory mechanism conferring this signal-specificity re-mains unknown (31ndash34)

In the present study we demonstrate that p87 and p101 do notequivalently function as G adapters Furthermore we reportthat Ras is critical for distinct regulation of p87p110 andp101p110 arguing for a specific role of Ras in GPCR-controlled class IB PI3K signaling

ResultsfMLP Selectively Activates PI3K Heterodimers As a first steptoward deciphering a molecular mechanism for PI3K signal-specificity downstream of GPCRs we examined the contributionof p87 and p101 in this process PI3K activity was induced bystimulating a prototypical GPCR expressed in HEK cellsformyl-methionyl-leucyl-phenylalanine (fMLP) receptor andmonitored by confocal laser scanning microscopy using a GFP-fused PH domain PH-domain containing proteins bind to themajor product of PI3K-activity PtdIns (345)P3 resulting intheir plasma membrane translocation thereby providing a directreadout for PI3K activity in cells (30 31 35 36) As expected

Author contributions BK and BN designed research BK AS TB and Prajwal per-formed research BK AS Prajwal CB MRA MS AG and EJ contributed newreagentsanalytic tools BK AS TB MRA MPW EJ and BN analyzed data andBK CH MPW EJ and BN wrote the paper

The authors declare no conflicts of interest

This article is a PNAS Direct Submission

1Present address Splicos 34090 Montpellier France

2Present address Institute of Pharmacology and Toxicology and Rudolf Virchow DFGResearch Center for Experimental Biomedicine University of Wurzburg 97070 WurzburgGermany

3To whom correspondence should be addressed E-mail berndnuernberguni-tuebingende

This article contains supporting information online at wwwpnasorgcgicontentfull0905506106DCSupplemental

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solitarily expressed p110 showed no GPCR-induced activation[supporting information (SI) Fig S1 A Upper] whereas p101p110 was robustly activated (Fig S1 A Lower) (35) In contrastto p101p110 GPCR-induced stimulation of p87p110 led onlyto a half-maximal activation of the kinase (Fig S1 AndashC) (31)Similar results were obtained on direct stimulation of bothheterodimers with G (Fig 1A) We excluded differences inexpression levels of p87 and p101 and improper dimerization ofp87 and p110 (Fig S2 A and B) thus the results indicate thatthe fMLP receptor discriminates between the 2 PI3K dimers bymeans of distinct efficiencies of G to stimulate the dimers

To determine the specific effect of G on p87p110activity we compared G-induced stimulation of PI3Kheterodimers using purified proteins reconstituted to phos-pholipid vesicles (Figs 1 B and S2C) G moderately stim-ulated monomeric p110 (EC50 of 214 357 nM) whereasdimerization of p110 with p101 resulted in a dramatic in-crease in the sensitivity of p110 toward G (EC50 of 12 22 nM and 60-fold improved efficiency Fig 1B) Surprisinglyp87 failed to mediate an appropriate sensitization of p110toward G The potency of G to induce p110 activationwas not affected (EC50 223 497 nM) although it increasedthe efficiency slightly These results certainly call into questionthe current view of equal G-mediated regulatory mecha-nisms for p87p110 and p101p110 In addition comparingthe in vitro data with corresponding results obtained in cellsrevealed a clear discrepancy in p87p110 activity Althoughthe degree of activation of p87p110 and monomeric p110 byG did not differ in vitro (Fig 1B) both GPCR-induced andG-induced stimulation clearly resulted in significant activityof p87p110 but not of p110 in cells (Figs 1 A and S1A)Thus we postulated that an additional regulator is involved inGPCR-induced p87p110 activation in vivo

Endogenous GTP-Bound Ras is Indispensable for Activation of p87p110 The second known PI3K activator the small GTPase Rasstimulates PI3K through direct interaction with p110 (Fig S3AndashC) (37 38) Thus Ras represents an excellent candidate forcoregulating p87p110 activity To assess the impact of Ras onGPCR-induced p87p110 activation we used a p110 variant(referred to as p110K251A) known to exhibit reduced H-Rasbinding (37) Coimmunoprecipitation assays revealed a negligiblebinding of p110K251A to constitutive active H-RasG12V comparedwith wild-type p110 (Fig S3D) and thus H-RasG12V could stim-ulate p110K251A only marginally (Fig S3E) Using this variantfMLP-stimulated receptors almost completely failed to activatep87p110K251A whereas conversely the degree of fMLP-inducedp101p110K251A stimulation remained unchanged compared withthe wild-type heterodimer (Fig 2 A and B) In a second indepen-dent and complementary approach we overexpressed the GTPase-activating protein (GAP) domain of neurofibromin 1 (NF1) toneutralize endogenous Ras activity NF1 stimulates the GTPaseactivity of all p110-interacting Ras isoforms thereby retaining Rasin its GDP-bound inactive form (Figs S3F and S4C) (38ndash40)Although the fMLP-induced activation of p101p110 was unaf-fected by NF1 the p87p110 heterodimer failed to activate in thepresence of NF1 resembling the situation seen with p110K251A(Fig 2 A and C) Equivalent results were obtained using overex-pressed G to stimulate PI3K (Fig S4 A and B) while NF1activity was confirmed in parallel aliquots of the transfected HEKcells (Fig S3F) Because blockage of the Rasndashp110 interaction ledto a loss of fMLP-induced p87p110 activity but not of p101p110activity our data implicate that fMLP receptorndashinduced activationof p87p110 is mediated by Ras and G In contrast p101p110is sufficiently activated by G with an insignificant impact of Rasfor activation Thus Ras is an indispensable coactivator of thep87p110 heterodimer

GPCR-Induced p87p110 Activity Is Enhanced by Ras OverexpressionTo extend our findings we coexpressed wild-type H-Ras in HEKcells (Fig S5) fMLP-induced p87p110 activity was significantlyenhanced (Fig S5 Middle) although the basal activity was unaf-fected Furthermore the overexpression of wild-type Ras led to amaximal fMLP-induced activation of p87p110 equal to that ofp101p110 (Fig S5C) This indicates specific roles for G and Rasduring receptor-induced activation of p87p110 This approachdoes not allow clarification of how Ras and G mechanisticallycontribute to the activation of p87p110 however

p87 Fails To Serve as a G Recruitment Adapter for PI3K To tacklethis issue we used an established lipid vesicle pull-down assay (24)to gain deeper insight into G interaction with monomeric orheterodimeric p110 G recruited p87p110 to phospholipidvesicles only marginally compared with p101p110 (Fig 3A) Toestimate the affinity of p87 toward G we copurified both entitiesfrom baculovirus-infected Sf9 cells Only very little G wasassociated with p87 resembling the situation for p110 whereasG was strongly bound to p101 (Fig 3B) The weak interactionbetween p87 and G appears to be insufficient for membranerecruitment of PI3K in cells (Fig 3C) These observations dem-onstrate that the PI3K adapter protein p87PIKAP unlikely serves asan adapter for G-dependent recruitment

H-Ras Recruits p110 to Membranes The recruitment of the cytosolicPI3K to the plasma membrane which contains its regulators aswell as its substrate is mandatory for the activation of this enzymeThus we hypothesized that active Ras may represent the missinglink required for p87p110 membrane translocation Indeed con-stitutively active H-RasG12V but not dominant negative H-RasS17Ninduced a cellular redistribution of YFP-labeled p110 to mem-branes indicating that Ras-GTP is indeed capable of recruiting thelipid kinase in living cells (Fig 4A) Furthermore analysis of fixed

Fig 1 Activation of p87p110 and p101p110 by G (A) Membranerecruitment of GFP-Grp1PH in living cells HEK cells were transfected withplasmids encoding G PI3K (p110 p87p110 or p101p110) and GFP-Grp1PH as indicated above The localization of GFP-Grp1PH is shown in repre-sentative starved (18 h) HEK cells out of 3 independent experiments (cLSMslices of 08 m) p101p110 activity led to a more pronounced redistributionof the PH domain compared with p87p110 activity (Scale bar 10 m) Thehistogram represents the statistic evaluation of the membrane translocationof GFP-Grp1PH in the corresponding experiments The data represent themean SD of 3 independent experiments analyzing 18 cells (B) Purified PI3K

(ie p110 p87p110 and p101p110) stimulated with increasing concen-trations of G using PtdIns (45)P2 as a substrate 32P-labeled PtdIns (345)P3

was separated by thin-layer chromatography and quantified with a FujifilmFLA-5000 imaging system p101 but not p87 affects the potency with whichG activates p110 Data points and EC50 values represent the mean of 3independent experiments

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HEK cells revealed a colocalization of H-RasG12V and YFP-p110at cellular membranes (Fig 4B) In control cells we demonstratedH-Rasndashdependent recruitment of p87p110 and p101p110 butnot of solitarily expressed p87 or p101 to cell membranes suggest-ing that recruitment of p87p110 is facilitated through a directinteraction of Ras with the catalytic p110 subunit (Figs S3A andS6) To further support the conclusion that membrane recruitment

of p87p110 by Ras represents its prominent function we used apreviously generated p110CAAX mutant known to constitutivelylocalize p110 to membranes (Fig S2B) (35) Consistently coex-pressed NF1 did not blunt G-induced stimulation of p87p110CAAX activity (Fig 4C)

NF1 Selectively Inhibits Adenosine-Induced Activation of p87p110 inMurine Bone MarrowndashDerived Mast Cells (BMMCs) To validate ourfindings in more relevant cells we used BMMCs from p110 nullmice p110-- BMMCs are devoid of both PI3K catalytic andregulatory subunits enabling specific analysis of either p87p110 or p101p110 on their complementation (33 34) Atten-uation of Ras activity by the coexpression of NF1 in PI3K-reconstituted BMMCs led to a loss of adenosine-dependentphosphorylation of PKBAkt in cells expressing p87p110 butnot in cells expressing p101p110 (Fig 5)

DiscussionThe signaling specificity of class IA PI3Ks is supported by theexistence of 3 distinct catalytic subunits whereas a comparablesource for signaling specificity within the class IB PI3Ks whichincludes only 1 isoform (ie PI3K) is missing (1 17ndash21 41)Here we have demonstrated that Ras in concert with G andthe regulatory PI3K subunits provides a basis for PI3Ksignal-specificity This conclusion stems from 3 fundamental andunexpected findings In contradiction to current thinking (i)

Fig 2 Interaction of PI3K with Ras is indispensable for fMLP-inducedactivation of p87p110 (A) Membrane recruitment of GFP-Grp1PH in livingcells HEK cells were transfected with plasmids encoding fMLP-R PI3K (p110p87p110 p101p110 p87p110K251A or p101p110K251A) NF1 and GFP-Grp1PH as indicated above Shown are representative starved (18 h) HEK cells(cLSM slices of 08 m) out of 3 independent experiments before and after theaddition of fMLP (1 M 3 min) Comparison of the PI3K activities shows equalp101p110 and p101p110K251A activities whereas p87p110K251A activity issignificantly reduced compared with its wild-type p110 counterpart Consis-tently in the presence of NF1 p101p110 activity is unaltered whereasp87p110 activity is lost (Scale bar 10 m) (B) Kinetics of GFP-Grp1PH

redistribution in the cells shown in A (C) Histogram showing the statisticalevaluation of 6 cells out of 6 independent experiments as shown in (A) and bargraphs showing mean SD values of the redistributed fluorescence on fMLPtreatment (1 M 3 min)

Fig 3 p87 does not function as a G adapter for PI3K membrane recruit-ment (A)G wastestedfor itsability to recruitp110 p87p110 orp101p110

(each with 200 ng of p110) to phospholipid vesicles Aliquots of pelleted phos-pholipid vesicles and supernatants were subjected to SDSPAGE followed byimmunoblotting Chemiluminescence signals were documented with a CCD cam-era Data are given as mean SD of duplicate determinations in 3 independentexperiments (Lower) Immunoblots of 1 representative experiment are shown(Upper) Only p101p110 was relevantly translocated to phospholipids by G(B) G was coexpressed with His-p110 His-p101 or His-p87 in Sf9 cells andpurified Following purification on Ni2-NTA Superflow resin bound proteinswere separated by SDSPAGE and analyzed by immunoblotting The amount ofG copurified with p110 and p87 is comparable whereas that copurified withp101 was higher (C) Subcellular distribution of fluorescently labeled PI3K sub-units HEK cells were transfected with plasmids encoding G and N-terminally orC-terminallyYFP-taggedPI3K subunits (p110p101orp87)as indicatedaboveThe localization of YFP-labeled PI3K subunits in representative starved (18 h)HEK cells (cLSM slices of 08 m) out of 3 independent experiments is shown Onlyp101 is sufficiently translocated to the plasma membrane by G (Scale bar 10m)

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interaction of p87 with G differs significantly from that ofp101 (ii) Ras is able to recruit p110 to membranes and (iii) Rasacts as an indispensable coregulator of p87p110

p87 Fails to Substitute for p101 Most surprising was our observationthat p87 failed to sensitize p110 for G-dependent activation andto function as a recruitment adapter These results were particularlyamazing because we discovered p87 as a homolog of p101 withsignificant sequence similarities not only within the predictedN-terminal p110 but also within the putative C-terminal G-binding region of p101 (30) In fact based on the significant degreeof homology within the G-binding region of p87 and p101 incombination with the initial results of a similar GPCR- or G-induced activation of p87p110 and p101p110 we and othersinitially assumed that p87 functioned as a second G-recognizingrecruitment adapter (31 32 34) However a sequence similarity ofonly 24 a lower degree of similarity within the putative G-

Fig 4 Active H-Ras recruits p110 to cell membranes (A) Subcellular distribu-tion of fluorescently labeled p110 in living cells HEK cells were transfected withplasmids encoding H-Ras (H-RasG12V or H-RasS17N) and YFP-tagged p110 asindicated above The localization of YFP-labeled p110 subunits in representa-tive starved (18 h) HEK cells (cLSM slices of 08 m) out of 3 independentexperiments is shown p110 translocated to the plasma membrane in the pres-ence of H-RasG12V (Scale bar 10 m) (B) Colocalization of fluorescently labeledp110 and immunostained H-Ras HEK cells were transfected with plasmidsencoding H-Ras (H-RasG12V or H-RasS17N) and YFP-tagged p110 as indicatedabove The localization of YFP-labeled p110 (yellow) and immunostained H-Ras(red) is shown in representative fixed HEK cells from 3 independent experiments(cLSM slices of 08 m) p110 colocalized at the plasma membrane withH-RasG12V (Scale bar 10 m) (C) Membrane recruitment of GFP-Grp1PH in livingcells HEK cells were transfected with plasmids encoding G PI3K (p87p110

or p87p110CAAX) GFP-Grp1PH and NF1 as indicated above Shown are repre-sentative starved (18 h) HEK cells (cLSM slices of 08 m) from 4 independentexperiments Unlike p87p110 activity p87p110CAAX activity was un-changed in the presence of NF1 (Scale bar 10 m) The right panels presentthe quantification of the membrane translocation of GFP-Grp1PH in the cor-responding experiments The data represent the mean SD of 4 independentexperiments analyzing 24 cells

Fig 5 Overexpressed NF1 blunts activation of p87p110 but not p101p110 in murine BMMCs (A) PKBAkt phosphorylation (pThr308 and pSer473)in BMMCs p110 and HA-p87 or HA-p101 were coexpressed with or withoutFlag-NF1 in p110 null BMMCs Transfected BMMCs were starved for 3 h andthen stimulated with 2 M adenosine (Ade) or 10 ngmL of murine stem cellfactor (SCF) for 2 min Expression of proteins was verified NF1-inducedattenuation of Ras activity was validated using anti-phosphoMAPK (pMAPK)antibodies Adenosine-induced p101p110 PKB phosphorylation was un-changed whereas p87p110 PKB phosphorylation was depleted in the pres-ence of NF1 At the same time the phosphorylation of MAPK triggered byadenosine or SCF was affected by NF1 (B) Quantification of adenosine-triggered phosphorylation of PKB (on pSer473 and pThr308) and pMAPK fromexperiments as shown in (A) Values from NF1 cotransfected cells are ex-pressed as percent of control (p110 null BMMCs reconstituted with theindicated PI3K complexes) Data are given as mean SEM (pSer473 n 5pThr308n 3 pMAPK n 4) P 02

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binding region compared with the predicted p110-binding regionof p101 and p87 and an attenuated activation of p87p110 byGPCRs or G (ref 31 and this study) prompted us to reconsiderour initial assumption We compared the function of p87p110 notonly with that of p101p110 but also with that of monomericp110 We found only a faint interaction of p87 with G in vitroaccounting for the inability of p87 to mediate G-induced mem-brane recruitment in cells as well as for the missing sensitization ofp110

Ras Has a Recruiting Effect on PI3K Our study demonstrates thatin living cells interaction of Ras with the catalytic p110 subunitprovokes membrane recruitment of p110 p87p110 or p101p110 This finding is striking because the Ras-dependentrecruitment of p110 has been a subject of intense debateAttempts to prove Ras-dependent p110 membrane recruitmenthave failed although membrane localization of Ras-GTP hasbeen shown to be necessary for its activating impact on p110(38) Contrarily genetic approaches suggest that p110-dependent cell mutagenesis is essentially regulated by Ras-driven membrane recruitment of p110 (42) We cannot excludethe possibility that previous attempts to directly show Ras-dependent p110 membrane recruitment might have failedbecause of methodologic limitations To avoid this we analyzedintact living cells and indeed observed a Ras-dependent mem-brane accumulation of p110 due to direct interaction Certainlyour result is supported by the concept that a membrane-boundstimulator recruits its cytosolic effector to bring it in closecontact with its substrate Furthermore our result is consistentwith studies demonstrating membrane translocation as a majorfunction of Ras on other effectors (43 44) Thus our data extendthe current knowledge of Ras action on PI3K showing that Rashas both activating and membrane-recruiting effects on p110

PI3K Heterodimers Are Specifically Regulated by GPCRs Finally wehave shown that active Ras acts as an indispensable costimulatorof p87p110 whereas G is sufficient for activation of p101p110 This conclusion is supported by 2 independent andcomplementary approaches first by interfering with Ras bindingby modification of p110 and second by modulating Ras activityBoth diminished the regulatory impact of Ras while leavingG-dependent regulation untouched Furthermore these re-sults are consistent with the observation that p87 failed to berecruited by G but instead was translocated by Ras viainteraction with p110 In addition using a constitutively mem-brane-bound p110 mutant we could strengthen our hypothesisthat recruitment represents the predominant function of Ras inthe activation of p87p110

Based on our data we have proposed a model for distinctregulatory mechanisms of GPCR-induced activation of the 2PI3K heterodimers (Fig 6) In this scenario p87p110 isspecifically regulated by Ras and G in an orchestrated fashionwhere Ras is indispensable for membrane recruitment and thusactivation of the lipid kinase In contrast p101p110 is essen-tially and sufficiently recruited and stimulated by G Thusdistinct interactions of the 2 PI3K heterodimers p101p110and p87p110 with their upstream regulators define specificregulation of these heterodimers

This instantly raises questions about this modelrsquos physiologicalrelevance Interestingly it has been recently proposed that Rasand G can simultaneously but differentially regulate distinctPI3K effectors on activation by the same GPCR in neutrophils(45) Essentially the data from that study imply that most of theGPCR-induced and PI3K-regulated cellular effects in neutro-phils are mediated via the G adapter p101 or via Ras bindingwhereas the GPCR-induced production of ROS is mediatedexclusively via Ras binding to p110 In addition we haverecently reported that adenosine-induced activation of the

2 PI3K heterodimers addresses distinct cellular effects inBMMCs (33) The underlying regulatory mechanisms for thisremain unclear however (34) Here we provide evidence thatRas may contribute to the differential coupling of PI3K het-erodimers to downstream cell responses It may be speculatedthat this is accomplished by directing the 2 PI3K heterodimersto different membrane compartments by Ras (46 47)

Taken together our findings point to a unique and indispensablerole of Ras for the GPCR-induced PI3K-dependent production ofROS as well as degranulation of mast cells Thus it is reasonableto speculate that ROS production and degranulation in contrast toother PI3K-dependent cellular effects may be specifically regu-lated by Ras and p87p110 heterodimer (Fig 6)

ConclusionIn conclusion we have identified distinct regulatory mechanismsupstream of the 2 PI3K dimers p87p110 and p101p110conferring isoform specificity within class IB PI3Ks A promisingfuture aim is to extend the assignment of these 2 PI3Kheterodimers to distinct cellular functions This could lead to thedevelopment of pharmacologic strategies for more specific in-tervention in pathophysiologically relevant signaling pathways

Experimental ProceduresConstruction of Expression Plasmids The p87 expression plasmids were gener-ated using mouse full-length cDNA (German Science Centre for Genome Re-search) Table S1 lists the primers restriction enzymes and vectors used for thecloning of p87 p110 YFP-H-Ras H-RasG12V and the Ras-GAP domain of NF1constructs H-Ras-wt and H-RasS17N in pcDNA3 as well as templates for theconstruction of YFP-H-Ras and H-RasG12V were generous gifts from M Schmidt

Fig 6 Hypothetical model of regulatory mechanisms of PI3K het-erodimers The scheme illustrates the activation processes of p87p110 (Left)and p101p110 (Right) Interaction of p101 with G is sufficient to translo-cate p101p110 to the plasma membrane as a prerequisite for its activationIn contrast p87p110 is not translocated to the plasma membrane by Ginstead translocation of p87p110 is accomplished by the interaction ofp110 with Ras-GTP Based on the requirement of Ras-dependent recruitmentof p87p110 Ras constitutes an indispensable activator in the activationprocess of this specific isoform Following recruitment of PI3K both p87p110 and p101p110 are allosterically activated Consistent with the presentknowledge these distinct mechanisms might be the basis for the specificregulation of different PI3K-dependent cellular effects (see the text fordetails) The data presented here provide evidence of differential regulatorymechanisms of the 2 PI3K heterodimers

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binding region compared with the predicted p110-binding regionof p101 and p87 and an attenuated activation of p87p110 byGPCRs or G (ref 31 and this study) prompted us to reconsiderour initial assumption We compared the function of p87p110 notonly with that of p101p110 but also with that of monomericp110 We found only a faint interaction of p87 with G in vitroaccounting for the inability of p87 to mediate G-induced mem-brane recruitment in cells as well as for the missing sensitization ofp110

Ras Has a Recruiting Effect on PI3K Our study demonstrates thatin living cells interaction of Ras with the catalytic p110 subunitprovokes membrane recruitment of p110 p87p110 or p101p110 This finding is striking because the Ras-dependentrecruitment of p110 has been a subject of intense debateAttempts to prove Ras-dependent p110 membrane recruitmenthave failed although membrane localization of Ras-GTP hasbeen shown to be necessary for its activating impact on p110(38) Contrarily genetic approaches suggest that p110-dependent cell mutagenesis is essentially regulated by Ras-driven membrane recruitment of p110 (42) We cannot excludethe possibility that previous attempts to directly show Ras-dependent p110 membrane recruitment might have failedbecause of methodologic limitations To avoid this we analyzedintact living cells and indeed observed a Ras-dependent mem-brane accumulation of p110 due to direct interaction Certainlyour result is supported by the concept that a membrane-boundstimulator recruits its cytosolic effector to bring it in closecontact with its substrate Furthermore our result is consistentwith studies demonstrating membrane translocation as a majorfunction of Ras on other effectors (43 44) Thus our data extendthe current knowledge of Ras action on PI3K showing that Rashas both activating and membrane-recruiting effects on p110

PI3K Heterodimers Are Specifically Regulated by GPCRs Finally wehave shown that active Ras acts as an indispensable costimulatorof p87p110 whereas G is sufficient for activation of p101p110 This conclusion is supported by 2 independent andcomplementary approaches first by interfering with Ras bindingby modification of p110 and second by modulating Ras activityBoth diminished the regulatory impact of Ras while leavingG-dependent regulation untouched Furthermore these re-sults are consistent with the observation that p87 failed to berecruited by G but instead was translocated by Ras viainteraction with p110 In addition using a constitutively mem-brane-bound p110 mutant we could strengthen our hypothesisthat recruitment represents the predominant function of Ras inthe activation of p87p110

Based on our data we have proposed a model for distinctregulatory mechanisms of GPCR-induced activation of the 2PI3K heterodimers (Fig 6) In this scenario p87p110 isspecifically regulated by Ras and G in an orchestrated fashionwhere Ras is indispensable for membrane recruitment and thusactivation of the lipid kinase In contrast p101p110 is essen-tially and sufficiently recruited and stimulated by G Thusdistinct interactions of the 2 PI3K heterodimers p101p110and p87p110 with their upstream regulators define specificregulation of these heterodimers

This instantly raises questions about this modelrsquos physiologicalrelevance Interestingly it has been recently proposed that Rasand G can simultaneously but differentially regulate distinctPI3K effectors on activation by the same GPCR in neutrophils(45) Essentially the data from that study imply that most of theGPCR-induced and PI3K-regulated cellular effects in neutro-phils are mediated via the G adapter p101 or via Ras bindingwhereas the GPCR-induced production of ROS is mediatedexclusively via Ras binding to p110 In addition we haverecently reported that adenosine-induced activation of the

2 PI3K heterodimers addresses distinct cellular effects inBMMCs (33) The underlying regulatory mechanisms for thisremain unclear however (34) Here we provide evidence thatRas may contribute to the differential coupling of PI3K het-erodimers to downstream cell responses It may be speculatedthat this is accomplished by directing the 2 PI3K heterodimersto different membrane compartments by Ras (46 47)

Taken together our findings point to a unique and indispensablerole of Ras for the GPCR-induced PI3K-dependent production ofROS as well as degranulation of mast cells Thus it is reasonableto speculate that ROS production and degranulation in contrast toother PI3K-dependent cellular effects may be specifically regu-lated by Ras and p87p110 heterodimer (Fig 6)

ConclusionIn conclusion we have identified distinct regulatory mechanismsupstream of the 2 PI3K dimers p87p110 and p101p110conferring isoform specificity within class IB PI3Ks A promisingfuture aim is to extend the assignment of these 2 PI3Kheterodimers to distinct cellular functions This could lead to thedevelopment of pharmacologic strategies for more specific in-tervention in pathophysiologically relevant signaling pathways

Experimental ProceduresConstruction of Expression Plasmids The p87 expression plasmids were gener-ated using mouse full-length cDNA (German Science Centre for Genome Re-search) Table S1 lists the primers restriction enzymes and vectors used for thecloning of p87 p110 YFP-H-Ras H-RasG12V and the Ras-GAP domain of NF1constructs H-Ras-wt and H-RasS17N in pcDNA3 as well as templates for theconstruction of YFP-H-Ras and H-RasG12V were generous gifts from M Schmidt

Fig 6 Hypothetical model of regulatory mechanisms of PI3K het-erodimers The scheme illustrates the activation processes of p87p110 (Left)and p101p110 (Right) Interaction of p101 with G is sufficient to translo-cate p101p110 to the plasma membrane as a prerequisite for its activationIn contrast p87p110 is not translocated to the plasma membrane by Ginstead translocation of p87p110 is accomplished by the interaction ofp110 with Ras-GTP Based on the requirement of Ras-dependent recruitmentof p87p110 Ras constitutes an indispensable activator in the activationprocess of this specific isoform Following recruitment of PI3K both p87p110 and p101p110 are allosterically activated Consistent with the presentknowledge these distinct mechanisms might be the basis for the specificregulation of different PI3K-dependent cellular effects (see the text fordetails) The data presented here provide evidence of differential regulatorymechanisms of the 2 PI3K heterodimers

20316 wwwpnasorgcgidoi101073pnas0905506106 Kurig et al

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and K Giehl respectively The construction of all of the other plasmids has beendescribed elsewhere (25 31 35)

Cell Culture Transfection and Infection HEK cells (German Resource Centre forBiological Material) were grown and transfected as described previously (3035 36) Fall armyworm ovary cells (Sf9 cells Invitrogen) were cultured andinfected as described previously (25 28 48)

Immunofluorescence and Fluorescence Imaging Immunofluorescence stainingwas carried out as detailed previously (36) with minor modifications (see SIText) Cell imaging was performed as described previously using a Zeiss LSM510-META confocal laser scanning microscope (30 35 36)

Analysis of GFP-Grp1PH Translocation The subcellular distribution of GFP-Grp1PH was evaluated as detailed elsewhere (36) Significance was assessedusing the paired Student t-test with P 05 P 01 and P 005

Purification and Copurification of Recombinant G12 and PI3K From Sf9 CellsThe purification of recombinant G12 and PI3K has been detailed elsewhere

(25 28 48) Copurification was performed as described previously (28) withmodifications (see SI Text)

Phospholipid Vesicle Pull-Down Assay Determination of G12 and PI3K

association on phospholipid vesicles was performed as described previously(24) with modifications (see SI Text)

BMMC Cell Culture Isolation Differentiation Nucleofection and StimulationBMMC cell culture isolation differentiation nucleofection and stimulationfrom PI3K null mice (2) was performed as described previously (33) withmodifications (see SI Text)

ACKNOWLEDGMENTS We thank Wibke Ballhorn Julie Mason and Ilse Meyerfor technical assistance BK thanks Oleg Fedorchenko for assistance with thecloning of p87 We are grateful to Dr Martina Schmidt and Dr Peter Gierschikrsquoslaboratory for providing experimental tools Valuable discussions with Drs PhilHawkins Klaus Schulze-Osthoff Len Stephens Roger Williams and colleaguesfrom our department are greatly appreciated This work was supported by theDeutsche Forschungsgemeinschaft the Swiss National Science Foundation(Grants 31EM30-126143 and 310030127574) and a Keystone Symposia Scholar-ship (from National Institutes of Health Grant 1R13CA139718-01 to BK)

1 Rommel C Camps M Ji H (2007) PI3K and PI3K Partners in crime in inflammation inrheumatoid arthritis and beyond Nat Rev Immunol 7191ndash201

2 Hirsch E et al (2000) Central role for G proteinndashcoupled phosphoinositide 3-kinase

in inflammation Science 2871049ndash10533 Sasaki T et al (2000) Function of PI3K in thymocyte development T cell activation

and neutrophil migration Science 2871040ndash10464 Laffargue M et al (2002) Phosphoinositide 3-kinase is an essential amplifier of mast

cell function Immunity 16441ndash4515 Wymann MP et al (2003) Phosphoinositide 3-kinase A key modulator in inflamma-

tion and allergy Biochem Soc Trans 31275ndash2806 Camps M et al (2005) Blockade of PI3K suppresses joint inflammation and damage

in mouse models of rheumatoid arthritis Nat Med 11936ndash9437 Barber DF et al (2005) PI3K inhibition blocks glomerulonephritis and extends lifes-

pan in a mouse model of systemic lupus Nat Med 11933ndash9358 Chang JD et al (2007) Deletion of the phosphoinositide 3-kinase p110 gene atten-

uates murine atherosclerosis Proc Natl Acad Sci USA 1048077ndash80829 Ruckle T Schwarz MK Rommel C (2006) PI3K inhibition Towards an lsquolsquoaspirin of the

21st centuryrsquorsquo Nat Rev Drug Discov 5903ndash91810 Marone R Cmiljanovic V Giese B Wymann MP (2008) Targeting phosphoinositide

3-kinase Moving towards therapy Biochim Biophys Acta 1784159ndash18511 Hirsch E et al (2006) Signaling through PI3K A common platform for leukocyte

platelet and cardiovascular stress sensing Thromb Haemost 9529ndash3512 Patrucco E et al (2004) PI3K modulates the cardiac response to chronic pressure

overload by distinct kinase-dependent and -independent effects Cell 118375ndash38713 Hirsch E et al (2001) Resistance to thromboembolism in PI3K-deficient mice FASEB

J 152019ndash202114 Macrez N et al (2001) Phosphoinositide 3-kinase isoforms selectively couple receptors

to vascular L-type Ca2 channels Circ Res 89692ndash69915 Hawkins PT Anderson KE Davidson K Stephens LR (2006) Signaling through Class I

PI3Ks in mammalian cells Biochem Soc Trans 34647ndash66216 Fruman DA Bismuth G (2009) Fine-tuning the immune response with PI3K Immunol

Rev 228253ndash27217 Okkenhaug K Ali K Vanhaesebroeck B (2007) Antigen receptor signaling A distinc-

tive role for the p110 isoform of PI3K Trends Immunol 2880ndash8718 Graupera M et al (2008) Angiogenesis selectively requires the p110 isoform of PI3K

to control endothelial cell migration Nature 453662ndash66619 Guillermet-Guibert J et al (2008) The p110 isoform of phosphoinositide 3-kinase

signals downstream of G proteinndashcoupled receptors and is functionally redundantwith p110 Proc Natl Acad Sci USA 1058292ndash8297

20 Ciraolo E et al (2008) Phosphoinositide 3-kinase p110 activity Key role in metabo-lism and mammary gland cancer but not development Sci Signal 1ra3

21 Jia S et al (2008) Essential roles of PI3K-p110 in cell growth metabolism andtumorigenesis Nature 454776ndash779

22 Stoyanov B et al (1995) Cloning and characterization of a G proteinndashactivated humanphosphoinositide-3 kinase Science 269690ndash693

23 Kurosu H et al (1997) Heterodimeric phosphoinositide 3-kinase consisting of p85 andp110 is synergistically activated by the subunits of G proteins and phosphotyrosylpeptide J Biol Chem 27224252ndash24256

24 Maier U et al (2000) G is a highly selective activator of phospholipid-dependentenzymes J Biol Chem 27513746ndash13754

25 Czupalla C et al (2003) Identification and characterization of the autophosphoryla-tion sites of phosphoinositide 3-kinase isoforms and J Biol Chem 27811536ndash11545

26 Tang X Downes CP (1997) Purification and characterization of G-responsive phos-phoinositide 3-kinases from pig platelet cytosol J Biol Chem 27214193ndash14199

27 Hawkins PT Stephens LR (2007) PI3K is a key regulator of inflammatory responses andcardiovascular homeostasis Science 31864ndash66

28 Maier U Babich A Nurnberg B (1999) Roles of non-catalytic subunits in G-inducedactivation of class I phosphoinositide 3-kinase isoforms and J Biol Chem 27429311ndash29317

29 Stephens LR et al (1997) The G sensitivity of a PI3K is dependent upon a tightlyassociated adaptor p101 Cell 89105ndash114

30 Voigt P Brock C Nurnberg B Schaefer M (2005) Assigning functional domains withinthe p101 regulatory subunit of phosphoinositide 3-kinase J Biol Chem 2805121ndash5127

31 Voigt P Dorner MB Schaefer M (2006) Characterization of p87PIKAP a novel regulatorysubunit of phosphoinositide 3-kinase gamma that is highly expressed in heart andinteracts with PDE3B J Biol Chem 2819977ndash9986

32 Suire S et al (2005) p84 a new G-activated regulatory subunit of the type IBphosphoinositide 3-kinase p110 Curr Biol 15566ndash570

33 Bohnacker T et al (2009) PI3K adaptor subunits define coupling to degranulation andcell motility by distinct PtdIns(345)P3 pools in mast cells Sci Signal 2ra27

34 Balla T (2009) Finding partners for PI3K When 84 is better than 101 Sci Signal 2e3535 Brock C et al (2003) Roles of G in membrane recruitment and activation of

p110p101 phosphoinositide 3-kinase J Cell Biol 16089ndash9936 Preuss I Kurig B Nurnberg B Orth JH Aktories K (2009) Pasteurella multocida toxin

activates G dimers of heterotrimeric G proteins Cell Signal 21551ndash55837 Pacold ME et al (2000) Crystal structure and functional analysis of Ras binding to its

effector phosphoinositide 3-kinase Cell 103931ndash94338 Suire S Hawkins P Stephens L (2002) Activation of phosphoinositide 3-kinase by Ras

Curr Biol 121068ndash107539 Ahmadian MR Hoffmann U Goody RS Wittinghofer A (1997) Individual rate constants

for the interaction of Ras proteins with GTPase-activating proteins determined byfluorescence spectroscopy Biochemistry 364535ndash4541

40 Rodriguez-Viciana P Sabatier C McCormick F (2004) Signaling specificity by Ras familyGTPases is determined by the full spectrum of effectors they regulate Mol Cell Biol244943ndash4954

41 Hirsch E Costa C Ciraolo E (2007) Phosphoinositide 3-kinases as a common platform formulti-hormone signaling J Endocrinol 194243ndash256

42 Zhao L Vogt PK (2008) Class I PI3K in oncogenic cellular transformation Oncogene275486ndash5496

43 Song C et al (2001) Regulation of a novel human phospholipase C PLC throughmembrane targeting by Ras J Biol Chem 2762752ndash2757

44 Stokoe D Macdonald SG Cadwallader K Symons M Hancock JF (1994) Activation ofRaf as a result of recruitment to the plasma membrane Science 2641463ndash1467

45 Suire S et al (2006) Gs and the Ras binding domain of p110 are both importantregulators of PI3K signalling in neutrophils Nat Cell Biol 81303ndash1309

46 Kranenburg O Verlaan I Moolenaar WH (2001) Regulating c-Ras function Cholesteroldepletion affects caveolin association GTP loading and signaling Curr Biol 111880ndash1884

47 Furuchi T Anderson RG (1998) Cholesterol depletion of caveolae causes hyperactiva-tion of extracellular signalndashrelated kinase (ERK) J Biol Chem 27321099ndash21104

48 Shymanets A Ahmadian MR Nurnberg B (2009) G-copurified lipid kinase impurityfrom Sf9 cells Protein Pept Lett 161053ndash1056

Kurig et al PNAS December 1 2009 vol 106 no 48 20317

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