Molecular Cell 21, 495–507, February 17, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.01.008

Structural and Mechanistic Insightsinto Ras Association Domainsof Phospholipase C Epsilon

Tom D. Bunney,1 Richard Harris,3

Natalia Lamuno Gandarillas,1 Michelle B. Josephs,1

S. Mark Roe,2 S. Caroline Sorli,1 Hugh F. Paterson,1

Fernando Rodrigues-Lima,4 Diego Esposito,5

Chris P. Ponting,6 Peter Gierschik,7

Laurence H. Pearl,2 Paul C. Driscoll,3,5

and Matilda Katan1,*1Cancer Research UK Centre for Cell and Molecular

Biology2Structural BiologyChester Beatty LaboratoriesThe Institute of Cancer ResearchFulham RoadLondon SW3 6JBUnited Kingdom3Department of Biochemistry and Molecular BiologyUniversity College LondonGower StreetLondon WC1E 6BTUnited Kingdom4Laboratoire de Cytophysiologie et Toxicologie

Cellulaire andUFR de BiochimieUniversite Denis Diderot-Paris 775005 ParisFrance5National Institute for Medical ResearchThe RidgewayMill HillLondon NW7 1AAUnited Kingdom6MRC Functional Genetics UnitDepartment of Human Anatomy and GeneticsUniversity of OxfordOxford OX1 3QXUnited Kingdom7Department of Pharmacology and ToxicologyUniversity of Ulm89069 UlmGermany

Summary

Ras proteins signal to a number of distinct pathways

by interacting with diverse effectors. Studies of ras/effector interactions have focused on three classes,

Raf kinases, ral guanylnucleotide-exchange factors,and phosphatidylinositol-3-kinases. Here we describe

ras interactions with another effector, the recently

identified phospholipase C epsilon (PLC3). We solvedstructures of PLC3 RA domains (RA1 and RA2) by NMR

and the structure of the RA2/ras complex by X-raycrystallography. Although the similarity between ubiq-

uitin-like folds of RA1 and RA2 proves that they are ho-mologs, only RA2 can bind ras. Some of the features of

the RA2/ras interface are unique to PLC3, while theability to make contacts with both switch I and II re-

*Correspondence: matilda.katan@icr.ac.uk

gions of ras is shared only with phosphatidylinositol-3-kinase. Studies of PLC3 regulation suggest that, in

a cellular context, the RA2 domain, in a mode specificto PLC3, has a role in membrane targeting with further

regulatory impact on PLC activity.

Introduction

The elucidation of ras interaction with its effectors hasbeen a topic of intense study for over 10 years (Down-ward, 2003; Herrmann, 2003). Phospholipase C epsilon(PLC3) is a relatively recent addition to the growing listof such effectors (Mitin et al., 2005). While most of thesuggested ras effectors have either kinase or guanylnu-cleotide exchange factor (GEF) activity, through inter-action with PLC3, ras has been implicated in regulatinganother class of enzyme, phosphoinositide-specific PLC,involved in the generation of well-characterized secondmessengers (Rhee, 2001).

Database searches have revealed that PLC3, in com-mon with other PLC families, incorporates the PLC cat-alytic domain and C2 domain; the presence of a PHdomain and EF hands has also been suggested (Winget al., 2003). Within the unique regions, PLC3 has a GEFdomain and two RA domains, the latter representing ras-association domain homologs present also in some raseffectors (Ponting and Benjamin, 1996). These uniquestructural elements among PLC enzymes suggest regu-latory links with ras-family GTPases. However, subse-quent studies indicated multiple regulatory mechanismsthat could be triggered by diverse stimuli (Mitin et al.,2005; Wing et al., 2003). Nevertheless, studies focusingon PLC3 interaction with ras demonstrate that PLC3 ful-fils most criteria necessary to be considered as an effec-tor of ras. For example, studies of the second RA domain(RA2) of PLC3 have shown its direct interaction with sev-eral ras family members in a GTP bound form (Kelleyet al., 2001, 2004; Song et al., 2001; Wohlgemuth et al.,2005). It was also shown that PLC activity of PLC3 canbe stimulated by activated ras when both moleculeswere introduced into COS cells and in a reconstitutionsystem in vitro (Kelley et al., 2001; Song et al., 2001). Fur-thermore, involvement of PLC3 in ras-mediated develop-ment of skin tumors in mice has been demonstrated (Baiet al., 2004). Using mice where PLC3 is geneticallymanipulated, it has been shown that mice lacking PLC3PLC activity exhibit delayed and reduced onset of skinpapillomas that fail to undergo malignant progressionto carcinomas.

Structurally characterized ras effectors include Rafprotein kinases, GEFs active on the ralA GTPase(RalGDS, Rgl, and Rlf), and phosphatidylinositol-3-ki-nase g (PI3Kg). Studies of isolated domains that interactwith ras from Raf kinase, Raf kinase homolog from yeast(Byr2), RalGDS, and closely related Rgl and Rlf havebeen performed using both NMR and X-ray crystallogra-phy (references in Herrmann [2003]). In the case of thePI3Kg, the structure of this domain was solved in thecontext of the full-length protein (Pacold et al., 2000).Structural studies of protein complexes with GTPases

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from the ras family, Hras and rap1A, revealed that thecommon ubiquitin fold from the effectors binds rasGTPases in a similar mode. However, while the overallstructures are similar, interaction surfaces are distinct,with important functional consequences. Independentlyof these structural studies, unique properties of ras/ef-fector interactions have been revealed by mutationalstudies of ras where different mutations within the effec-tor binding region selectively affect interactions withRalGDS, Raf kinase, and PI3K (Rodriguez-Viciana et al.,1997). This selectivity of ras mutants, although partial,has been exploited in studies aimed at identifying effec-tor molecules mediating different biological functions ofras (Cambell and Der, 2004). With identification of ras ef-fectors such as PLC3, it is important to understand theunique properties of this interaction that could facilitatedevelopment of better approaches to selectively uncou-ple different effectors. Another important aspect of un-derstanding ras/effector interactions is the elucidationof the different consequences that these interactionscould have for overall regulation of effector function.Presently, there are only a few studies that have attemp-ted to test two general concepts of ras/effector inter-actions supporting the role of ras as either primarily (1)involved in effector targeting to specific subcellularcompartments or (2) as the regulator of an effector activ-ity through conformational changes (Linnemann et al.,2002; Suire et al., 2002; Terai and Matsuda, 2005). Fur-ther studies of each of the ras effectors are needed toassess to what extent these two possible consequencesof ras binding contribute to its regulation. With theseaims in mind, here we describe structures of PLC3 RAdomains and functional studies revealing their role inthe enzyme regulation.

Results and Discussion

PLC3 RA Domains Form Independent Ubiquitin-likeFolds that Are Likely the Result of DNA Duplication

Database searches have previously identified a numberof open reading frames containing ubiquitin-like folds(Ponting and Benjamin, 1996). Initially classified as RB(ras binding) or RA (RalGDS/AF-6, ras-associating) do-mains, about 108 RA and 20 RB are currently listed inthe SMART database. However, only a subset of thesedomains is likely to form a productive interface with ras.To reflect a possible functional diversity of this commonfold, it has been recently suggested that the ubiquitin-like fold should be designated as the UB domain (Wohl-gemuth et al., 2005). For consistency with previousstudies on PLC3, however, we shall retain the originaldesignation as RA1 and RA2 domains (Kelley et al.,2001; Song etal., 2001). Functional studies have classifiedRA2 as binding ras in a GTP-dependent way. AlthoughRA1 was initially reported to bind ras in a nucleotide-independent fashion (Kelley et al., 2001), subsequentstudies suggest that it may not interact with ras (Wohl-gemuth et al., 2005).

To obtain structural insights into PLC3 RA domainsand elucidate structural determinants that underlie theirdifferent functional properties, we analyzed RA1 andRA2 proteins by 2D heteronuclear NMR spectroscopy.Initial analysis of the 15N labeled RA1(WT) and RA2(WT)demonstrated that only the spectrum of RA1(WT) pos-

sesses idealcharacteristicsconsistentwithamonomericglobular fold. The spectra of monomeric RA2(WT) at lowconcentrations, millimolar RA2 with point mutationsR2150L and Y2176L, and the RA2(WT) subspectrumwithin a monomeric RA1RA2(WT) tandem domain pairhave similar crosspeak numbers and an essentially iden-tical pattern of chemical shift dispersion (data notshown). These results indicate that RA2(WT) has a ten-dency toward self association but that R2150L andY2176L mutations, corresponding to residues predictedto be surface exposed, produce little if any perturba-tion to the overall structure of the monomeric RA2 do-main. 13C,15N-isotope-labeled samples of RA1(WT) andRA2(R2150L) were prepared, and full resonance assign-ments were obtained using triple resonance NMRmethods. On the basis of the analysis of 3D 15N- and13C-separated 1H-NOESY spectra, we determined the3D solution structures of these proteins. The lowest-energy structure and a best-fit superposition of thebackbone atoms of the 20 lowest-energy conformersare shown in Figure 1, demonstrating ubiquitin-like foldsof RA1 and RA2.

PLC3 RA domains share little sequence similarity withthe other ras binding domains, including those fromCraf, RalGDS, Byr2, and PI3Kg, for which 3D structuresare known (Figure 1C). Nevertheless, the various RA andRB domains can be aligned on the basis of their well-conserved secondary structure elements. The domainsexhibit remarkable similarity of the fold, as illustratedby the ribbon representations of RA1, RA2, CrafRBD,RalGDSRA, and PI3KgRBD structures; the arrange-ments of b1, b2, and a1, known to contribute to the rasbinding surface in Craf, RalGDS, and PI3Kg, are alsosimilar (Figure 1D). One notable difference between thePLC3 RA domains and the other RA/RBDs, however, isthe length of the loops between the secondary structureelements. In particular, in RA1 and RA2, the loop be-tween b3 and b4 is much longer and quite flexible, whilethe larger a1-b3 loop in RA2 is similar to the correspond-ing region only in PI3KgRBD. Further comparison of sur-face charge distributions (Figure 1D) reveals that thecharged residues in the interface that would face ras inthe favorable arrangement have a similar disposition inall but the RA1 domain. In RA1, the residues are predom-inantly negative and unlikely to support an interactionwith ras because of the latter molecule’s negative poten-tial within its effector binding surface. This difference,together with the absence of some critical residues in-volved in ras binding in RA2 (discussed in the followingsection), accounts for the inability of RA1 to bind ras.

Ras binding domains (RA and RB) are usually presentas a single domain in proteins identified in the SMARTand Pfam databases. To analyze whether the presenceof a tandem pair of RA domains in PLC3 could have func-tional implications, and to investigate the possibility thatthis structure was generated by duplication, we per-formed further sequence analysis and studied possibleinteractions between RA1 and RA2.

We analyzed the similarity between the RA1 and RA2sequences using PSI-BLAST and an inclusion E valuethreshold of 2 3 1023 that yielded significant similarity(E = 8 3 10213; 24% identity; 42% similarity). Analysisof multiple sequence alignments of human, mouse, rat,dog, chicken, and nematode orthologs using COMPASS

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Figure 1. Structure of PLC3 RA Domains and Comparison to Other RA/RBDs

(A and B) Ribbon diagrams of a representative structure of RA1 (Aa) and RA2 (Ba) showing the five-stranded b sheet (blue) and two a helices (red/

yellow). Superposition of the 20 best-fit NMR-derived structures is shown for RA1 (Ab) and RA2 (Bb). The structures are of RA1(WT) and

RA2(R2150L).

(C) Sequence alignment of ubiquitin-like folds from PLC3 (RA1 and RA2), Craf, RalGDS, Byr2, and PI3Kg. Secondary structure elements are high-

lighted, and amino acid residues determined to be important for ras binding are underlined; asterisk marks lysine residue introduced in PI3Kg by

mutagenesis.

(D) Ribbon diagrams of the PLC3 RA1 and RA2 compared to CrafRBD (PDB 1GUA), RalGDSRA (PDB 1LFD), and PI3KgRBD (PDB 1HE8). The

structures are oriented to show the prospective ras binding interface with b1, b2, and a1 labeled. Surface representations of the domains in

the same orientation are illustrated below. Electrostatic potentials are represented by positive (blue) and negative (red) charge; prominent, pos-

itively charged residues are labeled.

(PMID, 12547212) also established significant similarity(E = 7.4 3 10213) between the two RA domains. Thiswas sufficient to indicate that they represent tandem andhomologous RA domains (Figure 2Aa) that were likely to

have arisen by an internal gene duplication. The evolu-tionary relationship among these domains is furtheremphasized by an exon-intron boundary that is con-served between the two mammalian RA domains and

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Figure 2. PLC3 RA Domains Are Evolutionarily Related but Functionally Distinct

(A) (Aa) Sequence alignment of RA1 and RA2 from PLC3 orthologs from human (19923455), mouse (23956100), rat (12744799), dog (57107463),

chicken (50766642), and nematode worm (Caenorhabditis elegans) (25150381). Predicted (using PHD, PMID is 8345525) RA1 and RA2 have been

shaded according to an 80% consensus using CHROMA (PMID, 11590103). GenInfo accession numbers for each sequence are in brackets. (Ab)

Superposition of NMR structures of PLC3 RA1 (green) and RA2 (red).

(B) (Ba) 1H-15N HSQC spectrum of RA1RA2(WT). (Bb) Overlay of the 1H-15N HSQC spectra of RA1(WT) (red) and RA2(Y2176L) (blue). (Bc) Overlay

of 1H-15N HSQC spectra of RA1RA2(WT) in the absence (blue) and presence (red) of GMP.PNPHras(1–166,V12) at 1:1 molar ratio.

(C) (Ca) Average measured chemical shift changes for the amide HN and 15N resonances (Ddav) for each residue of RA2(R2150L) and RA2(Y2176L)

upon titration of GMP.PNPHras at 1:1 molar ratio. Residues that show ablation of crosspeak intensity are marked with a dot. (Cb) Magnitude of

chemical shift changes for RA2(R2150L) and RA2(Y2176L) mapped onto a ribbon representation of the solution structure of RA2(R2150L). Res-

idues that display the greatest chemical shift differences between free and bound states are colored in the darkest blue, and residues that show

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the second nematode RA domain (data not shown).Superposition of both domains (Figure 2Ab) furtherstresses the similarity between the protein folds thatextends beyond the similarity to other ubiquitin-likedomains.

Since the wild-type RA2 domain possessed a propen-sity to dimerize, and since we have shown that the RA1and RA2 domains are structurally related and homolo-gous, we investigated the possibility that the RA do-mains interact when expressed as tandem domains.However, when the combined NMR data of the isolateddomains are compared with the RA1RA2 tandem pair,no major differences are revealed (Figure 2B), suggest-ing that, within the tandem pair, RA1 and RA2 behaveas loosely tethered but otherwise independent domains.Using the same experimental approach, we also investi-gated the possibility that RA1 could interact with RA2upon ras binding or could contribute to ras bindingwithin the RA1RA2 tandem pair. Comparison of the1H-15N HSQC spectra of 15N-labeled RA2(R2150L),RA2(Y2176L), or RA1RA2(WT) with increasing concen-trations of unlabeled ras reveals complex formation,albeit with different dissociation kinetics: RA2(Y2176L)shows slow exchange behavior; RA2(R2150L), fast ex-change; and RA1RA2(WT), a pattern of selective cross-peak broadening (intermediate exchange) entirely local-ized to the RA2 domain (Figure 2Bc). Figure 2Ca displaysa plot of the average measured chemical shift changesfor the amide HN and 15N resonances (Ddav) for each res-idue in RA2(R2150L) and RA2(Y2176L) bound to ras. Thehighly similar patterns of chemical shift differences forRA2(R2150L) and RA2(Y2176L) and loss of crosspeakintensity for RA2(WT) in the tandem domain, shownschematically and mapped onto the ribbon representa-tion of the solution structure of RA2 (Figure 2C), suggestthat they bind Hras in a structurally similar mode. Thedata suggest that ras binds RA2(Y2176L) relativelystrongly with a slow off rate, and RA2(R2150L) bindsras weakly under the conditions of the NMR experiment.Moreover, in the RA1RA2 construct, RA1 does not bindras or interact with RA2, even as a subsequent event af-ter RA2/ras binding. Strictly, these data do not precludethe possibility that RA1 interacts with the C-terminal tailof ras or will interact with ras when RA1 is present in thecontext of the PLC3 molecule. Nevertheless, experi-ments with full-length PLC3 with deleted RA1 or RA2 do-mains support a conclusion that they are independentdomains, with RA2 being critical for stimulation by ras(Figure S1A and Figure 5C, see below).

Structural and Functional Insights into Interactions

of PLC3 RA2 with RasStructural studies of interaction between ras and its ef-fectors have demonstrated that, despite general similar-ity, there are significant differences in the specific sidechain interactions, electrostatic charge distribution atthe interface, and relative orientation of RA/RB domainsto ras; furthermore, the binding properties of the do-

mains differ, and binding constants and energies arequite diverse (Herrmann, 2003). The unique propertiesof these interactions are also reflected in different selec-tivity of the ras mutants in the switch regions for the ef-fectors and relative preference to different members ofthe ras family. To further probe the specific propertiesof the complex formed between RA2 and ras, we em-ployed X-ray crystallography and subsequent structure-function analysis (Figures 3 and 4).

We were able to crystallize and solve the structure ofthe complex of monomeric RA2(Y2176L) with ras at 1.9 Aresolution (Figure 3A). Electron density was associatedwith the whole RA2 molecule except for the b3–b4loop; the NMR data for free RA2 suggest that this loopdisplays flexibility in solution, and this could accountfor this region being undefined in the crystal structure.Overall, the complex is structurally similar to other ras-effector complexes with an intermolecular b sheet beingthe main structural feature. The interaction surface (Fig-ures 3B and 3C) shows the involvement of residues fromb1, b2, and the a1-b3 loop of RA2 and residues from bothswitch I and switch II of ras. F2138 from the b1 strand ofRA2 and V2152 forms a hydrophobic pocket togetherwith ras residues Y64, I36, and M67. A similar hydropho-bic surface is seen for ras interacting with the RBD ofPI3Kg (Pacold et al., 2000). I36, E37, and D38 are ras res-idues involved at the ras interaction interface of all mam-malian effectors; however, the interacting residues ineach effector and the strength of interaction vary (Herr-mann, 2003). A further specificity is provided by the res-idues flanking the core effector-recognition motif. In theRA2/ras complex, several other ras residues (e.g., Q25,E31, and R41) make contacts with RA2. Interestingly, rasR41 makes a polar contact with RA2 E2147. We can findno other example of an acidic residue in an RBD makingcontact with a positive residue in ras. Strikingly, a polarcontact is made between RA2 K2154 and a residuewithin ras switch II, E63. This type of interaction has onlybeen seen in the case of PI3Kg with ras, where the cor-responding K234 makes contacts with ras Y64 and E63(Pacold et al., 2000). Thus, the crystal structure of theRA2/ras complex reveals unique properties comparedto other RA/RBD interactions with ras and also somefeatures that otherwise are only shared with PI3KgRBD.In particular, involvement of switch II from ras is com-mon to RA2 and PI3KgRBD, while, in complexes formedwith other RA/RBDs, only switch I contacts have beenobserved.

We superimposed the NMR solution structure ofRA2(R2150L) with the crystal structure of RA2(Y2176L)bound to ras (Figure 3D). As with other ras effectors, theconformational changes are relatively small. The largestshift corresponds to the b2 strand that forms the inter-face with ras. Furthermore, there appears to be an or-dering of the loop between a1 and b3 that partially foldsinto an a helix. This area corresponds to the region (res-idues 2183–2189) where NMR analysis reveals largeshifts upon ras binding (Figure 2Ca). Interestingly, in

no evidence of chemical shift perturbation are in white. Residues whose crosspeaks disappear upon binding are shown in red. (Cc) Map of RA2

residues whose backbone amide NH crosspeaks are broadened beyond detection upon addition to RA1RA2(WT) of a small excess of

GMP.PNPHras, depicted as a red coloration on the solution structure of RA2(R2150L). In this experiment, there are no detectable changes to

crosspeaks of the RA1 domain in the presence of ras.

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Figure 3. Structural Perspectives of the Complex between Ras and RA2

(A) Ribbon representations show RA2(Y2176L) on the left and Hras(1–166,V12) on the right.

(B) View of the complex interface with contacting amino acid side chains highlighted.

(C) Schematic representation of the complex interface. Lines with arrow depict side chain interactions. Lines with no arrow depict main chain

interactions. Dashed lines depict hydrophobic contacts. Ras switch I and switch II amino acid residues are distinguished on the representation.

(D) Superposition of the RA2(R2150L) solution structure (red) with the RA2(Y2176L) structure in complex with ras (pink).

PI3Kg but not in other RA/RBDs, the corresponding a1–b3 loop is ordered upon ras binding, which contributes toa specific orientation of PI3Kg toward ras (Pacold et al.,2000).

Previous functional studies of PLC3 provided somecharacterization of RA2/ras interactions (Wing et al.,2003). We studied this interaction more extensively bycombining cellular studies and experiments with puri-fied components in vitro; we analyzed different ras bind-ing proteins and a large panel of ras family members aswell as RA2 and ras proteins with specific point mutants(Figure 4). Furthermore, the structural determination ofRA2/ras complex provided the framework for interpret-ing the effects of different mutations on ras binding andtheir selectivity for different members of the ras family.

Initially, using ITC, we could confirm the dissociationconstant for the ras/CrafRBD to be in the nanomolarrange, whereas for RalGDSRA/ras and RA2/ras it is inthe low micromolar range (Figure 4Aa) (Wohlgemuthet al., 2005). In an attempt to correlate the binding prop-erties determined in vitro with the function of the variousRA/RB domains in cells, we comicroinjected MDCK cellswith vectors expressing Kras(V12) and GFP-tagged

RA/RB domains; in the absence of ras, these domainsare present in the cytoplasm. Fixed cells were antibodylabeled for ras expression, and the intrinsic GFP fluores-cence of the fusion proteins was visualized (Figure 4Ab).CrafRBD and PLC3 RA2 clearly colocalize with Kras atthe plasma membrane. The RA1 does not colocalize withras at the membrane; this was expected, since their in-teraction is not detectable by NMR, pull-down, or ITC(Figures 2 and 4E, Table 1). The data in Figure 4Ab alsosupport previous assumptions that translocation of thefull-length PLC3 by ras is due to the interaction with theRA2 domain (Song et al., 2001; Sorli et al., 2005), sincewe show here that the isolated RA2 has the ability to in-teract with the membrane in a ras-dependent manner.

To gain further insight into the residues of ras impor-tant for RA2 binding and to assess the possibility of aras effector mutant specific for PLC3, we tested bindingof the RA/RB domains to ras mutants in the pull-downassay (Figure 4B); in this type of assay, the data are likelyto reflect changes in off rates rather then differences inaffinity. The ras(E37G) mutation has been long regardedas specific for RalGDS binding and activation (Cambelland Der, 2004), but recent advances in our knowledge

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of ras effectors suggest other proteins can bind and po-tentially be activated by this ras mutant (Rodriguez-Vici-ana et al., 2004). PLC3 is one such effector (Figure 4B)(Kelley et al., 2001). It has been also suggested thatthe ras(D38N) mutation allows activation of PLC3 (Kelleyet al., 2001), which could potentially confer selectivity forthis effector. The binding of ras(D38N) to RA2 is compat-ible with the surface interactions revealed by our crystalstructure. Substituting D38 for asparagine would permitthe interaction with RA2 T2151 but remove the interac-tion with K2171. Since neither repulsive charges nor ste-ric hindrances are introduced, RA2 and ras(D38N) wouldstill bind, albeit to a lesser extent than in the wild-type.We confirmed this binding and found ras(D38N) to bespecific for PLC3 when compared to CrafRBD andRalGDSRA (Figure 4B). However, when other proteinsare considered and titrated to higher concentrations(Figure 4C), it can be seen that PI3Kg can similarlybind to this ras mutant. Although ras(D38N) could beuseful for selectively discriminating between differentgroups of effectors, we would suggest caution when in-terpreting data from ras effector mutant phenotypes oncells. It is likely that ras effector mutants have more thanone target, and a comprehensive study will await deter-mination of the full plethora of ras effectors.

We also analyzed mutations in the RA2 domain thatwere suggested from our crystal structure to be impor-tant for ras binding (Figure 4D, Table 1). By adding pos-itive surface charge at critical locations, we were able toincrease the affinity of the RA2 domain for ras. TheRA2(Q2148K) mutation had a 1.4-fold higher affinity forras and the RA2(Q2140K) mutation a 2.8-fold increase.In contrast, adding negative charge in RA2(Q2148E) re-duces the affinity for ras 17.5-fold. Since these gluta-mine residues make no direct interactions with ras(Figure 3C), the effect of mutating them to positivelycharged residues reflects general influence on the sur-face charge distribution of RA2 that contributes to thebinding. The RA2(Q2140K) mutation corresponds tothe V223K mutation in PI3Kg that increases the affinityof the protein for ras (Pacold et al., 2000). Therefore,the introduction of lysine into b1 strand of RA2 couldcreate a polar interaction with ras E37 that might explainthe corresponding increase in affinity. The RA2(R2150L)mutation has the most profound effect on ras binding. Inthe pull-down assay, this protein was completely unableto form a stable complex and in ITC experimentsshowed a 130-fold lower affinity for ras. R2150 formsboth main chain and side chain contacts with ras S39and provides critical interactions within the complex.The RA2(Y2174L) mutation (affecting interaction withras E31) greatly reduces RA2/ras binding. Surprisingly,the RA2(K2171L) mutation has little effect on ras binding(Figure 4D). K2171 was suggested on the basis of its mu-tation to glutamate to be important for ras binding (Kel-ley et al., 2001). However, a positive to hydrophobic mu-tation has little effect on reducing ras affinity for RA2.

To assess the preference with regard to RA/RBDbinding of a wide range of ras family members, we com-pared the binding of 15 ras proteins to the RA/RBDs ofCraf and RalGDS (Figure 4E). Both domains havebroad-range specificity, binding to classical ras (H, K,and N), Rras (R, TC21, and M), and rap (1A, 1B, 2A,and 2B) proteins. CrafRBD binds weaker to rheb, rin,

and rit, while neither domain binds to ralA or ralB.Whereas CrafRBD shows highest affinity for the classi-cal ras proteins, RalGDSRA has highest affinity for theraps. The PLC3RA2 domain has a much narrower spec-ificity. It binds strongest to the classical ras proteins andmuch more weakly to some other members of the family.To confirm this specificity for the classical ras proteins,we carried out ITC on ras family members with RA2 (Ta-ble 1). From the dissociation constants, it can be seenthat RA2 binds with an 8-fold higher affinity to Hrasover rap1, a 10-fold higher affinity over rap2, and a 16-fold higher affinity than Rras. RalA binds very weakly,which would suggest it is not a physiological bindingpartner for the RA2 domain.

The preference of RA2 for classical ras proteins canbe explained by the differences in the effector bindingdomain between ras family members. As described pre-viously for CrafRBD (Nassar et al., 1996), the different af-finities of RA2 for ras and rap can also be partially ex-plained by the charge reversal of ras E31, which islysine in rap. The introduction of positive charge at thisposition would abrogate favorable charge interactionswith RA2 K2173. The evidence supporting the role ofras to rap charge reversal is provided by the analysisof ras mutations (Figure 4B); the D30E, E31K ras mutanthas a lower affinity for RA2, as shown in the pull-downassay. Another important residue present in ras is E63,which forms an ion pair/hydrogen bond with RA2K2154 (Figure 3C). In rap, the equivalent residue is a glu-tamine, which would not form the ion pair interactionwith RA2 and hence should lead to a weaker affinity.Similarly, Rras has a lower affinity than ras for RA2.This can be partially explained by the change of gluta-mate at position 31 in ras for aspartate in Rras. Thiswould allow for an important polar contact at the inter-face. Furthermore, the change of R41 for threoninemight reduce the impact of the main chain polar contactto RA2 E2147.

Our data (Figure 4E and Table 1) support previousfindings that classical ras is a potent activator of PLC3in cells (Kelley et al., 2001, 2004; Song et al., 2002). How-ever, studies of regulation of different effectors demon-strated that, in the cellular context, many factors otherthan effector affinity for a ras family GTPase are involvedin determining effector activation (Downward, 2003;Herrmann, 2003). Nevertheless, combined with cellularstudies, in vitro data will contribute to elucidating mech-anisms that could lead to activation in the absence ofhigh-affinity binding between ubiquitin-like folds andthe effector binding regions of ras. In the case of PLC3,such studies are needed to clarify whether and howother previously implicated ras family GTPases, rapand ralA, directly regulate PLC3 in vivo (Kelley et al.,2004; Schmidt et al., 2001; Song et al., 2002).

The Role of RA Domains in PLC3 MembraneRecruitment and in Activation as an Autoinhibitory

Switch on Lipase ActivityUsing protein preparations of a PLC3 variant (truncated,Figure 5A) previously shown to be stimulated by rhoA(Seifert et al., 2004), we show that Kras could also di-rectly stimulate activity of this protein in a reconstitutionassay in vitro (Figure 5Ba). To better understand the roleof the RA2 domain in recruiting and/or activating PLC3,

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Figure 4. Analysis of Interactions between Ras and PLC3 RA Domains

(A) (Aa) ITC analysis of CrafRBD, RalGDSRA, and PLC3 RA2(WT) with Hras(V12, 1–166). (Ab) Comicroinjection of GFP-RA/RB domains with

Kras(V12) into Madin Darby canine kidney (MDCK) cells. Localization of ras is visualized using specific antibodies (left) and superimposed

(merge, middle) with fluorescence of GFP or GFP-RA/RB domains (right).

(B) Pull-down analysis of RA/RB domains using various immobilized ras effector domain mutants. The indicated mutations were introduced into

Hras(V12) and proteins bound to either GTP (T) or GDP (D).

Phospholipase C Epsilon RA Domains503

we deleted this domain in the context of the full-lengthPLC3 (Figure 5A) and analyzed PLC activity in trans-fected COS cells (Figure 5C). If the RA2 domain hasa critical role in these events, then its deletion shouldprevent stimulation of PLC3 by ras and EGF; this is be-cause EGF, at least in part, stimulates PLC3 throughras (Kelley et al., 2004). Figures 5Ca and 5Cb showthat RA2 is essential for Kras- and EGF-driven activationof PLC3. To further investigate the role of RA2 in stimu-lation of PLC3 by factors other than ras, we cotrans-fected the wild-type PLC3 and the RA2 deletion con-struct with rhoA and found that rhoA stimulates PLC3

independently of RA2 (Figure 5Cc). This observationalso rules out the possibility that RA2 deletion con-structs would be incapable of full activation due to mis-folding of the PLC3 molecule.

To elucidate the mechanism of PLC3 regulation by ras,we further analyzed the events of recruitment and acti-vation. Two scenarios of PLC3 regulation can be envis-aged in which (1) PLC3 is constitutively active in the cy-toplasm and needs translocation to the membrane tobring it to its substrate or (2) the enzyme is inhibited,and both recruitment and subsequent activation are re-quired to give maximal substrate turnover. Studies ofPLC3 translocation (Song et al., 2001; Sorli et al.,2005), the requirement for lipid modified ras for PLC3

stimulation in the reconstitution system in vitro (Fig-ure 5Bb), and the RA2 translocation by ras (Figure 4Ab)support the role of this domain in membrane targeting.To analyze the possible role of RA2 in further PLC3 acti-vation, we engineered a PLC3 variant with a C-terminalCAAX box to achieve constitutive membrane localiza-tion in transfected cells, as previously shown in studiesof other ras effectors (Cambell and Der, 2004). PLC3(WT)and PLC3(CAAX) show distinct subcellular localization;the cytoplasmic localization of the wild-type constructcontrasts with the peripheral staining shown by thePLC3(CAAX) variant (Figure 5Ce). Furthermore, the

Table 1. Thermodynamic Quantities for the Binding of Ras Family

GTPases with the PLC3 RA Domains and Their Mutants

PLC3

Domain GTPasea n

KD

(mM)

2DHo

(kcal mol21)

DS

(cal mol21 K21)

RA2(WT) Hras (V12) 0.7 1.4 10.4 28.2

RA1RA2(WT) Hras (V12) 1.1 2.3 10.3 28.8

RA2(R2150L) Hras (V12) 0.8 184 12.2 223.9

RA2(Y2176L) Hras (V12) 1.0 1.1 10.5 27.9

RA2(Q2140K) Hras (V12) 0.8 0.5 19.5 235.7

RA2(Q2148K) Hras (V12) 0.4 1.0 12.5 213.9

RA2(Q2148E) Hras (V12) 0.2 24.5 21.3 215.5

RA2(WT) Rras (V38) 0.5 22.9 7.7 24.7

RA2(WT) rap1A (V12) 0.4 11.5 7.9 24.0

RA2(WT) rap2B (V12) 0.5 13.8 5.3 4.5

RA2(WT) ralA (V23) 0.5 197 14.4 231.2

RA1(WT) Hras (V12) 0 0 0 0

a All GTPases were truncated variants corresponding to amino acids

1–166 in Hras.

membrane-localized PLC3 CAAX variant has enhancedactivity when compared to the wild-type, but EGF stim-ulation leads to appreciable further enhancement of ac-tivity (Figure 5Cd). The implication is that PLC3 is notconstitutively active at the membrane, and additionalsteps are required to fully activate the enzyme. We alsoanalyzed the RA2 deletion mutant in the context ofPLC3(CAAX) construct. Interestingly, addition of CAAXbox to the RA2 deletion construct not only rescued theRA2 deletion by bypassing the translocation step, butthe mutant PLC3(DRA,CAAX) also showed enhancedbasal activity equivalent to the EGF-stimulated activityof wild-type PLC3; the activity could be further stimu-lated to a hyperactivated state by addition of EGF (Fig-ure 5Cd). Cotransfection with ras had an effect similarto EGF stimulation (data not shown). In experiments inwhich rhoA was used to stimulate PLC3, we observeda similar enhancement of activity of the RA2 deletionmutant (Figure 5Cc) and, to some extent, of the RA1 de-letion mutant (data not shown).

Since our data suggest that PLC3 is not fully activatedeven when present at the membrane, we hypothesizedthat the RA domains could inhibit PLC activity by pre-venting optimal access of the substrate to the catalyticcenter. To investigate the possibility that the domain’sabsence leads to a more active PLC3, we prepared anengineered enzyme in which the RA domains could becleaved off in vitro. We generated a variant of truncatedPLC3 with a TeV protease cleavage recognition se-quence in the linker between the C2 and RA1 domains(Figure 5A). The engineered enzyme was purified and in-cubated with TeV protease and the protein subjected togel filtration chromatography to remove the cleaved RAdomains from the remainder of the enzyme. For compar-ison, an equivalent sample of the enzyme was processedwithout treatment with TeV protease. The enzyme gen-erated by the removal of RA domains was shown tohave higher catalytic activity in vitro (Figure 5D). To elim-inate the possibility that the protease is activating theenzyme by a mechanism unrelated to the TeV recogni-tion sequence, we utilized a similar assay in vitro, includ-ing the wild-type truncated protein for comparison; forthis analysis, the RA domains were not removed by gelfiltration. Nevertheless, the wild-type protein was actu-ally partially inhibited by the addition of TeV, whereasthe engineered enzyme was activated by the protease(data not shown). The RA domains could therefore in-hibit the catalytic domain in an intramolecular fashion.

Previous studies of the mechanisms controlling rasand rho effectors suggest that these mechanisms couldbe diverse and include both translocation and subse-quent activation. It is also possible that activation ofeffectors could be regulated either by releasing intra-molecular inhibitory constraints or through allostericchanges directly affecting catalysis. The emerging pat-tern suggested for several rho effectors (mDia, WASP,and Pak) is that the binding of rho GTPases releasesan autoinhibition that exists in the resting state (Otomo

(C) Pull-down analysis using immobilized Hras (V12) (left) and Hras(V12, D38N) and increasing concentrations of purified ras binding proteins

(right). In addition to isolated RA/RB domains, PI3Kg was included as a full-length protein.

(D) Pull-down analysis of Hras(V12) binding to immobilized RA2 mutants.

(E) Pull-down analysis of RA/RB domain binding to immobilized ras family proteins. All GTPases were in activated form equivalent to Hras(V12).

PLC3 variant (1258–2225) is also included for a subset of ras proteins.

Molecular Cell504

Figure 5. Regulation of PLC3 Activity In Vitro and In Cells

(A) Schematic representation of PLC3 constructs.

(B) Reconstitution of PLC3 activation by rho and ras in vitro. Activity of PLC3 (truncated, TR) was measured in the absence (gray bar) and pres-

ence of GDP (white bars) or GTPgS (black bars). Membrane preparations were from Sf9 cells expressing either rhoA or Kras (Ba). Nras was pu-

rified either in lipid-modified form (L) from membranes of Sf9 cells or after expression in bacteria (B) (Bb). SD is represented by error bars.

(C) (Ca–Cd) PLC activity analyzed by production of total inositol phosphates (IP) in COS cells transfected with the indicated PLC3 constructs; non-

transfected cells (mock) were used as a control. PLC activity was measured in the absence (black bars) or presence (white bars) of cotransfected

Kras (Ca), rhoA (Cc), or EGF (Cb and Cd). PLC activity of PLC3(CAAX) and control PLC3(SAAX) variants is expressed as % of PLC3(WT) (Cd, right).

SD is represented by error bars. (Ce) Subcellular localization of GFP-tagged constructs of PLC3 (WT) and PLC3(CAAX) in transfected COS cells.

(D) (Da) In vitro PLC activity, measured by IP3 production, of truncated PLC3 with the engineered TeV cleavage site with (black bars) or without

(white bars) treatment with TeV protease and gel filtration separation of the cleaved RA1RA2 domains. The activity corresponds to fractions

(7–9) containing proteins within the 80–100 kDa range, while RA1RA2 protein is present in later fractions. (Db) PLC activity of TeV-cleaved

PLC3 was analyzed as in (Da) with the addition of IgG (control) or purified RA1 (triangles) and RA2 (squares).

Phospholipase C Epsilon RA Domains505

Figure 6. Model of Two-Step Mechanism of

PLC3 Translocation and Activation

et al., 2005). The rho binding site is bipartite with the siteof high affinity and the site involved in autoinhibition be-ing present in close proximity. A similar mechanism hasbeen proposed for Raf kinase in which the high-affinityRBD and the proximal autoinhibitory domain, CRD, in-teract with ras (Thapar et al., 2004). In the case of classI PI3K enzymes, the main inhibitory impact on catalyticsubunit is imposed by a separate, regulatory subunit.At present, there is no experimental evidence to supportor disprove that potential further inhibitory impact withinthe catalytic subunit could involve the RBD domain,while recent studies of stimulation of free p110g cata-lytic subunit by ras (Pacold et al., 2000; Suire et al.,2002) suggest an allosteric mechanism. The interactionof p110g with ras not only involves extensive interac-tions with the RBD but also further, direct interactionswith the catalytic domain; it is suggested that, insteadof releasing an inhibitory constraint, the conformationalchanges in the catalytic domain could directly affect therate of catalysis (Pacold et al., 2000; Suire et al., 2002).The translocation of p110g by ras has not been ob-served (Suire et al., 2002). In the case of RalGDS, it hasbeen shown that the CAAX box can replace RA withoutan enhancement of activity, suggesting that this domainis not autoinhibitory (Linnemann et al., 2002). However,how further interactions of ras and RalGDS(DRA,CAAX)lead to an increase of GEF activity toward ralA is notclear. The data presented here for PLC3 suggest that,despite similarity between PI3Kg and PLC3 in ras bind-ing properties, the mechanism of activation could be dif-ferent; this could be due to the way that these similarubiquitin-like folds are integrated into the complex, mul-tidomain structures of these proteins and the nature ofinteractions that underpin a low-activity state in thesetwo classes of signaling molecules. We suggest (Fig-ure 6) that the role of RA2 in PLC3 is to participate intranslocation by ras and also, possibly together with

RA1, to contribute to intramolecular inhibition in theresting state. Further, RA2-independent interactions arealso likely to be involved in activation and could includeras itself and other direct activators such as rho actingthrough additional binding sites within PLC3.

Experimental Procedures

Protein Expression and Purification

All constructs were prepared in pTriEx4 (Novagen) unless otherwise

stated in the text. Cloning and purification of GTPases were essen-

tially as described (Frankel et al., 2005; Sorli et al., 2005). RA domain

constructs (RA1 amino acids 2006–2114, RA2 amino acids 2131–

2246) were expressed in E. coli and purified by chelating chromatog-

raphy, TeV cleavage, and gel filtration chromatography. Labeled

proteins for NMR studies were prepared essentially as outlined (Ple-

vin et al., 2004). Hras (amino acids 1–166, V12), used for ITC exper-

iments, NMR titration, and X-ray crystallography, was described

previously (Pacold et al., 2000). For PLC activity assays in COS cells,

the vector pCMVscript-PLC3FLAG (Kelley et al., 2001) was used. The

variations on this construct (DRA2, amino acids 2110–2225 re-

moved, and CAAX-tag from Hras or Nras [residues 170–189]) were

obtained by PCR sewing (overlapping PCR) and verified by se-

quencing. For in vitro activation assays, a truncated version of the

rat PLC3 (amino acids 1258–2225) (Seifert et al., 2004) was cloned

into pIVexMBP (Roche) and purified as a maltose binding protein fu-

sion. All posttranslationary modified ras isoforms were prepared

from the pTriEx4 vector system by conversion to baculoviruses fol-

lowing manufacturers’ instructions. All GST-fusion proteins were ex-

pressed from pGex vectors (GE Healthcare) as outlined (Sorli et al.,

2005). All mutagenesis of ras and RBD proteins was carried out us-

ing the QuikChange Site-Directed Mutagenesis Kit (Stratagene), fol-

lowing the manufacturer’s instructions.

NMR and X-Ray Crystallography

Further information on the NMR methodology for solving the 3D so-

lution structures, the structural statistics for the final lowest-energy

bundle of representative set of 20 conformers (Table S1), and NMR

titrations of ras with RA1 and RA2 proteins are presented in the Sup-

plemental Data. The data collection, structure determination, and re-

finement statistics for ras/RA2 complex by X-ray crystallography

Molecular Cell506

(Table S2) together with experimental details are also presented in

the Supplemental Data.

Pull-Down Assay

The pull-down assay was used either to immobilize ras proteins and

pull down RA/RB proteins or the reverse. The practicalities of the as-

say differ depending on the proteins used. To bind tagged ras to the

beads, 50 mg of ras with an N-terminal S tag was immobilized using

40 ml of S protein agarose beads (Novagen) in a nucleotide loading

buffer (NLB) containing 100 mM Tris.Cl, 15 mM EDTA, and 1 mM

DTT (pH 8.0). Incubation was carried out at 4ºC for 30 min with con-

stant agitation. The beads were washed once in 500 ml of NLB.

GTPase-charged beads were incubated with 500 ml of NLB contain-

ing 1 mM pf either GDP or GTP for 30 min at 25ºC and the reaction

stopped with the addition of 100 mM MgCl2. Subsequently, 10 mg

of GST-tagged RBD was added in binding buffer (BB) containing

50 mM Tris.Cl, 150 mM NaCl, 1 mM TCEP, 1 mM MgCl2, 1% (v/v) Tri-

ton X-100, and 2 mgml21 BSA (pH 7.5) and incubated at 25ºC for

30 min with constant agitation. The beads were washed three times

in wash buffer (WB) containing 50 mM Tris.Cl, 150 mM NaCl, 1 mM

TCEP, 1 mM MgCl2, and 1% (v/v) Triton X-100 (pH 7.5). Beads

were boiled in Laemmli buffer and subjected to SDS-PAGE. GST-

tagged proteins were detected by Western blotting using an anti-

GST antibody (Santa Cruz). Blots were visualized by ECL (GE Health-

care).

When carried out in reverse, 50 mg of an S-tagged RBD was immo-

bilized on 40 ml of S protein agarose in WB. Ras (100 mg) was loaded

with nucleotide in 200 ml of NLB containing 1 mM GDP or GTP. The

reaction was stopped by the addition of 100 mM MgCl2. The volume

was made up to 1 ml with BB, and 50 ml was added to the immobi-

lized RBD and the volume made up to 500 ml with BB. The pull-

down was carried out further as shown above except that anti-pan-

Ras antibody (Transduction Labs) was used to detect ras bound to

RBDs in the assay.

Isothermal Titration Calorimetry

Heats of interaction were measured on an MSC system (Microcal)

with a cell volume of 1.458 ml. GTPases were loaded with the nonhy-

drolyzable GTP analog, GMP.PNP, following previously published

methods (Herrmann et al., 1996). Proteins were dialysed for 16 hr

in ITC buffer (25 mM Tris.Cl, 50 mM NaCl, 5 mM MgCl2, and 1 mM

TCEP [pH 7.5]). RBD proteins were loaded in the sample cell at

100 mM and titrated with GTPases in the syringe (1 mM). The titra-

tions were performed while samples were being stirred at 260 rpm

at 25ºC. A total of 20 injections was carried out, with 15 ml injected

each time and a 4 min interval between each injection to allow the

baseline to stabilize. The data were fitted with a single site model

to calculate the number of binding sites (n), the binding constant

(Ka), the change in enthalpy (DHo), and change in entropy (DS) using

Origin software (Microcal).

Measurements of PLC Activity and Studies of Subcellular

Localization

Determination of PLC activity in transfected COS-7 cells, microinjec-

tion of plasmids, visualization of expressed proteins, and in vitro as-

says for PLC activity were as previously described (Illenberger et al.,

2000; Sorli et al., 2005).

Supplemental Data

Supplemental Data include Supplemental Experimental Procedures,

Supplemental References, one figure, and two tables and can be

found with this article online at http://www.molecule.org/cgi/full/

content/21/4/495/DC1/.

Acknowledgments

Many thanks to A. Smith and H. King for excellent DNA sequencing

and to A. Paul for protein analytical assistance. We are grateful to

R. Williams for PI3Kg protein. We acknowledge the assistance of

G. Kelly at MRC-NIMR, Mill Hill for 800 MHz NMR, T. Piechulek for

help with the reconstitution assay, C. Prodromou for help with ITC,

and B.S. Magalhaes for help with figures. This work is funded by

a grant from Cancer Research UK. C.P.P. is funded by the MRC

(UK). Work in the F.R.-L. laboratory is supported by ARC (Associa-

tion pour la Recherche sur le Cancer).

Received: September 1, 2005

Revised: November 24, 2005

Accepted: January 3, 2006

Published: February 16, 2006

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Accession Numbers

The atomic coordinates of the final 20 simulated annealing RA1 and

RA2 conformers, and the list of experimental restraints (accession

codes 2BYE and 2BYF, respectively), have been deposited at the

RCSB Protein Data Bank. Chemical shifts for resonance assign-

ments for RA1 and RA2 have been deposited at the BioMagResBank

(accession codes 6624 and 6635). The Protein Data Bank deposition

code for the crystal structure of RA2/ras complex is 2c5l.