Cyclase-associated protein 1 (CAP1) is a prenyl-bindingpartner of Rap1 GTPaseReceived for publication, January 5, 2018, and in revised form, February 22, 2018 Published, Papers in Press, April 4, 2018, DOI 10.1074/jbc.RA118.001779

Xuefeng Zhang‡1, Shufen Cao§1, Guillermo Barila‡2, Martin M. Edreira‡3, Mamta Wankhede‡, Nyla Naim‡,Matthias Buck§, and Daniel L. Altschuler‡4

From the ‡Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh,Pennsylvania 15261 and §Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University,Cleveland, Ohio 44116

Edited by Henrik G. Dohlman

Rap1 proteins are members of the Ras subfamily of smallGTPases involved in many biological responses, includingadhesion, cell proliferation, and differentiation. Like all smallGTPases, they work as molecular allosteric units that are activein signaling only when associated with the proper membranecompartment. Prenylation, occurring in the cytosol, is an enzy-matic posttranslational event that anchors small GTPases at themembrane, and prenyl-binding proteins are needed to mask thecytoplasm-exposed lipid during transit to the target membrane.However, several of these proteins still await discovery. In thisstudy, we report that cyclase-associated protein 1 (CAP1) bindsRap1. We found that this binding is GTP-independent, does notinvolve Rap1’s effector domain, and is fully contained in itsC-terminal hypervariable region (HVR). Furthermore, Rap1prenylation was required for high-affinity interactions withCAP1 in a geranylgeranyl-specific manner. The prenyl bindingspecifically involved CAP1’s C-terminal hydrophobic �-sheetdomain. We present a combination of experimental and compu-tational approaches, yielding a model whereby the high-affinitybinding between Rap1 and CAP1 involves electrostatic and non-polar side-chain interactions between Rap1’s HVR residues,lipid, and CAP1 �-sheet domain. The binding was stabilized bythe lipid insertion into the �-solenoid whose interior was occu-pied by nonpolar side chains. This model was reminiscent of therecently solved structure of the PDE�–K-Ras complex; accord-ingly, disruptors of this complex, e.g. deltarasin, blocked theRap1–CAP1 interaction. These findings indicate that CAP1 is ageranylgeranyl-binding partner of Rap1.

Rap1 proteins are members of the Ras subfamily of smallGTPases involved in many biological responses, e.g. cell– cell(1) and cell– extracellular matrix attachment (2, 3), cytoskeletaldynamics (4 –6), endocytosis/exocytosis (7–10), polarity (11),cell proliferation (12–16), apoptosis (17–19), differentiation(20 –22), and migration/invasion (23). Like all small GTPases,they work as molecular allosteric units switching between inac-tive and active conformations in a regulated fashion withkinetic rate constants modulated by guanine-exchange factorsand GTPase-activating proteins (24). In their GTP-boundactive conformation, they bind and prompt the activation ofeffectors for signal propagation (25). All these nucleotide-de-pendent events are associated with the core G-domain (26),involving residues 1–166 in Ras/Rap1 proteins. Beyond thisdomain, the C terminus of these proteins is responsible for theirtrafficking and anchoring to a membrane-bound compartmentand consists of �20 residues, the hypervariable region (HVR),5and ends with a C-terminal CAAX motif (27).

Although at steady state members of the Ras subfamily ofproteins reside at membrane environments, they are synthe-sized in the cytosol and require a series of posttranslationalevents for membrane association (28). Cytosolic prenyltrans-ferases are first responsible for adding a prenyl group, i.e. C15farnesyl (Far) in Ras and C20 geranylgeranyl (GerGer) in Rap1,as an irreversible thioether linkage to the cysteine resi-due in the C-terminal CAAX motif (29). Once prenylated,they move to the endoplasmic reticulum (ER) for furtherprocessing via ER-resident enzymes RceI (30, 31), a proteo-lytic activity responsible for releasing the terminal AAX, andIcmt (32, 33), a methyltransferase modifying the newlyformed C-terminal isoprenylcysteine. However, prenylcys-

This work was supported by National Institutes of Health Grant R01GM099775 (to D. L. A. and partially to M. B). The authors declare that theyhave no conflicts of interest with the contents of this article. The content issolely the responsibility of the authors and does not necessarily representthe official views of the National Institutes of Health.

This article contains Figs. S1 and S2 and Table S1.1 Both authors contributed equally to this work.2 Present address: Center for Research on Reproduction and Women’s

Health, University of Pennsylvania Health System, Philadelphia, PA 19104.3 Present address: IQUIBICEN-CONICET, Instituto en el Departamento de

Química Biológica de la Facultad de Cíencias Exactas y Naturales, de laUniversídad de Buenos Aires - Consejo Nacional de Investigaciones Cientí-ficas y Técnícas, C1428EGA, Ciudad Autónoma de Buenos Aires, Argentina.

4 To whom correspondence should be addressed: Dept. of Pharmacology andChemical Biology, School of Medicine, University of Pittsburgh, W1346 Bio-medical Science Tower, 200 Lothrop St., Pittsburgh, PA 15261. Tel.: 412-648-9751; Fax: 412-648-1945; E-mail: altschul@pitt.edu.

5 The abbreviations used are: HVR, hypervariable region; CAP1, cyclase-asso-ciated protein 1; Far, farnesyl; GerGer or GG, geranylgeranyl; ER, endoplas-mic reticulum; GDI, GDP-dissociation inhibitor; PDE, phosphodiesterase;CaM, calmodulin; REP, Rab escort protein; GDS, GDP-dissociation stimula-tor; C-ter, C-terminal sequence; hCAP1, human CAP1; aa, amino acids;GTP�S, guanosine 5�-3-O-(thio)triphosphate; RBD, Rap-binding domain;GGTase I, geranylgeranyltransferase; NBD-FPP, {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate; MST,microscale thermophoresis; OG, Oregon Green; sh-V, scrambled control;r.m.s.d., root mean square deviation; MD, molecular dynamics; TSH, thy-rotropin; Ni-NTA, nickel-nitrilotriacetic acid; GGPP, geranylgeranyldiphosphate; HEPPSO, �-hydroxy-4-(2-hydroxyethyl)-1-piperazinepro-panesulfonic acid monohydrate; TCEP, tris(2-carboxyethyl)phosphine;LED, light-emitting diode; PM, plasma membrane.

croARTICLE

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teines provide only a low-affinity interaction with membranes(34, 35), and it was recognized early on that a “second signal”was required for stable membrane interaction, i.e. palmitoyla-tion in H-Ras/N-Ras, a reversible thioester linkage to upstreamcysteine residue(s) that increases hydrophobicity, or a polybasicdomain in the K-Ras/Rap1 HVR that provides electrostaticcontacts with negatively charged phospholipids (36 –38).Because of the differential localization of Golgi-resident acyl-transferases and cytosolic esterases, dynamic acylation/deacy-lation allows vesicle-mediated transport of these isoforms viathe secretory pathway to guarantee their steady-state localiza-tion in different membrane compartments (39, 40).

However, traversing the hydrophilic cytosol poses prob-lems for pools of nascent prenylated proteins en route to theER endomembrane, for retrograde transport of deacylated–prenylated proteins, and for prenylated polybasic domain–containing proteins on their way to plasma membrane.Recently, new chaperone proteins with prenyl-bindingdomains, able to mask the thermodynamically unfavorableexposure of the isoprenyl group to solvent, were described (e.g.Cdc42-Rho/RhoGDI (41, 42), Rab/RabGDI/REP (43), Rnd/14-3-3 (44), Ras/PDE�/Arl2–3 (45), K-Ras/CaM (46), Rab/PRA1(47), Ras/galectins (48), and N-Ras/VPS35 (49)). Most notably,newly identified SmgGDS variants differentially bind nonpre-nylated and prenylated forms, therefore facilitating processing

and trafficking, respectively (50, 51), thus raising the possibilitythat prenylation of these polybasic domain– containing pro-teins could be subject to regulation.

In this study, we report the identification of cyclase-associ-ated protein 1 (CAP1) (52) as a new partner of Rap1. Binding isnucleotide-independent and involves Rap1’s C-terminal HVR.The C20 geranylgeranyl modification is required for high-affin-ity interaction with CAP1, involving specifically its C-terminalsolenoid-like �-sheet domain, providing a hydrophobic tunnel.The structure is broadly reminiscent of the recently solvedcomplex of PDE� with K-Ras (53), and accordingly inhibitors ofthis complex, e.g. deltarasin (54), blocked the Rap1–CAP1interaction. These studies indicate that CAP1 is a novel gera-nylgeranyl-binding partner of Rap1.

Results

Rap1b interacts with CAP1

hCAP1 was isolated in a two-hybrid screening utilizing con-stitutively active Rap1b as bait against a human brain cDNAlibrary. Along with the canonical effector RalGDS, CAP1 rep-resented the most frequent isolate with multiple clones varyingin size, all including the C-terminal 123 amino acid fragment(aa 353– 475) (Fig. 1A). The N-terminal domain of CAP1 (aa1–318), however, did not interact with Rap1b under similar

Figure 1. Rap1b interacts with CAP1. A, two-hybrid isolation of a C-terminal fragment of human CAP1. The activation domain fusion plasmid expressing theC terminus of hCAP1 was cotransformed with empty vector pGBKT7 (Vector) or bait plasmid expressing G12V-Rap1b (G12V) and plated on selection mediumlacking Trp, Leu, His, and Ade (QDO; top) versus medium lacking only Trp and Leu (�LT; bottom). B, direct CAP1–Rap1b interaction. Immobilized GST orGST-Rap1b proteins (Coomassie Blue stain shown in lower panel) were incubated with [35S]methionine-labeled in vitro translated CAP1. After extensive washes,samples were resolved by SDS-PAGE. Once dried, radioactivity was visualized by fluorography using a phosphorimaging system (representative image of n �2). C, in vivo Rap1b and CAP1 interaction. Empty vector (myc-V) or pCMV-myc-CAP1 was cotransfected with HA-Rap1b in HEK293T cells. After 48 h, cell lysates(normalized for equal HA-Rap1b signal) were immunoprecipitated with an anti-myc antibody (9E10), and the presence of Rap1b in the immunocomplex wasassessed by Western blotting with an HA-specific antibody. D, in vivo Rap1b and CAP1 interaction. HEK293T cells were transfected with myc-CAP1 plasmidalong with GST empty vector or GST-Rap1b mammalian expression plasmids. After 48 h, cell lysates (normalized for equal myc-CAP1 signal) were pulled downwith GSH-Sepharose beads. The presence of CAP1 in the complex was assessed by Western blotting with a myc-specific antibody. A representative experiment(n � 3) is shown for C and D. E, colocalization of CAP1 and Rap1b. PCCL3 thyroid follicular cells stably expressing GFP-Rap1b were stained with anti-CAP1antibodies, and fluorescence microscopy was used to assess intracellular colocalization of CAP1 (red) and Rap1b (green) (representative image; n � 3).

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conditions (data not shown). To address whether the interac-tion was direct, Escherichia coli– expressed GST or GST-Rap1bwas immobilized on beads, and its ability to bind CAP1 wastested in vitro. As shown in Fig. 1B, [35S]methionine-labeledCAP1 associated specifically with GST-Rap1b, confirming adirect interaction. Similar studies were performed in cells uponcotransfection into HEK cells. Co-immunoprecipitation withHA-Rap1b (Fig. 1C) and pulldown assays of GST/GST-Rap1b(Fig.1D) indicated a specific interaction with myc-CAP1. Addi-tionally, fluorescence microscopy was used to assess intracellu-lar Rap1b-CAP1 colocalization. PCCL3 thyroid follicular cellsstably expressing or transiently transfected with GFP-Rap1bwere stained with anti-CAP1 antibody to monitor endogenousCAP1 protein. Colocalization of CAP1 (red) and Rap1b (green)is observed with accumulation in the cytosol and at perinuclearsites as well as, with lower intensity, in peripheral actin-richlamellipodia (Fig. 1E). Taken together, these results indicatethat Rap1b physically interacts with CAP1.

CAP1 is not a Rap1 effector

Different approaches were used to determine whether CAP1binding uses the canonical Rap1 nucleotide-dependent switch.Lysates expressing HA-Rap1b were loaded in vitro with GDP orGTP�S, and binding was assessed upon incubation with myc-CAP1 immobilized on beads. Although the canonical RalGDS-RBD domain behaved as expected for an effector protein, i.e.Rap1-GTP�S– dependent interaction (Fig. 2B), CAP1 bindsequally to Rap1-GDP and Rap1-GTP�S (Fig. 2A). Similarly,HA-CAP1 interacted with both constitutively active (G12V)and dominant negative (S17N) Rap1b proteins (Fig. 2C), con-sistent with a nucleotide-independent association. More-over, the interaction was not disrupted by introduction ofeffector domain mutations in switch I (Fig. 2D), known tointerfere with effector binding (55). Collectively, these re-sults indicate that CAP1 binding to Rap1b does not involvethe G-domain, i.e. the nucleotide-dependent switch andeffector-binding domains. Thus, CAP1 is not a prototypicalRap1 effector protein.

Rap1 binds CAP1’s C-terminal domain

Full-length CAP1 can be divided in two main subdomains, ahelical N-terminal and a �-sheet C-terminal subdomain (Fig.3A). To identify the domain in CAP1 involved in the interactionwith Rap1, we prepared constructs expressing both N-terminal(N-CAP; 1–318) and C-terminal fragments (C-CAP; 319 – 475).Unexpectedly, although full-length CAP1 interacted with Rap1,both N-CAP and C-CAP failed to do so (Fig. 3B). Prompted bythe results from the original two-hybrid screen identifying theC-terminal fragment as a positive partner, our studies focusedon this domain. The X-ray crystal structure of C-CAP1 (56)indicated the formation of a domain-swapped dimer, mediatedby intertwined �-hairpins involving the last two �-strands ofeach monomer (Fig. 3C). To assess whether C-CAP dimeriza-tion could be responsible for masking the Rap1-binding site,those strands were deleted. As shown in Fig. 3D, C-CAP� (aa319 – 448) strongly bound Rap1. Thus, consistent with the two-hybrid assays, these results confirmed that Rap1 interacts spe-cifically with the C-terminal fragment of CAP1 encompassingthe �-sheet domain.

Rap1 isoprenylation modulates affinity interaction

Preliminary studies indicated no interaction betweenCAP1 and the deletion construct Rap(1–167) (not shown);therefore, in the absence of any evidence for the involvementof Rap1’s effector domain in the interaction with CAP1, ourefforts switched to the C terminus of the GTPase. Cys181

in the C-terminal CAAX domain is the target residue forisoprenylation, as noted above, a posttranslational enzymat-ic event required for small GTPases biology. To addresswhether Rap1 isoprenylation plays a role in the interactionwith CAP1, a C181G mutation was introduced, and itseffects on binding were assessed as described above. Fig. 4Ashows that the lack of an isoprenyl moiety negatively impactsthe interaction with CAP1. Based on the actual sequence ofthe CAAX box, isoprenylation results in the addition of a C15farnesyl (i.e. CVLS in H-Ras) or C20 geranylgeranyl group(i.e. CQLL in Rap1). To assess whether the isoprenylationeffect is lipid-specific, a farnesylated Rap1 construct (i.e.

Figure 2. CAP1 is not a Rap1b effector protein. A, nucleotide-independent Rap1–CAP1 interaction. Lysates expressing HA-Rap1b were loaded in vitro withGDP or GTP�S, and a binding assay was performed upon incubation with myc-CAP1 immobilized on beads. B, Rap1 activation was monitored by GST-RalGDS-RBD pulldown assay. C, HA-CAP1 interacted equally with WT, constitutively active (G12V), and dominant negative (S17N) Rap1 proteins. D, effector domain (ED)mutations in switch I did not affect the interaction between CAP1 and Rap1b. Representative experiments (n � 3) are shown. V, empty vector.

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Rap-CVLS) was tested for CAP1 binding. As shown in Fig.4A, the shorter C15 farnesyl moiety in Rap-CVLS is not ableto replace the native C20 geranylgeranyl and rescue the inter-action. Consistent with this observation, CAP1 does notinteract with H-Ras (Fig. 4B).

The reduced CAP1–Rap1 interaction in the absence of ageranylgeranyl might represent a direct role for this lipid inbinding or, alternatively, indicate that unprocessed cytosolicRap1 is no longer colocalizing with CAP1 in cells. To directlyassess a role for isoprenylation in binding, purified Rap1 wasin vitro isoprenylated with purified geranylgeranyltransferase(GGTase I), and the reaction was monitored utilizing NBD-FPP, a fluorescent substrate (57), as shown in Fig. 4C. More-over, as evidenced by phosphodeficient S179A and phosphomi-metic S179D mutants, phosphorylation of Ser179 by PKA, justupstream of the substrate Cys181, does not interfere with theability of GGTase I to isoprenylate Cys181 (see next section).Binding of CAP1 to lipidated and nonlipidated Rap1 was thenassessed by His-affinity coprecipitation utilizing lysates fromC-CAP�–transfected cells. Results shown in Fig. 4D indicate aleft shift for the lipidated sample, qualitatively indicating a rolefor the isoprenyl group in binding. Microscale thermophoresis(MST) was then utilized for the quantitative determination ofequilibrium dissociation constants; purified C-CAP� proteins

were labeled in vitro (maleimide NT647) and titrated withincreasing amounts of purified Rap1. Consistent with the pull-down assays (Fig. 4D), results from the MST assay (Fig. 4E)indicate a �10� increase in affinity upon isoprenylation (Rap1KD, �5.6 0.6 �M; Rap1-GG KD, �0.46 0.07 �M). To directlytest the role of Rap’s C terminus, a peptide encompassing theHVR and CAAX domain (166NRKTPVPGKARKKSSCQLL184)was chemically synthesized and labeled at its N terminus withOregon Green (OG) followed by in vitro isoprenylation withpurified GGTase I. Labeled peptides (OG-HVR and OG-HVR-GG) were titrated with purified C-CAP�, and affinities wereestimated by MST. Fig. 4F shows the results from this ex-periment: although, compared with the proteins, a muchlower affinity is observed with the free peptides (OG-HVRKD, �156 42 �M; OG-HVR-GG KD, �8.6 0.8 �M), a similareffect of isoprenylation on affinity (�20�) is manifest. More-over, when the same C-terminal sequence is expressed as a GFPfusion (GFP-C-ter) and isoprenylated in vitro (GFP-C-ter-GG),both a high-affinity interaction with C-CAP� and the effects ofisoprenylation are reconstituted (Fig. 4, G and H; GFP-C-terKD, �9 1.3 �M; GFP-C-ter-GG KD, �0.17 0.08 �M),approaching the values observed in full-length Rap1 protein.These results demonstrate a role for the geranylgeranyl groupfor the Rap1–CAP1 interaction affinity. To test whether the

Figure 3. Rap1 interacts with the C-CAP domain. A, scheme of the CAP1 constructs used in this study. AC, adenylyl cyclase; PR, proline-rich; D, dimerization;FL, full length. B, both GST-N-CAP1 and GST-C-CAP1 failed to interact with Rap1b. The top panel shows purified GST-CAP1 fragments (Coomassie); the bottompanel shows associated Rap1b (HA) after GST-CAP1 pulldown from HA-Rap1b-G12V– expressing cells. C, C-CAP1 dimer formation from X-ray crystal structure(Protein Data Bank code 1K8F). D, C-terminal deletion, myc-C-CAP� (aa 319 – 448), restores binding to immobilized His-Rap1. Representative experiments (n �3) are shown for B and D.

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effect of the lipid depends on its ability to interact with thenonpolar environment of the �-sheet pocket as described abovefor other prenyl-binding proteins, we tested the effect ofdeltarasin, a recently identified inhibitor of the PDE�–K-Rasinteraction that binds with high affinity to the prenyl-bindingpocket of PDE� (58). As shown in Fig. 4I, deltarasin is able toinhibit the binding of OG-HVR-GG to C-CAP� (IC50, �3.6 1.2 �M). Thus, the combined results show that the Rap1 C ter-minus is sufficient for CAP1 binding, and lipid modification

increases affinity by interaction with the prenyl-binding pocketof CAP1.

Rap1 Ser179 phosphorylation does not directly affect bindingto CAP1

Rap1b is phosphorylated by PKA at residue Ser179 (59), justupstream of the isoprenylated Cys181. To assess potentialeffects of Rap1 Ser179 phosphorylation on the interaction withCAP1, we performed GST pulldown assays upon stimulation

Figure 4. Rap1 isoprenylation modulates interaction with CAP1. A, Rap1 lysates from HEK293T cells (i.e. HA-Rap1b-G12V, HA-Rap1b-G12V-GGLL, andHA-Rap1b-G12V-CVLS) were incubated with Ni-NTA-agarose–prebound His-C-CAP� for 1 h at 4 °C. After washes, associated proteins were resolved bySDS-PAGE and blotted with anti-HA antibody. B, HA-Rap1b and HA-Ras lysates from HEK293T cells were incubated with Ni-NTA-agarose–prebound His-C-CAPand His-C-CAP�. His pulldown was performed as above. Associated proteins were revealed by anti-HA antibody (top panel). His-C-CAP and His-C-CAP� totalproteins were revealed by anti-His tag antibody (bottom panel). Representative experiments (n � 3) are shown for A and B. C, purified Rap1 was in vitroisoprenylated with purified GGTase I utilizing NBD-FPP (top) as substrate. Total proteins were separated by SDS-PAGE (bottom panel; Coomassie), and fluores-cent isoprenylated Rap1 proteins (His-Rap1*) were visualized under UV light (top panel). This experiment was repeated twice with similar results. D, immobilizedin vitro isoprenylated (S179D-GG) and control His-Rap1-S179D (S179D) were incubated with normalized myc-C-CAP� lysates (Input lanes). The top panel showsthe binding results as revealed by anti-myc antibody. The bottom panel shows quantitation of these blots upon densitometric analysis via ImageJ. Curves areexpressed as a function of normalized lysate volume. A representative experiment (n � 3) is shown. E, MST analysis of in vitro labeled His-C-CAP�-NT647 withpurified (Rap1) and in vitro prenylated Rap1 (Rap1-gg) used as titrants. F, MST analysis of labeled Rap1b HVR (OG-HVR) and in vitro prenylated (OG-HVR-gg)peptides and purified His-C-CAP� as titrant. G, MST analysis of purified GFP-Rap1b HVR (GFP-C-ter) and control GFP proteins used as probes against purifiedHis-C-CAP� as titrant. H, MST analysis of in vitro prenylated (GFP-C-ter-gg) as probe against purified His-C-CAP� as titrant. I, MST analysis of the effect ofdeltarasin titrant on the binding of OG-HVR-GG to C-CAP� (conditions equivalent to �80% maximum based on F). Error bars for all MST studies representmean S.E. (n � 3), and data analyses were performed using NanoTemper Analysis software.

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with forskolin, a cAMP-elevating and thus PKA-stimulatingagent. Cells were cotransfected with GST-Rap1 or GST controland epitope-tagged myc-CAP1 or myc-C-CAP�. Upon for-skolin stimulation, phosphorylation of pSer179-GST-Rap1bwas confirmed by blotting, and a decrease in the amountof Rap1–CAP1/C-CAP�–associated protein was observed(Fig. 5A). Preincubation with the PKA inhibitor H89 blockedpSer179-GST-Rap1 phosphorylation and stabilized the GST-Rap1–CAP1 interaction (Fig. 5B), consistent with a PKA-de-pendent action of forskolin. Moreover, the negative effect offorskolin on the GST-Rap1–CAP1 interaction is lost in phos-phodeficient GST-Rap1-S179A and fully mimicked by GST-Rap1-S179D (Fig. 5C) whose phosphomimetic properties werethoroughly characterized in our laboratory (60). These resultsindicate that forskolin/PKA–mediated Rap1 Ser179 phosphor-ylation negatively modulates its interaction with CAP1 incells.

To directly quantitate the effect of Rap1 Ser179 phosphoryla-tion on CAP1 binding, we utilized WT and Rap1-S179D astitrants on saturation binding MST assays with NT647-labeledC-CAP� as probe. Contrary to the effects observed in vivo, nosignificant phosphorylation-mediated differences are observed

in the binding curves using either lipidated or nonlipidatedRap1 proteins (Fig. 5D). Thus, these results indicate that Ser179

phosphorylation does not directly act on the Rap1–CAP1 con-tacts, but rather other factor(s) are required to manifest its neg-ative effect in cells.

CAP1 is required for proper Rap1 membrane localization

To investigate potential effects of CAP1 in Rap1b localiza-tion, an shCAP1 approach was utilized. Scrambled sequenceand shCAP1-specific sequence against rat CAP1 were sub-cloned in a vector expressing an independent dsRed unit.As shown in Fig. 6A, almost complete down-regulation ofCAP1 expression was observed in shCAP1- versus scrambledcontrol (sh-V)–transfected (red) cells. To monitor Rap1localization, experiments were repeated in the presence ofGFP-Rap1, and green fluorescence was assessed in dsRed-transfected shCAP1 cells. As shown in Fig. 6, B and C, CAP1down-regulation is accompanied by an almost complete lossof membrane GFP-Rap1. These results indicate a critical rolefor CAP1 in the proper plasma membrane localization ofRap1.

Figure 5. Rap1 Ser179 phosphorylation modulates interaction in cells but not in vitro. A, HEK293T cells were co-transfected with GST-Rap1 or GST controland epitope-tagged myc-CAP1 or myc-C-CAP�. Upon forskolin stimulation (20 �M, 20min), GST pulldown assays were performed, and associated proteinsassessed by Western blots with specific antibodies. B, PKA inhibitor H89 (20 �M) blocked pSer179-GST-Rap1 phosphorylation and stabilized the GST-Rap1–CAP1interaction. C, The negative effect of forskolin on GST-Rap1–CAP1 interaction can be mimicked by phosphomimetic GST-Rap1-S179D and is lost in phospho-deficient GST-Rap1-S179A. Representative experiments (n � 3) shown in A–C. D, MST analysis of labeled His-C-CAP�-NT647, purified Rap1 (Rap-WT andRap-S179D), and in vitro prenylated (Rap-WT-GG and Rap-S179D-GG) proteins as titrant. Error bars for all MST studies represent mean S.E. (n � 3), and dataanalyses were performed using NanoTemper Analysis software.

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Molecular modeling and dynamics simulations suggestdifferential binding affinities toward differently modifiedpeptides

Currently no crystal structures of the C-CAP protein areavailable when it is complexed with Rap1b C-terminal peptides,and solution NMR awaits optimization of conditions to yieldgood spectra of the protein. Molecular modeling and dynamicssimulations represent a suitable tool to make predictionsof possible interactions. Because the farnesylated and gera-nylgeranylated peptides are highly hydrophobic due to attach-ment of the lipid group and the C-CAP domain presents a tun-nel with an opening on both sides, we manually positioned thelipidated peptide as deep into the tunnel as possible, makingfavorable polar and charge interactions between the peptideRKKSSC residues at the N- or C-terminal opening of the tunnel(see “Experimental procedures”). The systems were then brieflyminimized, solvated, and equilibrated for 5 ns using all-atommolecular dynamics using standard protocols. This was fol-lowed by a 95-ns production run. The equilibrated structuresare shown in Fig. 7, A and B, for the N-terminal insertion and inFig. S1 for the C-terminal insertion.

The behavior of the peptides in the tunnel and at the tunnelmouth was analyzed by root mean square deviation (r.m.s.d.)measurements using the position of lipid tail or the C� atom ofCys181 in the initial structures as a reference, and the plots areshown in Fig. S2. It is clear that the behavior is dramaticallydifferent, depending on the nature of the peptide and directionof tunnel insertion. Briefly, peptides modified by farnesyl, espe-cially the C-terminal inserted peptide, slide toward the mouthof the tunnel where they stay but are highly dynamic. By con-trast, GerGer-modified peptides are relatively stable in the tun-nel when inserted from the N-terminal side, whereas they slideslightly when inserted from the C-terminal side. Rapid transi-tions in sliding of the lipid/peptide in the tunnel (e.g. at 95 ns inFig. S2, panel A3) arise from stochastic fluctuations in the sim-ulations and are normal, especially in relatively long simula-tions. Because transitions can also revert back (e.g. at 90 ns inFig. S2, panel A4), we need to consider the area under ther.m.s.d. curve to get an overall qualitative measure of structuralstability. By those criteria, an N-terminal insertion of GerGer-

Rap1 and Far-Rap1 is preferred. The persistence of contacts ateither mouth of the tunnel was analyzed by the frequency keyresidues are in proximity of the peptide (with 5-Å cutoffbetween nonhydrogen atoms of residues). These data are sum-marized in Table S1 for all 10 simulations. In the case of thefarnesyl-peptide simulation, they show relatively persistentcontacts (�60% of time) between Ser179, Lys178, and Arg176

with C-CAP N-terminal residues Val341, Glu343, and Asp344;however, contacts on C-terminal insertion are only strong forArg176 but not Lys178 or Ser179, also reflected in lesser contactfrequency for residues at the C-terminal mouth of the tunnel(�30% or less). The GerGer-modified peptide, inserted fromthe N-terminal side, behaves similarly to the Far-peptide butadds more contacts with Cys181 and the C-CAP protein resi-dues Leu322, Trp329, and Glu346. The simulations with theGerGer peptides were also run with Ser179 when it is phosphor-ylated and given a �2 charge, showing contacts with N-termi-nal C-CAP residues 322–324 as well as 344 and 346 with higherfrequency. At the C-terminal mouth, contacts with residue436 – 438 and 450 – 452 are significantly more stable. Thus,although the data appear to show a preference of the GerGerpeptide for N-terminal insertion, both modes of insertion aremoderately stabilized by Ser179 phosphorylation.

We also ran separate simulations with the nonlipidated pep-tide RKKSSCQLL and with deltarasin inhibitor molecules.Again, the manual positioning sought to optimize polar inter-actions with the C-CAP polar tunnel residues for the former(on both N-terminal (Fig. 7C) and C-terminal insertions) andC-CAP tunnel/mouth aromatic ring/nonpolar side-chain resi-dues with the rings of deltarasin in the latter case (Fig. 7D). Forthe nonlipidated peptides, the peptide residues were not stablewhen inserted into the tunnel from either end and slid outwithin 30 ns, albeit slightly faster from the C-terminal side.Another difference is that, once it had slid out from the C-ter-minal side, the peptide actually detaches/interacts only withresidue 452 at 50% frequency (others at 10%). By stark con-trast, the N-terminal inserted peptide, although no longer res-ident near the center of the tunnel, maintains a stable confor-mation at the tunnel mouth by forming extensive contactsamong residues Arg176, Lys177, Cys181–Leu184, and C-CAP res-

Figure 6. CAP1 depletion alters Rap1 localization. A, CAP1 staining (green channel) in samples of PCCL3 cells transfected with control sh-V (sh-VEC) orsh-CAP1 for 72 h. Transfected cells (red channel) in the field are shown with * in the green channel. B, GFP-Rap1– expressing PCCL3 cells were transfected withsh-CAP1 or sh-V control plasmids. 72 h posttransfection GFP-Rap1 intracellular distribution was assessed by confocal microscopy. Representative fields areshown in A and B. C, quantification of GFP-Rap1 membrane delocalization upon CAP1 depletion. PM/total pixel intensity ratio (green channel) was determinedin random red� cells from experiment shown in B as described under “Experimental procedures.” Data were analyzed, and significance was tested using atwo-tailed Student’s t test (� level was defined as 0.05). ***, p 0.0001. All data points are shown. Lines indicate the mean value.

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idues 320/322/329 and 341/343/344/346. The fact that Leu182

and Leu183 can be accommodated is remarkable and suggeststhat the N-terminal mouth of the tunnel has considerable non-polar character. A similar behavior is observed with the highlyhydrophobic deltarasin inhibitor, which moves out very quicklyfrom the CAP tunnel, either N- or C-terminally inserted, withinless than 10 ns but then is maintained in the proximity of bothN- and C-terminal mouths of C-CAP tunnel. However, theconformation is highly dynamic, and only a few contacts areformed (more so in the N-terminal than in the C-terminal case).In summary, the MD simulations are consistent with the deepinsertion of GerGer-modified peptide into the C-CAP tunnel,especially from the N-terminal side. Far-modified peptides arenot stable in the tunnel, and unlipidated peptides also slide outbut are maintained at the tunnel mouth. A similar observationis made with deltarasin inhibitor. The latter two cases, unlipi-dated peptides and deltarasin, may highlight interactions tooptimize for more tightly binding inhibitors/competitors to thenatural Rap1b interaction with C-CAP.

Discussion

Here, we report the identification of CAP1 as a novel partnerof the small GTPase Rap1b. The interaction does not involvethe canonical nucleotide-dependent switch and is resistant toknown effector domain mutants, indicating that CAP1 is not aRap1 effector protein. The interaction instead involves Rap1’sC-terminal HVR and its lipid moiety in a geranylgeranyl-spe-cific manner. Thus, CAP1 represents a novel Rap1 prenyl-bind-ing protein.

CAP1, which was originally isolated in yeasts (Srv2) (61, 62),is highly conserved in evolution (63). Mammalian CAP1 has475 aa with several recognizable domains: an N-terminal olig-omerization domain, a helical folded domain, a WH2 domainsurrounded by two polyproline (P1 and P2) motifs, and a C-ter-minal �-solenoid domain (52, 63). Our studies indicate CAP1’sC-terminal �-solenoid is responsible for Rap1 binding.

Although full-length CAP1 forms hexameric structures incells (64, 65), its C-terminal �-solenoid domain is dimeric invitro (56). X-ray crystallography shows each monomer presentsa right-handed �-helical organization with six coils forming anelliptical solenoid whose interior is moderately well-packed

with mostly nonpolar side chains, leaving a tunnel that runsthrough the entire protein. Distal to the solenoid, each mono-mer terminates with a �-hairpin (antiparallel strands �8-�9)responsible for intersubunit domain swapping and formationof an intertwined dimer. Contrary to full-length CAP1, wecould not observe an interaction with the full-length C-CAPfragment isolated in the two-hybrid assay. We initially thoughtthat the strand-exchanged C-CAP dimer might be responsiblefor capping and blocking the entry site to the hydrophobic sole-noid, and deletion of the last �30 aa, including the �-hairpin,indeed unmasked a high-affinity Rap1 interaction domain.

The CAP1 C-terminal 27 aa are involved in binding mono-meric G-actin (66, 67). Moreover, recent mutagenesis studiesdemonstrated a more extended interaction surface, includingthe C-terminal dimerization domain as well as conserved sol-vent-exposed hydrophobic residues in the solenoid domain ofboth units in the dimer (66, 68). These results suggest that Rap1binding and G-actin binding to C-CAP might be mutuallyexclusive; however, pulldown of CAP1 from cell lysates showsan almost stoichiometric amount of bound G-actin that is notaffected by overexpression of Rap1.6 Thus, we propose thateither Rap1’s effect on dimerization/oligomerization dynamics,the deletion of the C-terminal �-hairpin, and/or the presence ofN-terminal sequences (e.g. full-length CAP1 or fusion two-hy-brid library constructs) exposes a binding domain at the N-ter-minal opening of C-CAP, allowing prenylated Rap1 binding.

Molecular models, built with both Far- and GerGer-modi-fied Rap1b C-terminal peptides, confirm these findings at theresidue level. Farnesylated peptides are not stable in the tunneland begin to slide out when inserted either from the N- or theC-terminal opening. In contrast, geranylgeranylated peptidesare stably maintained in the tunnel over 100 ns of all-atomsimulations, especially when inserted from the N terminus.Although the favorable entry site for inserted peptides from theN- versus C-terminal opening largely derives from the interac-tions that are possible between Rap1b residues and the tunnelmouth, the preference for GerGer- over Far-modified peptideslikely originates from the extent of hydrophobic contacts that

6 X. Zhang, S. Cao, G. Barila, M. M. Edreira, M. Wankhede, N. Naim, M. Buck, andD. L. Altschuler, unpublished results.

Figure 7. MD simulation and models for lipidated and unmodified peptides and for deltarasin inhibitor. Interactions of RKKSSC-Rap1b-GerGer (A) and-Far (B) prenylated peptides with the N-terminal opening of C-CAP after 5 ns of all-atom dynamics simulation are shown. Both peptides are phosphorylated onSer179. Although the GG lipid tail is maintained along nearly the entire length of the tunnel, the Far-modified peptide substantially slides out of the tunnel,consistent with weaker binding experimentally (the Far group is initially �10 Å shorter than GerGer). Several salt bridges exist between peptide Lys/Argresidues and C-CAP Asp/Glu residues side chains at the N-terminal mouth. In the case of unprocessed peptide and deltarasin inhibitor, initial structures afterminimization are shown (C and D). This peptide may be stabilized by the nonpolar side chains in the tunnel (labeled in C), whereas the carbon rings of theinhibitor deltarasin may be stabilized by Phe/Trp and Ile upon initial insertion into N-terminal C-CAP as shown (D). Note that D shows the CAP1 tunnel entrancefrom the N-terminal side, different from the side-on view of the other panels (A–C).

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can be formed in the tunnel (probably proportional to tunnellength occupied). Although in principle shorter Far-modifiedpeptides may insert from both sides to cover the entire tunnel,this is unlikely as it would require a very high concentrationof Rap1b GTPases. Thus, consistent with the experimentalresults, in the presence of only one lipidated HVR per CAP,Far-lipidated peptide would not provide enough contacts, leav-ing a considerable nonpolar region of the tunnel unoccupied.

MD simulations also suggest how nonlipidated and unpro-cessed HVR peptide may interact with C-CAP. Although notstable near the center of the tunnel, the peptide is maintained atthe N-terminal mouth of the tunnel by both charge– charge andnonpolar side-chain interactions. A similar number of interac-tions are not available at the C terminus of C-CAP, and thepeptide dissociates. We also carried out simulations with thehydrophobic inhibitor deltarasin, inserted at either end intothe tunnel after optimizing initial interactions. On both sides,the inhibitor quickly left its initial position (10 ns) and movedto the mouth of the tunnel. However, the inhibitor thenremained at the mouth of the tunnel for the 100-ns simulation.By contrast to the peptides, the deltarasin inhibitor is largelynonpolar except for a linking oxygen and two linking and tworing nitrogens. The only protonated nitrogen available forhydrogen bonding is at the most solvent-exposed part of thetunnel. The contacts between deltarasin and the CAP1 centerare mostly hydrophobic, although ring stacking with a Phe andTrp at the N-terminal entrance would make this site morefavorable and likely somewhat specific. The structure ofdeltarasin bound to PDE� has not yet been solved, but from ourmodel of the inhibitor bound to CAP1, it seems plausible thatthe inhibitor works by blocking the tunnel entrance, hence pre-venting the CAP1–Rap interaction.

It should be noted that, although the simulations are rela-tively lengthy at 100 ns, the positions of the peptides and inhib-itor in the tunnel are first hand-modeled and then minimized.This treatment would need to be dramatically extended toobtain statistically significant results (four to eight repeat sim-ulations) or even more computationally expensive, so-calledumbrella-sampling simulations, to estimate free energies ofbinding. However, such calculations are beyond the scope ofthis report and our computational resources. The computa-tional studies are, however, remarkably consistent with theexperimental results across the series of peptides, and the pre-dictions should be valuable in suggesting C-CAP mutants orHVR variants (e.g. testing GTPase isoforms with similar termi-nal residues) for future investigations.

CAP1 depletion results in Rap1 mislocalization from theplasma membrane, consistent with a role in stabilizing Rap1 atthe membrane or as a chaperone for delivery of prenylated Rap1to the membrane. Recently, several small GTPase chaperonessharing a common �-sandwich fold have been reported. Con-trary to the GDP-bound specificity of RhoGDI (42) or the non-specific prenyl–lipid interaction in PDE� (69), our data showedthatCAP1,withadifferentstructure,binds inanucleotide-inde-pendent manner specifically to only geranylgeranylated Rap1;no interaction was observed with native farnesylated H-Ras orfarnesylated Rap1-CVLS. Rap1’s G-domain by itself (aa 1–167)does not interact with CAP1 and the lipidated HVR peptide is

able to fully recapitulate the affinity of the interaction with full-length Rap1. The different affinities observed for free peptideversus full-length Rap1b or GFP-C-ter reflects low-affinity con-tacts involving Rap1’s G-domain or GFP or, most likely, aneffect on decreased conformational entropy, i.e. the distribu-tion of conformational states populated by the HVR tail.Whether other geranylgeranylated small GTPases can also bindCAP1 and/or the HVR sequence provides all specificity and/orit communicates allosterically with the G domain (70) remainsto be investigated.

Rap1b is phosphorylated by PKA at Ser179 with cell-specificassociated responses. We have shown in endocrine cellsthat Rap1-GTP and pSer179 are synergistically required forcAMP-dependent cell proliferation (71–73). In contrast, it wasrecently reported that in some cell types Rap1 phosphorylationmight negatively impact its prenylation pathway, an effectinvolving a novel family of prenyl-binding proteins (74). Smg-GDSs represent a family of alternatively spliced novel prenyl-binding proteins not involving a �-sandwich fold; although thelargest isoform binds unprocessed Rap1 and seems to help inthe prenylation pathway, the small isoform binds prenylatedRap1, facilitating its final translocation to membranes (51).Interestingly, phosphorylation of unprocessed Rap1 seems toinhibit its interaction with SmgGDS, affecting its prenylationand further trafficking (75). Although we observed a negativeeffect on the Rap1–CAP1 interaction in cells, pSer179 did notmanifest any effect on binding when tested in vitro with eitherlipidated or nonlipidated Rap1 proteins or HVR-derived pep-tides. Overall, this is consistent with the molecular models andsimulations where pSer179 showed only a slight effect, mostlikely organizing the neighboring charged Arg/Lys side chainsof the HVR to point in the direction of the negative chargedC-CAP residues at the mouth of the tunnel.

These results indicate that, for the effects observed in vivo,another cellular factor(s) might be required. Mammalian CAP1itself can be phosphorylated in cells at many sites (76). How-ever, all these events are PKA-independent and reside outsidethe C-CAP domain and are thus unlikely to affect Rap1b–CAP1interaction. It has recently been reported that Rap1 phosphor-ylation creates a novel 14-3-3– binding site responsible forkinase suppressor of Ras (KSR) recruitment leading to B-Raf/ERK activation (77). Alternatively, a pSer179-mediated allos-teric communication to switch regions (60) might be responsi-ble for local conformational effects, resulting in effectorcoupling and activation. Even though the Rap1–CAP1 interac-tion is GTP-independent, the model predicts that Rap1-GTPmight interact with effectors scaffolded in the CAP1 complex.This hypothesis is currently under investigation.

As discussed above, Rap1 has been proposed to be involved inseveral actin-dependent processes. It is localized in a Rab11�

perinuclear compartment and plasma membrane lamellipodia(78). Similarly, CAP1 localizes in F-actin–rich regions (67,79 – 81), and it purifies from cells as a hexameric complexbound (1:1) to monomeric G-actin (82, 83). Its N-terminaldomain is responsible for a cofilin-mediated actin-severingactivity (64, 65, 84), suggesting a role in F-actin disassembly. ItsC terminus is responsible for binding G-actin (66, 67) and incollaboration with profilin participates in nucleotide exchange

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on G-actin-ADP (85, 86), consequently indicating a role inF-actin assembly. Thus, like Rap1, the current model placesCAP1 function in the context of cellular events requiring activeactin dynamics, i.e. cell polarity, migration, and receptor-medi-ated endocytosis (63).

The role of Rap1 in tumor migration and invasion was con-firmed in many tissues (23), and numerous recent reports dem-onstrate that CAP1 affects tumor migration and that its over-expression correlates with invasiveness in metastasis (88 –100).Thus, it is tempting to speculate that the novel Rap1–CAP1complex might provide a mechanistic link to integrate thesebiological responses.

Experimental procedures

Materials

TSH, forskolin, H89, and GTP�s were from Sigma. GSH-agarose was from GE Healthcare. Ni-NTA-agarose was fromQiagen. Antibodies against HA (HA.11) and myc (9E10) werefrom Covance. Anti-GST (A5800) antibody was from Invitro-gen. Anti-phospho-(Ser/Thr) PKA substrate antibody was fromCell Signaling Technology. Anti-CAP1 antibody was from Pro-teintech. NBD-FPP and GGPP were from Jena Bioscience.

Cell lines and transfections

PCCL3, a normal TSH-dependent rat thyroid follicular cellline, was grown in 5% Coon’s modified F-12 medium (Sigma)supplemented with 5% FBS and a combination of four hor-mones, TSH (1 mIU/ml), insulin (1 �g/ml), transferrin (5�g/ml), and hydrocortisone (1 nM), as described before (73).HEK293T cells were maintained in Dulbecco’s modified Eagle’smedium (Cambrex) supplemented with 10% FBS. Cells werekept at 37 °C in a 5% CO2, 95% humidified air environment.Transfections were performed using a Lipofectamine 3000transfection kit (Invitrogen) or X-tremeGENE HP DNA trans-fection reagent (Roche Applied Science), adjusting the totalamount of DNA plasmid to 0.5–1 �g/well as directed by themanufacturers.

DNA constructs

pCGN-HA-Rap1, GST-Rap1, HA-Rap1-G12V, HA-Rap1-G12V/S179A, and GFP-Rap1 constructs were already de-scribed (59, 101). Effector domain mutants (provided by Dr.Stork) were introduced in HA-Rap1-G12V. Human CAP1 genewas amplified by PCR using human cDNA as template. ThePCR products were digested with XbaI and BamHI and theninserted into pCGN-HA vector, generating HA-CAP1. myc-CAP1, myc-C-CAP (aa 319 – 475), and myc-C-CAP� (aa 319 –448) were prepared by inserting SalI-XhoI PCR fragments intopCMV-myc vector using HA-CAP1 as template. PCR productsfrom myc-C-CAP and myc-C-CAP� were digested with NdeI-XhoI and subcloned into pET-28c, generating His-C-CAP andHis-C-CAP�. HA-Rap1-G12V-CVLS was subcloned as a PstI-BamHI PCR fragment. GFP-Rap1b-C-ter was prepared bydigesting full-length GFP-Rap1b with BglII, removing most ofRap1b from its N terminus. The remaining fragment wasblunted with Klenow and religated, generating GFP-Rap1b-C-ter. shCAP1 plasmid (targeting aa 1074 –1092) was generated

by subcloning the following annealed complementaryoligos into BamHI-EcoRI– digested pSIREN-dsRed vector(Clontech): 5�-GATCCGCACGACATTGCAAATCAAGGA-AGCTTGCTTGATTTGCAATGTCGTGCTTTTTTG-3� and5�-AATTCAAAAAAGCACGACATTGCAAATCAAGCAAG-CTTCCTTGATTTGCAATGTCGTGCG-3�.

Yeast two-hybrid screening

MatchmakerTM GAL4 Two-Hybrid System 3 and pretrans-formed human brain cDNA library were purchased from Clon-tech. Rap1-G12V/S179D bait was released from pCGN-Rap1-G12V/S179D with SpeI-Klenow (blunt)/BamHI and subclonedinto pGBKT7 at NcoI-Klenow (blunt)/BamHI sites. Approxi-mately, 1.48 � 107 colonies were screened following the man-ufacturer’s instructions.

Protein purification and in vitro binding

BL21/DE3 competent E. coli cells transformed with theappropriate pGEX or pET28c plasmids were grown until �0.8A600 and induced for 16 h at 24 °C with 0.5 mM isopropyl 1-thio-�-D-galactopyranoside. After centrifugation, cells were lysed,and supernatants were subjected to affinity chromatography onGSH-Sepharose (GE Healthcare) or Ni-NTA-agarose (Qiagen),respectively. For in vitro binding, E. coli– expressed GST orGST-Rap1b was immobilized on beads, and binding wasassessed with in vitro translated CAP1, generated frompET28c-CAP1 by TNT T7 Quick (Promega) and [35S]methio-nine (PerkinElmer Life Sciences) following the manufacturers’directions. Binding assays were performed in 50 mM Tris/Cl,pH 7.5, 50 mM NaCl, and 0.1%Tween 20 for 1 h at room tem-perature, and complexes were washed four times with bindingbuffer. Samples were resolved by 10% SDS-PAGE, transferred,dried, and exposed to film.

GST pulldowns

Cells were cotransfected with GST fusion and HA- or myc-tagged mammalian expression vectors. Upon lysis in immuno-precipitation buffer, GST proteins were pulled down withGSH-Sepharose beads, and associated proteins were resolvedby SDS-PAGE and blotted with anti-myc or -HA antibodies.

His pulldowns

Ni-NTA-agarose–prebound His-tagged proteins were incu-bated with cell lysates at 4 °C for 1 h. Beads were spun down andwashed four times, and associated proteins were resolved bySDS-PAGE and blotted with anti-myc or -HA antibodies.

Immunoprecipitation

pCMV-myc-CAP1 and HA-Rap1b were cotransfected inHEK293T or PCCL3 cells. After 48 h, cells were lysed with abuffer containing 50 mM Tris/Cl, pH 7.5, 50 mM NaCl, 0.5%Nonidet P-40, 10% glycerol, and protease inhibitors. Lysateswere incubated for 1 h at 4 °C with anti-c-myc-agarose(Thermo Scientific) beads followed by four washes with lysisbuffer.

Rap1 activation assay using RalGDS-RBD

Cells transfected with a plasmid expressing HA-Rap1b werelysed with a buffer containing 25 mM Tris/Cl, pH 7.5, 150 mM

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NaCl, 1% Nonidet P-40, 5% glycerol, 5 mM MgCl2, and proteaseinhibitors. Lysates were clarified by centrifugation at 13,000rpm for 10 min at 4 °C. Nucleotide loading was performed byincubation in the presence of 10 mM EDTA and either GTP�S(100 �M) or GDP (1 mM); the reaction was terminated after 30min at 30 °C by addition of MgCl2 (60 mM). Purified GST-Ral-GDS-RBD precoupled to GSH-Sepharose beads (10 �g) wasadded to the supernatants and incubated at 4 °C for 60 min withagitation. Beads were washed four times in the same lysis buffer.After the final wash, Laemmli sample buffer was added to thesamples. Proteins were fractionated by 12% SDS-PAGE andtransferred to a polyvinylidene difluoride membrane forblotting.

Immunofluorescence staining and confocal microscopy

Stable PCCL3-GFP-Rap1b cells were cultured on glasscoverslips in 6-well plates. For immunofluorescence stain-ing, cells were rinsed with phosphate-buffered saline (PBS;pH 7.4), fixed in 3.7% formaldehyde in PBS for 10 min atroom temperature, permeabilized in 0.5% Triton X-100 inPBS for 20 min, and washed in 0.1 M glycine in PBS for 10min. The coverslips were washed five times for 5 min eachwith PBS and incubated in PBS containing 1% bovine serumalbumin (BSA) and 1% goat serum for 30 min followed byanti-CAP1 and secondary antibodies. After five washes inPBS containing 1% BSA for 10 min, coverslips were mountedin PermaFluorTM (Thermo Fisher Scientific) and examinedby confocal microscopy (Olympus Fluoview FV1000) utiliz-ing the appropriate filters. Images were analyzed with thebuilt-in software and ImageJ.

GGTase I expression and purification

Plasmids p316 (pGATEV-Ftase-�) and p310 (pET-28a-GGTase-I-�) were a kind gift from Dr. Kirill Alexandrov (Uni-versity of Queensland, Australia). p316 and p310 were cotrans-formed in BL-21(DE3) competent cells. GGTase I expressionand purification followed the protocol for His-tagged proteinsmentioned above. Proteins were eluted in 1� PBS containing200 mM imidazole. Imidazole was removed through dialysisagainst PBS buffer.

In vitro prenylation

In vitro prenylation of Rap1 proteins was performed in abuffer containing 5 mM MgCl2, 10 �M ZnCl2, 2 mM DTT, and 10�M GDP in PBS. Typically, a 50-�l mixture contained 15 �M

purified Rap1 protein, 2 �M GGTase I, and 15 �M GGPP orNBD-FPP. Rap1 proteins and GGTase I were independentlyprewarmed at 37 °C for 5 min. Mixed samples were incubated at37 °C for 20 min. Free GGPP was removed through centrifugalfilter units.

Microscale thermophoresis

Labeling of His-C-CAP� proteins with NT647-maleimidefluorescent dye followed the protocols provided by Nano-Temper. Briefly, 20 �M purified proteins were mixed with 60�M dye in a volume of 100 �l. Labeling mixture was incubatedfor 30 min at room temperature in the dark. Free dye wasremoved through the column from the kit. The thermophoresis

measurements were performed in a Monolith NT.115 instru-ment with blue/red channels (NanoTemper) using Premiumcoated capillaries (NanoTemper, catalog number MO-K005).For experiments with OG-HVR peptide binding to His-C-CAP�, samples were prepared in MST binding buffer (50 mM

Na-HEPPSO, pH 7.8, 5 mM TCEP, 0.05% Tween 20, 0.05% Plu-ronic F-127, and 0.1 mg/ml BSA). The peptides were used at afinal concentration of 25 nM, and measurements were per-formed at 14% LED and 60% MST power. For experiments withNT647-maleimide–labeled His-C-CAP� binding to His-Rap1,samples were prepared in MST binding buffer, 1� PBS, 0.05%Tween 20, 0.05% Pluronic F-127, and 0.1 mg/ml BSA. Thelabeled His-C-CAP� was used at a final concentration of 100nM, and measurements were performed at 95% LED and 60%MST power. For experiments with His-C-CAP� binding toGFP-Rap-C-ter, samples were prepared in the same MST bind-ing buffer as labeled His-C-CAP�, GFP-Rap-C-ter was used ata final concentration of 15 nM, and measurements were per-formed at 40% LED and 60% MST power. Dose responses intriplicates (mean S.E.) were analyzed with the unit softwareor upon import into GraphPad Prism.

Rap1 localization upon CAP1 depletion

GFP-Rap1– expressing PCCL3 cells were transfected with ashCAP1 and sh-V control plasmids. The vector used, pSIREN,had an independently driven cassette for expression of a redfluorescent protein (dsRed), thus marking transfected cells.Cells were harvested 72 h posttransfection and processedfor immunofluorescence detection of endogenous CAP1expression and GFP-Rap1 intracellular distribution by con-focal microscopy. Regions of interest were drawn (polygontool in ImageJ) on plasma membrane (PM) and whole cellson random red�/green� cells. Background-subtracted pixelintensity in the green channel was measured in the selectedregions of interest, and the PM/total ratio was calculated foreach cell. Data were analyzed, and significance was testedusing a two-tailed Student’s t test (� level was defined as0.05).

Molecular modeling and dynamics simulations

Simulations were performed on the human C-CAP in com-plex with the processed and unprocessed C terminus of humanRap1b. Specifically, for the processed peptide, amino acids176RKKSSC181 were modeled in an extended conformationwhere the C-terminal carboxylic acid group is methylated andthe cysteine is linked to a farnesyl or geranylgeranyl lipid group.The structures of the complex were built by placing the farnesylor geranylgeranyl into the higher resolution (lower B-factor)monomer unit of the C-CAP protein, taking residues 319 – 452of the asymmetric dimer crystal structure (Protein Data Bankcode 1K8F, residues 319 – 475; this removes the C-terminal�-strands of C-CAP that are domain-swapped in the dimer).The lipid group was inserted either in the N- or the C-terminalopening of the C-CAP �-solenoid to the farthest extent possibleand oriented so that the charged or polar groups of RKKSSpoint in the direction of oppositely charged groups or polargroups that surround the tunnel opening. Simulations were alsorun with lipidated peptides in which the Ser179 side-chain

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hydroxyl was modified with a phosphorous, PO3�2 group. In the

case of the unprocessed Rap1b C-terminal peptide, amino acids176RKKSSCQLL184 were also inserted into the N- or C-terminaltunnel opening as deeply as possible. However, because theC-terminal group (treated as protonated), Cys–SH, and Glnand Ser side chains are polar, the peptide was placed near theside of the tunnel that contains polar residues, for example,making initial contacts with Ser394, Cys375, and Cys356 forN-terminal insertion. Similarly, no structure for a CAP1 pro-tein (of PDE)–inserted deltarasin is available, and we placed themolecule into the tunnel to initially make contacts between itsrings and C-CAP residues Phe382, Ile342, and Trp329. The poten-tial function parameters for the farnesyl and geranylgeranylgroups as well as for deltarasin (PubChem CID 73292904) weregenerated by the CHARMM generalized force field (CGenFF)online tool (102). Otherwise the CHARMM36 potential func-tion, including the CMAP correction, which also includesphosphoserine, was applied to the system (103–105). TheTIP3P model (106) was used for water. Each system was firstbriefly minimized to remove possible clashes and then solvatedin water within a box of pre-equilibrated solvent so that noprotein atom was closer than 1.0 nm to the box edge. The sys-tem was then neutralized and maintained at a near-physiolog-ical ion concentration of 0.15 M NaCl by randomly replacingwater molecules. In these all-atom simulations, the electro-static interaction was treated by the particle-mesh Ewaldmethod (107) for a rectangular periodic boundary box. The vander Waals interaction was truncated at 1.2 nm. The time stepwas set as 2 fs. Temperature was coupled by using a Langevinthermostat at 300 K, whereas pressure was 1 bar controlled by asemi-isotropic Langevin scheme. Energy minimization wasdone on the protein complex and waters, respectively, and onthe entire system after solvation. All simulations of C-CAP incomplex with peptides were run initially for 5 ns using theNAMD 2.10 package (87). The simulations were transferred tothe Ohio Supercomputer Center and run for another 95 ns. Thesimulations of C-CAP in complex with deltarasin were run onthe Ohio Supercomputer Center for 50 ns. The trajectorieswere analyzed using distance measurements between nonhy-drogen atoms of C-CAP to the nearest nonhydrogen atoms ofpeptide or inhibitor. Distances within a cutoff of 5 Å werecounted in each trajectory frame, examined at 100-ps intervals.After superposition on C� atoms of C-CAP, we calculated thedistance between the C-terminal peptide or lipid carbon atominserted initially into the tunnel and its subsequent position,measured as distance as a function of simulation time (Fig. S2).To remove directionality and to calculate the distance betweentwo points at time t � 0 and at a later time in x,y,z coordinatespace, the deviation distance is the r.m.s.d.

Author contributions—X. Z., S. C., G. B., M. B., and D. L. A. concep-tualization; X. Z., S. C., G. B., M. B., and D. L. A. formal analysis;X. Z., M. B., and D. L. A. supervision; X. Z., M. B., and D. L. A. fund-ing acquisition; X. Z., S. C., G. B., M. M. E., M. W., N. N., and M. B.methodology; X. Z., S. C., G. B., M. B., and D. L. A. writing-originaldraft; X. Z., M. B., and D. L. A. writing-review and editing; S. C. andG. B. visualization; G. B., M. M. E., M. W., N. N., and M. B.resources.

Acknowledgments—We thank Drs. Pat Casey (Duke, North Carolina)and Kirill Alexandrov (Queensland, Australia) for GGTase I proteinand GGTase I expression plasmids, respectively, and Dr. Phil Stork(Vollum Institute, Oregon) for the effector domain constructs. We alsothank Dr. Zhen Lu Li (Case Western Reserve University, Ohio) for helpwith the simulation setup and analysis. This project used the Univer-sity of Pittsburgh Peptide and Peptoid Synthesis Core for peptide syn-thesis and fluorophore conjugation services. Computational resourcesat the Case High Performance Computing Cluster and Ohio Super-computing Center are acknowledged as well.

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CAP1 binds prenylated Rap1

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Nyla Naim, Matthias Buck and Daniel L. AltschulerXuefeng Zhang, Shufen Cao, Guillermo Barila, Martin M. Edreira, Mamta Wankhede,Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

doi: 10.1074/jbc.RA118.001779 originally published online April 4, 20182018, 293:7659-7673.J. Biol. Chem. 

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VOLUME 293 (2018) 7659 –7673DOI 10.1074/jbc.AAC118.005290

Correction: Cyclase-associated protein 1 (CAP1) is aprenyl-binding partner of Rap1 GTPase.Xuefeng Zhang, Shufen Cao, Guillermo Barila, Martin M. Edreira,Kyoungja Hong, Mamta Wankhede, Nyla Naim, Matthias Buck,and Daniel L. Altschuler

PAGE 7659

Dr. Kyoungja Hong was inadvertently omitted from the author list.The corrected list is shown above.

ADDITIONS AND CORRECTIONS

J. Biol. Chem. (2018) 293(36) 13849 –13849 13849© 2018 Zhang et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.