Ras Gtpase Activating (Rasgap) Activity of the Dual Speciﬁcity GAP Protein Rasal Requires Colocalization and C2 Domain Binding to Lipid Membranes

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Ras GTPase activating (RasGAP) activity of the dual speciﬁcity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes

Begoña Sota,b, Elmar Behrmannc,1, Stefan Raunserc, and Alfred Wittinghofera,2

Departments of aStructural Biology and cPhysical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany; and bDepartment of Structure of Macromolecules, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientíﬁcas, 28048 Madrid, Spain

Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved November 16, 2012 (received for review January 30, 2012)

Rasal, belonging to the GAP1 subfamily of Ras GTPase-activating by a process that is mechanistically unclear, in particular be- proteins (RasGAPs) with dual RasGAP/RapGAP specificity, is epige- cause isolated Rasal in solution shows RapGAP but almost no netically silenced in several tumor types. Surprisingly, the isolated RasGAP activity (15). protein has GAP activity on Rap but not on Ras. Its membrane C2 domains are around 130 residues in length and fold in an recruitment is regulated by interaction with calcium and lipids, eight-stranded antiparallel β sandwich. Three loops, named cal- which simultaneously induces its RasGAP activity through a yet cium binding loops (CBL1, CBL2, and CBL3), are responsible 2+ unknown mechanism. Here we show that the interaction of Rasal for binding Ca ions (18). Specifically, CBL1 and -2 contain with membranes induces Rasal RasGAP activity by spatial and con- aspartate residues that serve as bidentate ligands for either two 2+ formational regulation, although it does not have any effect on its or three Ca ions. Binding of phospholipids to C2 domains is 2+ RapGAP activity. Not only is colocalization of Rasal and Ras in the mediated by Ca , which interacts with the negatively charged membrane essential for RasGAP activation, but direct and Ca-de- head groups of the phospholipids, mostly phosphatidylserine pendent interaction between the tandem C2 domains of Rasal and (PS) (18). Residues situated in the CBLs determine the selec- lipids of the membrane is also required. Whereas the C2A domain tivity for certain lipids. Members of the GAP1 family possess two binds specifically phosphatidylserine, the C2B domain interacts consecutive C2 domains, C2A and C2B, a situation frequently BIOCHEMISTRY with several phosphoinositol lipids. Finally we show, that similar found in exocytosis-associated proteins, like Synaptotagmin I. to the C2 domains of synaptotagmins, the Rasal tandem C2 In this study we characterize the lipid binding specificity of the domains are able to sense and induce membrane curvature by C2 domains of Rasal and show their capability to sense and/or the insertion of hydrophobic loops into the membrane. induce membrane curvature. Furthermore, we unravel the mechanism of Rasal RasGAP activation by membrane binding. We as is a GTP-binding protein (G protein in short) of the Ras show that, whereas colocalization of both Rasal and Ras in the Rsubfamily. It is involved in a wide range of important cellular membrane is required for the activity, additional binding of the processes. When Ras binds GTP, it is in an active conformation C2 domains is necessary for full activation. “ ” ( on state) and hence able to bind to and activate downstream Results effectors. The hydrolysis of GTP to GDP inactivates Ras (“off” state). GTPase-activating proteins (RasGAPs) are responsible Rasal Binds Phosphatidylserine and Phosphatidylinositol Phosphates. fi for accelerating the intrinsically slow GTPase activity of Ras. To study the effect of lipids on Rasal RasGAP activity, we rst fi Oncogenic Ras mutants incapable of hydrolyzing GTP are found characterized the lipid binding speci city of Rasal C2 domains. – in 20–30% of human tumors (1). Frequently mutated residues Rasal lipid vesicle interaction was measured by cosedimentation are the P-loop glycine and the catalytic Gln61 (2, 3). Mutations experiments (15, 19). Using similar methods, Walker et al. (15) or posttranslational modifications of RasGAPs, leading to de- had previously shown that Rasal C2 domains bind phosphati- regulation or inactivation of the proteins, also contribute to tumor dylcholine (PC) and phosphatidylserine (PS), but do not bind formation in cells containing wild-type (WT) Ras. This is partic- phosphatidylinositol (PI) or phosphatidylethanolamine (PE). ularly evident in the case of the tumor suppressor neurofibromin, However, in our hands Rasal did not efficiently bind to PC or a RasGAP, which is mutated or deleted in neurofibromatosis PC:PS (85:15 molar ratio) lipid vesicles (Fig. 1A). Rasal C2 type I (4, 5). Furthermore, the RasGAP family member Rasal domains (C2A–C2B) are quite homologous to the C2A–C2B (Ras-GTPase–activating-like protein) is epigenetically silenced domains of synaptotagmins, that bind PS and several phospha- by methylation in multiple tumors (6–10). tidylinositol phosphates (PIPs) (20). Thus, we measured the Rasal belongs to the Gap1 subfamily, which consists of Rasal, binding of Rasal to lipid vesicles containing PC, PS, and different Capri, tetrakisphosphate binding protein (GAP1IP4BP) and PIPs (PI3P; PI3,4P; PI4,5P; and PI3,4,5P), at a molar ratio GAP1m. Members of the Gap1 family contain two N-terminal C2 similar to physiological membrane composition (PC:PS:PIP + domains (C2A and C2B), a GAP domain, and a pleckstrin ho- 75:15:10) in the presence of 1 mM Ca2 . This calcium concen- mology domain (PH) connected to a Bruton’s tyrosine kinase tration is high enough to ensure that the lipid binding is not (Btk) motif. Rasal, Capri, and GAP1IP4BP are dual-specificity limited by calcium concentration, but not too high to induce GAPs, able to stimulate the slow GTPase reaction of both Ras and Rap in vivo (11–13). Another interesting feature of Rasal and Capri is that their GAP activity is regulated by intracellular Author contributions: B.S., S.R., and A.W. designed research; B.S. and E.B. performed 2+ Ca levels (14, 15). Their C2 domains are able to bind phos- research; B.S., E.B., and S.R. analyzed data; and B.S. and A.W. wrote the paper. 2+ pholipids at high Ca concentrations, although their PH domains The authors declare no conflict of interest. m seem to be inactive for lipid binding (14). In contrast, GAP1 and This article is a PNAS Direct Submission. IP4BP GAP1 have inactive C2 domains for lipid interaction, but 1Present address: Institute of Medical Physics and Biophysics, Charité, Universitätsmedizin bind to membranes through their PH domain (16, 17). Thus, a Berlin, 10117 Berlin, Germany. receptor-mediated increase in intracellular calcium concentration 2To whom correspondence should be addressed. E-mail: alfred.wittinghofer@mpi- recruits Rasal and Capri, which both reside as soluble proteins dortmund.mpg.de. in the cytoplasm, to the plasma membrane. Association with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the membrane increases RasGAP activity of Rasal and Capri 1073/pnas.1201658110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1201658110 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 PIP was removed, only 20–50% of Rasal was bound to the lipid vesicle (Fig. 1A). Thus, a combination of PS and PIPs is needed for efficient binding. This interaction depends on calcium, as the presence of EDTA inhibits the binding (SI Appendix, Fig. S2). To compare Rasal affinity for PS and PIP, Rasal binding to PS:PC (10:90) and PI3P:PC (10:90) at different total lipid concentration was measured (SI Appendix, Fig. S3), showing that Rasal pos- sesses a similar affinity for both lipids. The resultant apparent dissociation constants (Kds) were 35 ± 23 and 29 ± 18 μM for PI3P and PS, respectively, which are in a similar range of other C2 domain affinities for lipids (21–24). For synaptotagmins and other proteins with tandem C2A–C2B domains, it has been shown that C2B is responsible for binding to PIPs, mostly by a calcium-independent mechanism (25). To check if this is also the case for Rasal C2 domains, we measured the binding of RasalΔC2A, which lacks the C2A domain, to PC: PS:PIP lipid vesicles (Fig. 1B and SI Appendix, Fig. S1). Similar to other C2A–C2B proteins, the absence of one C2 domain decreased the binding to vesicles (25, 26). Only 30–60% of total protein interacted with the vesicles containing PC:PS:PIP. In- terestingly, these values did not decrease when PS was removed (PC:PIP vesicles), suggesting that the C2B domain binds pref- erentially to PIPs rather than to PS. ln contrast to synaptotagmin, binding of C2B to PIPs is calcium dependent, as it is inhibited by the presence of EDTA (SI Appendix, Fig. S2).

RasGAP Activation of Rasal by Colocalization of Ras and Rasal. It was previously reported that isolated Rasal is not an active RasGAP in solution (15). Furthermore, in cells expressing Rasal, the RasGAP activity was detected only upon increasing the intracellular calcium concentration, which in turn is responsible for Fig. 1. Rasal interacts with PS and PIPs. A total of 1 μM Rasal WT (A)or Rasal membrane localization. However, the mechanism of acti- RasalΔC2A (B) was incubated (30 min, 25 °C) with MLVs at 500 μM lipid vating the RasGAP activity through Rasal lipid binding is un-

concentration, in 50 mM Tris·HCl, 150 mM NaCl, 1 mM CaCl2. Percentage of known, in particular whether the activation is due to lipid or binding was calculated by cosedimentation. calcium binding. One possible mechanism would be the allosteric activation of the GAP domain by C2 domains binding to calcium and/or lipid. Thus, we measured the RasGTPase activity in the fusion of the vesicles in the condition used (SI Appendix,Fig. presence of recombinant soluble Rasal. As expected and pre- S7). Indeed, Rasal bound efﬁciently to vesicles containing PS viously reported, soluble Rasal was not able to activate the GTP and PI3P; PI3,4P; or PI4,5P although binding to PI3,4,5P hydrolysis of Ras. Furthermore, the addition of calcium (1 mM containing liposomes was much weaker compared with the other CaCl2) had no effect on the RasGAP activity (Fig. 2A), ruling phosphoinositides (Fig. 1A and SI Appendix, Fig. S1). When PS or out this ion as a direct (allosteric) RasGAP regulator.

Fig. 2. Rasal RasGAP function is activated by colocalization with Ras in lipid vesicles. GTPase activity of 100 μM Ras-GTP (A) in the absence of GAP or in the presence of 2.5 μM Rasal or 2.5 μM Rasal and 1 mM CaCl2. (B) In the presence of 2.5 μM Rasal, 1 mM

CaCl2 and 100 μM soluble lipids (phosphatidylinositol 3-phosphate diC8 [PI(3)P diC8) and 1,2-dioctanoyl- sn-glycero-3-phospho-L-serine (PS diC8)] or in the presence of Rasal bound to PC:PS:PI3P (75:15:10 molar ratio) vesicles, 3 mM total lipid concentration

and 1 mM CaCl2.(C) GTPase activity when 100 μM Ras-GTP and 2.5 μM Rasal are bound to the same vesicles (PC:DOGS-NTA:PS:PI3P at 52.5:30:10:7.5 molar ratio, 3 mM total lipid concentration) or when Ras and Rasal are bound to different vesicles. A total of 100 μM Ras-GTP bound to 3 mM PC:DOGS-NTA 50:50 vesicles and 2.5 μM Rasal bound to PC:PS:PI3P 65:30:15. (D) GTPase activity of 100 μM Rap-GTP catalyzed by 25 nM Rasal in solution and when both proteins are bound to the same vesicles (PC:DOGS- NTA:PS:PI3P at 52.5:30:10:7.5 molar ratio, 3 mM total lipid concentration).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1201658110 Sot et al. Downloaded by guest on September 27, 2021 To test a possible effect of anionic polar lipid head groups, lipids. The single mutants D21A and D202A, that eliminate two we measured the GTPase activity in the presence of PS and PI3P of the aspartic acid residues implicated in calcium interaction, with short aliphatic chains of eight carbons, which are soluble in have previously been used (15) to inhibit lipid interaction. To aqueous buffers. Rasal was still inactive in their presence (Fig. more drastically affect calcium binding, we designed a double 2B). Finally, we wanted to explore the possibility that Rasal [D21A/D202A, here called Rasal(DD) mutant] and a quadruple needs the insertion of C2 domains in the lipid vesicle for allo- mutant with two additional mutations of aspartic acids [D21A/ steric activation. Therefore, we bound Rasal to lipid vesicles D76R/D149R/D202A, here called Rasal(DDDD) mutant]. The (PC:PS:PI3P 75:15:10) (SI Appendix, Fig. S4A) and measured mutations were picked based on the high sequence homology GTP hydrolysis of soluble Ras. However, there was no activation with synaptotagmin 1 (SI Appendix, Fig. S6A) and the crystallo- of RasGAP function, indicating that Rasal is not appreciably graphic structure of its C2A domain bound to calcium (36). activated by the presence of these lipids (Fig. 2B). As expected, Rasal(DD) and Rasal(DDDD) mutants did not Because a direct and strong allosteric activation mechanism can bind vesicles containing PS and PI3P (SI Appendix, Fig. S6B). To be excluded, the most plausible possibility is that Rasal is not an colocalize both mutants with Ras in lipid vesicles, we took ad- active GAP due to very low affinity for Ras. In fact, fluorescence vantage of the unexpected capability of Ni-NTA-DOGS to bind IP4BP polarization experiments with Rasal homolog GAP1 and GAP domains. Rasal WT could bind PC:NTA-DOGS vesicles fluorescently labeled Ras·mGppNHp, a standard method to (SI Appendix, Fig. S6C), unless they did not contain PS or PI3P. measure affinity between G proteins and its cognate GAP (27–29), This interaction was not mediated by the C2 domains, as Rasal gave no binding saturation signal, even at high GAP concentration (DD) and Rasal(DDDD) mutants, as well as RasalΔC2AB (100 μM) (SI Appendix,Fig.S5), indicating a rather low binding IP4BP mutant, lacking the C2 domains, were able to bind these vesicles. affinity, although GAP1 is an active RasGAP in solution (11, GAP1IP4BP isolated GAP domain could also interact with the 13). The colocalization of both proteins in the same membrane PC:NTA-DOGS vesicles (SI Appendix, Fig. S6C), leading us to would induce close proximity and facilitate an effective in- the conclusion that the Ni-NTA-DOGS binding site is in the teraction. In vivo, Ras is attached to cellular membranes due to GAP domain. This binding did not interfere with GAP activity, lipidation of its C terminus. For Ras isoforms H- and N-Ras, this as the GAP domain of the closely related GAP1IP4BP colocalized fi modi cation involves farnesylation of cysteine 181 and further with Ras in PC:NTA-DOGS vesicles possessed the same Ras- processing of the C-terminal Caax box (30). To mimic Ras in- GAP activity as that of the unbound protein with free Ras in teraction with membranes, we used the C-terminal His6-tag of our SI Appendix D Materials and Methods solution ( , Fig. S6 ). Rasal(DD) and Rasal(DDDD) Ras construct ( ), which is expected to bind mutants of Rasal colocalized with Ras in PC:NTA-DOGS:PS: to vesicles containing Ni-NTA lipids, such as 1,2-dioleoyl-sn-glycero- BIOCHEMISTRY N PI3P vesicles were much less active than Rasal WT under the 3-{[ -(5-amino-1-carboxylpentyl)iminodiacetic acid]succinyl} same conditions (Fig. 3). The structure of the proteins appears to (nickel salt) (Ni-NTA-DOGS). This method has been successfully be unperturbed because the mutants remained active RapGAPs used for several proteins (31, 32), including the small GTP binding (SI Appendix, Fig. S6E). However, the extent of Ras activation by protein Arf6 (33). When both proteins were bound to PC:DOGS- SI Appendix B the mutants on lipid membranes was still higher compared with NTA:PS:PI3P (52.5:30:10:7.5) vesicles ( ,Fig.S4), soluble components. This shows that for full activation, coloc- there was indeed a strong activation of the GTPase activity of Ras fi C alization of Ras and Rasal is not suf cient and requires direct (Fig. 2 ). As expected when both enzyme and substrate are at- interaction of the C2AB domains to lipids. tached to membrane vesicles, the reaction kinetics did not follow a normal monoexponential equation. The association rate constant Rasal C2AB Domains Induce Curvature in Lipid Vesicles. Rasal C2 decreases with time due to enzyme and substrate segregation at domains have high sequence homology with synaptotagmins. These longer times as previously observed (33–35). To check whether it fi proteins are implicated in neurotransmitter exocytosis and are able is suf cient to localize the proteins to membranes, or whether to promote membrane fusion. Their interactions with membranes, colocalization in the same membrane compartment and thus mediated by calcium binding, promote the insertion of four hy- proximity is required, we measured the GTP hydrolysis when drophobic loops (two per C2 domain) into the membrane. This Rasal and Ras were bound to different vesicles. We thus pre- insertion induces membrane curvature, which triggers membrane incubated Rasal with vesicles without DOGS-NTA, and RasGTP with vesicles without PS and PI3P. When both populations were mixed, Rasal did not show any RasGAP activity, as shown in Fig. 2C. Members of the Gap1 family such as Rasal and GAP1IP4BP possess GAP activity for both Ras and Rap (11). Interestingly, it has been shown, using cells overexpressing Rasal and Rap (11), that RapGAP activity of Rasal in vivo appeared to be independent of its translocation to plasma membrane. To check the possible effect of Rasal and Rap colocalization in membranes using the system described above, we bound Rasal and Rap1B(1–180)-His6 to vesicles. As can be seen in Fig. 2D, it did not induce a relevant increase of Rasal RapGAP activity, arguing that for the latter activity, colocalization does not provide any advantage.

Allosteric Stimulation from Lipid Binding of C2 Domains. It has been previously shown that the tandem C2 domains increase the af- ﬁnity of GAP1IP4BP, a close homolog of Rasal, for Ras (13), although it is unclear whether this is due to their direct partici- pation in Ras binding or to conformational changes in the GAP domain by the presence of C2AB. Although we have shown here for Rasal that colocalization seems to be essential for activity, it might still be possible that membrane binding of C2 domains and their correct orientation has an additional direct effect on Fig. 3. Binding of C2 domains to lipid vesicles contributes to Rasal RasGAP RasGAP activity. To test for such a direct effect, it was necessary activity. (A) RasGAP activity of Rasal WT, (DD) and (DDDD) (2.5 μM) colo- to have a system where both proteins colocalized in the same calized in PC:DOGS-NTA:PS:PI3P 52.5:30:10:7.5 vesicles with 100 μM Ras-GTP. membrane but where the C2AB domains were unable to bind To compare, RasGAP activity of Rasal in the absence of lipids is also shown.

Sot et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 fusion (26). Interestingly, the hydrophobic residues able to induce residues V22, V78, L149, and V206 are essential for changing membrane curvature are also conserved in Rasal (as V22, V78, the curvature of the membrane. L149, and V206) (SI Appendix,Fig.S6A). This opens the possibility of Rasal C2 domains also being “active domains,” able to sense and Discussion or induce membrane curvature and therefore to be implicated in Rasal expression is down-regulated in several types of cancer, the membrane fusion or fission events. To test this possibility, we consequence of which should be enhanced Ras activation in performed two kinds of experiments previously applied to syn- cancer cells containing WT Ras. However, whereas Rasal has aptotagmin 1 (26). We first measured Rasal binding to liposomes a consistent RapGAP activity, its RasGAP activity, which is of different diameters. If the C2 domains induce curvature due highly regulated by the interaction of its C2 domains with calcium to the insertion of the hydrophobic loops, they should show and lipids, seems to be biologically at least as important. We thus a preference of binding to curved membranes. We prepared size- set out to explore the molecular mechanism of this regulation and fractionated liposomes of ∼170, 115, 85, and 65 nm diameters the contribution of the Rasal C2 domains. Here we show that the (experimentally measured by dynamic light scattering) and C2A domain binds PS, and the C2B domain interacts with several checked that the calcium concentration used (1 mM) for Rasal PIPs. This kind of selectivity for both types of negative lipids has binding did not induce vesicle fusion (SI Appendix, Fig. S7). Rasal also been described for synaptotagmins (22) and Rabphilin3A WT in the presence of calcium showed a higher affinity for (38, 39), although only for Rabphilin3A a calcium-dependent PIP smaller vesicles (Fig. 4 and SI Appendix, Fig. S8A), with the binding has been described (39). A comparison of Rasal C2B and highest affinities for liposomes of 85 and 65 nm. In the presence of Rabphilin3A C2A domain sequences suggests that Rasal C2B PIP EDTA, when the binding to liposomes is calcium independent, we specificity is provided by negative residues inside the CBL2 loop did not observe any preference for smaller vesicles. RasalΔC2A, (see SI Appendix for a longer discussion). which lacks two of the four hydrophobic residues, needs a bigger The results presented here show that calcium and membrane curvature to significantly increase its membrane binding. Thus, by interaction activates Rasal RasGAP activity by (i) temporal, (ii) decreasing the number of bulky hydrophobic residues a very low spatial, and (iii) conformational regulation. The (i) temporal lipid packing is required to increase the probability of insertion. regulation is introduced by the necessity of a high intracellular Finally, a Rasal mutant, called Rasal(AAAA), with the four bulky calcium concentration (15) for Rasal membrane binding, which hydrophobic residues (V22, V78, L149, and V206) mutated to will allow calcium release-dependent Rasal-mediated Ras in- alanine, had no preference for curved membranes. activation. The (ii) spatial regulation is controlled by the low We went on to test the ability of Rasal to induce tubulation of affinity of Rasal for Ras. The colocalization of Ras and Rasal in Folch lipid liposomes (100 nm diameter) using electron mi- close proximity in the same membrane would increase their local croscopy (EM). These lipids have been used instead of PC:PS: concentration, allowing their interaction only in spatially defined PI3P liposomes before (26, 37), because the latter are unstable areas of the cell. Such colocalization would increase 1,000 times under EM conditions (26). Rasal is able to bind Folch lipid the effective protein concentration (40–42). Furthermore, the liposomes (SI Appendix, Fig. S8B), and EM reveals that the Rasal selectivity for PIPs could also be important for this spatial “ ” protein induces membrane tubulation (Fig. 5 A and C and SI regulation although cell biology and in vivo experiments would Appendix, Figs. S9 and S10). Remarkably, the depletion of free be required to completely elucidate its physiological relevance. + fi Ca2 by EDTA inhibits the tubulation, suggesting that it is in- Rasal can bind PIPs as well as PS, and with similar af nity. Al- + duced by a Ca2 -mediated interaction of Rasal with the mem- though PS is a more abundant lipid than PIPs, the later are fi brane (Fig. 5 B and D)(SI Appendix, Figs. S9 and S10). Control enriched in speci c membranes or domains. Different PIPs are experiments in absence of protein showed that this effect is not distributed in different cellular compartments and could there- due to the buffer conditions (Fig. 5 E–G). To check whether the fore control where Rasal will inactivate Ras. Walker et al. (15) + Ca2 -dependent tubulation is induced by the insertion of hy- showed that Rasal interacted with the plasma membrane. How- drophobic residues of C2 domains, we performed the same ever, the authors did not check if Rasal also localized to other experiments using Rasal(AAAA), which lacks the hydrophobic subcellular membranes. Walker et al. (15) also showed that Rasal residues thought to be responsible for tubulation. Although the did not associate with the plasma membrane after EGF receptor protein seems to localize to the liposomes (SI Appendix, Fig. stimulation, inferring that the association was not controlled by S8B), and it causes their clustering, it does not induce tubulation PI3K activation and leading to the conclusion that Rasal did not (Fig. 5 H and I and SI Appendix, Figs. S9 and S10). This suggests bind PI3,4P. Although more experiments are needed to com- that the tandem C2 domains and the conserved hydrophobic pletely rule out a contribution of PI3,4P, it is a possibility that an increase of this lipid concentration is not required for the membrane binding of Rasal, as it is already highly controlled by calcium. This suggests that PI4,5P, a constitutive and abundant PIP (25), could interact with Rasal C2B domain in the plasma membrane. As it has been suggested before for GAP1m (43), its capability to associate with PIP2 would target RASAL out of lipid rafts, because PIP2 does not accumulate in rafts (44). As Ras has been shown to be absent from rafts after GTP loading, PIP2– Rasal interaction would target Rasal to activated Ras in the lipid- unordered phase. Furthermore, PI4,5P is enriched in membrane structures prone to be endocyted, which can be related to Rasal capability to sense and induce membrane curvature (see below). The (iii) conformational regulation is introduced by the fact that a complete Rasal RasGAP activation needs the interaction of C2AB domains with lipids in the membrane. It can be speculated that, as Rasal affinity for Ras is low, a favorable orientation for the interaction with Ras is achieved by the binding of the C2 domains to lipids. In addition, an allosteric activation similar IP4BP Fig. 4. Rasal binding preference for curved vesicles. The binding of 1 μM to the one whereby GAP1 regulates RapGAP activity via Rasal WT, RasalΔC2A, and Rasal(AAAA) to LUVs of different diameter (260 the C2 domains (13) cannot be ruled out. Thus, in contrast to IP4BP μM total lipid concentration), in the presence of 1 mM CaCl2 or 1 mM EDTA GAP1 , C2AB domains of Rasal are required for both Ras- was measured by cosedimentation. GAP and RapGAP activities.

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+ Fig. 5. Ca2 -dependent tubulation of vesicles by Rasal. (A–H) Electron micrographs of negatively stained vesicles incubated with the indicated additives for + 30 min at RT. (A and C) Vesicles tubulate in the presence of 1 mM Ca2 and 10 μM Rasal WT. (B and D) Tubulation is prevented when EDTA is used to complex + + Ca2 ions. (E–G) Control experiments showing that neither 1 mM Ca2 nor 1 mM EDTA alone have an effect on the vesicles. (H and I) Mutation of the hydrophobic residues leads to vesicle clustering instead of tubulation in the presence of 1 mM Ca2+ and 10 μM protein. (Scale bars, 100 nm.)

An interesting and intriguing observation is the capability of (50). However, in contrast to ArfGAP1, membrane curvature does Rasal to sense and induce membrane curvature in vitro. Al- not increase the RasGAP (SI Appendix,Fig.S11) activity of Rasal. though cell biology studies are required to confirm such behavior Its activity is rather tightly controlled by colocalization, C2 do- in vivo, the results presented here open a unique avenue toward main binding to lipids, and high calcium concentration. Rasal biology. It is tempting to speculate that Rasal is involved in + Ca2 -mediated membrane fusion processes like endo- or exocytosis. Materials and Methods In fact, all tandem C2 domain-containing proteins able to Protein Expression and Purification. Rasal WT and mutants were purified as bind lipid membranes are involved in exocytosis. Most recep- previously described (13). A construct encoding N-Ras(1–179) with a C-terminal tors implicated in Ras activation are endocytosed and eventu- His6-tag was cloned in a ptac expression vector and expressed in Escherichia coli ally recycled (45). Endocytosis has been shown to be directly CK600K cells. Rap1B(1–180) with a C-terminal His-tag was cloned in pet-21d expression vector and expressed in BL21 DE3 Codon + RIL cells. These proteins stimulated by interaction of activated Ras with the Rab5-GEF fi 2+ fi fi RIN1 (Ras interaction proteins 1) (46, 47). Furthermore, Ras were puri ed by Ni -NTA af nity chromatography followed by gel ltration chromatography. They are referred to as Ras and Rap throughout this study. directly initiates clathrin-independent endocytosis named macropinocytosis (48). Thus, Rasal could stabilize bud struc- Vesicle Preparation. Refer to SI Appendix for lipid description and further tures in these processes. As PI(4,5)P is necessary and enriched information. The multilamellar vesicles (MLVs) were prepared rehydrating for endocytosis (dependent and independent) (45, 48), the pre- in a buffer containing 150 mM NaCl, and 25 mM Tris·HCl (pH 7.5) a dried sence of this lipid would increase Rasal accumulation and in mixture of lipids. The suspension was then subjected to nine cycles of turn Ras deactivation in a negative feedback loop. Even if Rasal freezing and thawing and used directly for the experiments. is not directly implicated in budding, the C2AB domains could For the preparation of fixed-sized large unilamellar vesicles (LUVs), MLVs (500 be used as membrane curvature sensors, driving Rasal to mem- μM lipids) of PC:PS:PI3P: rhodamine-PE at 87:7.5:5:0.5 molar ratio were passed brane regions of high curvature to turn off Ras-GTP. A similar 10 times through a 200 nm filter (Whatman) and later another 20 times kind of mechanism has been described before for ArfGAP1, a through a 200-, 100-, 50-, or 30-nm filter, using the Avanti Mini-Extruder (Avanti GTPase-activating protein of the small G protein Arf1. Arf1 Polar Lipids). The average hydrodynamic diameter of the liposomes shown in interacts with coat protein I (COPI), a coat complex that Fig. 4 was determined by dynamic light scattering, being 170, 115, 85, and 65 promotes the budding of small transport vesicles between nm, respectively (SI Appendix,Fig.S7). The total concentration of the obtained LUVs was measured using the absorbance of rhodamine at 543 nm. endoplasmic reticulum and the Golgi (49). ArfGAP1 is an active GAP only when it is bound to lipid membrane (50). It Cosedimentation Assays. Vesicles (MLVs or LUVs) were incubated with the + binds lipids by the ArfGAP1 lipid packing sensor (ALPS) motif, different proteins used in this work. Ca2 or EDTA was added to a final a high sensitive membrane interacting domain (51), and there- concentration of 1 mM. After 30 min at room temperature the liposomes fore it only binds to highly curved membranes, like transport were centrifuged in a TLA-45 rotor at 125,000 × g for 30 min (MLVs) or 1 h 30 vesicles and buds of this transport vesicle. Thus, it will inactivate min (LUVs) at 10 °C. The pellets were resuspended in buffer, up to the same Arf1 function as budding promoter only once the bud is formed volume as the supernatant. Equal amounts of the supernatants and

Sot et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 resuspended pellets volumes (usually 10 μL) were analyzed by SDS/PAGE. perature, in the presence of 1 mM CaCl2 or EDTA. Samples were prepared Proteins were subsequently visualized by Coomassie staining and quantified for electron microscopy according to Ohi et al. (54) with minor changes (SI using ImageJ program. The percentage of binding was calculated as the Appendix). Specimens were imaged at room temperature using a JEM1400 pellet protein signal percentage of the total protein (pellet + supernatant). electron microscope (JEOL) equipped with a LaB6 electron source. The microscope was operated at an acceleration voltage of 120 kV. Images were GTP Hydrolysis. Nucleotide exchange was performed according to the pro- taken at a magnification of either 10,400× or 53,800× and recorded on cedure described by ref. 52. Reverse-phase HPLC was used to monitor the a 2,000 × 2,000 14 μm-per-pixel FastScan-F214 CCD camera (TVIPS). kinetics of GTP hydrolysis as described before (53). The standard buffer for all experiments contained 25 mM Tris pH 7.5 and 100 mM NaCl. A total of ACKNOWLEDGMENTS. We thank Carolin Kroerner for Rap1B(1-180)-His fi 100 μM Ras-GTP and 2.5 μM Rasal were used. For more details about the cloning and puri cation and Carsten Kötting for the generous gift of the ptac-Ras-His plasmid. S.R. is grateful to R. S. Goody for continuous support. different conditions used, refer to SI Appendix. This work was supported by an Alexander von Humboldt Foundation Re- search Fellowship (to B.S.), Deutsche Forschungsgemeinschaft Grant RA Electron Microscopy. A total of 500 μM Folch lipid LUVs (100 nm diameter) 1781/1-1 (to S.R.), Fonds der chemischen Industrie Grant 684052 (to E.B.), were incubated with 10 μM Rasal WT or mutants for 30 min at room tem- and the Max Planck Society (S.R. and E.B.).

1. Downward J (2003) Targeting RAS signalling pathways in cancer therapy. Nat Rev 27. Leonardy S, et al. (2010) Regulation of dynamic polarity switching in bacteria by a Ras- Cancer 3(1):11–22. like G-protein and its cognate GAP. EMBO J 29(14):2276–2289. 2. Bos JL (1989) ras oncogenes in human cancer: A review. Cancer Res 49(17):4682–4689. 28. Ismail SA, et al. (2011) Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for 3. Malumbres M, Barbacid M (2003) RAS oncogenes: The first 30 years. Nat Rev Cancer farnesylated cargo. Nat Chem Biol 7(12):942–949. 3(6):459–465. 29. Veltel S, Gasper R, Eisenacher E, Wittinghofer A (2008) The retinitis pigmentosa 2 4. Dasgupta B, Dugan LL, Gutmann DH (2003) The neurofibromatosis 1 gene product gene product is a GTPase-activating protein for Arf-like 3. Nat Struct Mol Biol 15(4): neurofibromin regulates pituitary adenylate cyclase-activating polypeptide-mediated 373–380. signaling in astrocytes. J Neurosci 23(26):8949–8954. 30. Brunsveld L, Waldmann H, Huster D (2009) Membrane binding of lipidated Ras peptides 5. Cichowski K, Jacks T (2001) NF1 tumor suppressor gene function: Narrowing the GAP. and proteins—the structural point of view. Biochim Biophys Acta 1788(1):273–288. Cell 104(4):593–604. 31. Dietrich C, Schmitt L, Tampé R (1995) Molecular organization of histidine-tagged 6. Kolfschoten IG, et al. (2005) A genetic screen identifies PITX1 as a suppressor of RAS biomolecules at self-assembled lipid interfaces using a novel class of chelator lipids. activity and tumorigenicity. Cell 121(6):849–858. Proc Natl Acad Sci USA 92(20):9014–9018. 7. Jin H, et al. (2007) Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating 32. Oh KJ, et al. (2006) A membrane-targeted BID BCL-2 homology 3 peptide is sufficient protein RASAL defines a new mechanism of Ras activation in human cancers. Proc for high potency activation of BAX in vitro. J Biol Chem 281(48):36999–37008. Natl Acad Sci USA 104(30):12353–12358. 33. Ismail SA, Vetter IR, Sot B, Wittinghofer A (2010) The structure of an Arf-ArfGAP + – 8. Ohta M, et al. (2009) Decreased expression of the RAS-GTPase activating protein RASAL1 complex reveals a Ca2 regulatory mechanism. Cell 141(5):812 821. is associated with colorectal tumor progression. Gastroenterology 136(1):206–216. 34. Berry H (2002) Monte carlo simulations of enzyme reactions in two dimensions: – 9. Seto M, et al. (2011) Reduced expression of RAS protein activator like-1 in gastric Fractal kinetics and spatial segregation. Biophys J 83(4):1891 1901. cancer. Int J Cancer 128(6):1293–1302. 35. Takakuwa Y, Nishino H, Ishibe Y, Ishibashi T (1994) Properties and kinetics of mem- 10. Bell A, Bell D, Weber RS, El-Naggar AK (2011) CpG island methylation profiling in brane-bound enzymes when both the enzyme and substrate are components of the human salivary gland adenoid cystic carcinoma. Cancer 117(13):2898–2909. same microsomal membrane. Studies on lathosterol 5-desaturase. J Biol Chem 269(45): – 11. Kupzig S, et al. (2006) GAP1 family members constitute bifunctional Ras and Rap 27889 27893. GTPase-activating proteins. J Biol Chem 281(15):9891–9900. 36. Sutton RB, Davletov BA, Berghuis AM, Südhof TC, Sprang SR (1995) Structure of the fi + 12. Kupzig S, Bouyoucef-Cherchalli D, Yarwood S, Sessions R, Cullen PJ (2009) The ability rst C2 domain of synaptotagmin I: A novel Ca2 /phospholipid-binding fold. Cell – of GAP1IP4BP to function as a Rap1 GTPase-activating protein (GAP) requires its Ras 80(6):929 938. 37. Gerlach H, et al. (2010) HIV-1 Nef membrane association depends on charge, curva- GAP-related domain and an arginine finger rather than an asparagine thumb. Mol ture, composition and sequence. Nat Chem Biol 6(1):46–53. Cell Biol 29(14):3929–3940. 38. Coudevylle N, Montaville P, Leonov A, Zweckstetter M, Becker S (2008) Structural 13. Sot B, et al. (2010) Unravelling the mechanism of dual-specificity GAPs. EMBO J 29(7): determinants for Ca2+ and phosphatidylinositol 4,5-bisphosphate binding by the C2A 1205–1214. domain of rabphilin-3A. J Biol Chem 283(51):35918–35928. 14. Lockyer PJ, Kupzig S, Cullen PJ (2001) CAPRI regulates Ca(2+)-dependent inactivation 39. Montaville P, et al. (2008) The PIP2 binding mode of the C2 domains of rabphilin-3A. of the Ras-MAPK pathway. Curr Biol 11(12):981–986. Protein Sci 17(6):1025–1034. 15. Walker SA, et al. (2004) Identification of a Ras GTPase-activating protein regulated by 40. Kholodenko BN, Hoek JB, Westerhoff HV (2000) Why cytoplasmic signalling proteins receptor-mediated Ca2+ oscillations. EMBO J 23(8):1749–1760. should be recruited to cell membranes. Trends Cell Biol 10(5):173–178. 16. Cozier GE, et al. (2000) GAP1IP4BP contains a novel group I pleckstrin homology 41. Kuriyan J, Eisenberg D (2007) The origin of protein interactions and allostery in co- domain that directs constitutive plasma membrane association. J Biol Chem 275(36): localization. Nature 450(7172):983–990. 28261–28268. 42. Gureasko J, et al. (2008) Membrane-dependent signal integration by the Ras activator 17. Lockyer PJ, et al. (1999) Identification of the ras GTPase-activating protein GAP1(m) as Son of sevenless. Nat Struct Mol Biol 15(5):452–461. a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo. Curr Biol 9(5): 43. Yarwood S, Bouyoucef-Cherchalli D, Cullen PJ, Kupzig S (2006) The GAP1 family of 265–268. GTPase-activating proteins: spatial and temporal regulators of small GTPase signal- 18. Rizo J, Südhof TC (1998) C2-domains, structure and function of a universal Ca2+ ling. Biochem Soc Trans 34(Pt 5):846–850. – -binding domain. J Biol Chem 273(26):15879 15882. 44. Hanzal-Bayer MF, Hancock JF (2007) Lipid rafts and membrane traffic. FEBS Lett 19. Török Z, et al. (1997) Evidence for a lipochaperonin: Association of active protein- 581(11):2098–2104. folding GroESL oligomers with lipids can stabilize membranes under heat shock 45. Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78: – conditions. Proc Natl Acad Sci USA 94(6):2192 2197. 857–902. fi fi 20. Vrljic M, et al. (2011) Post-translational modi cations and lipid binding pro le of insect 46. Han L, Colicelli J (1995) A human protein selected for interference with Ras function – cell-expressed full-length mammalian synaptotagmin 1. Biochemistry 50(46):9998 10012. interacts directly with Ras and competes with Raf1. Mol Cell Biol 15(3):1318–1323. 21. Guerrero-Valero M, Marín-Vicente C, Gómez-Fernández JC, Corbalán-García S (2007) 47. Tall GG, Barbieri MA, Stahl PD, Horazdovsky BF (2001) Ras-activated endocytosis is me- fi The C2 domains of classical PKCs are speci c PtdIns(4,5)P2-sensing domains with diated by the Rab5 guanine nucleotide exchange activity of RIN1. Dev Cell 1(1):73–82. fi – different af nities for membrane binding. J Mol Biol 371(3):608 621. 48. Donaldson JG, Porat-Shliom N, Cohen LA (2009) Clathrin-independent endocytosis: A + fi 22. Radhakrishnan A, Stein A, Jahn R, Fasshauer D (2009) The Ca2 af nity of synapto- unique platform for cell signaling and PM remodeling. Cell Signal 21(1):1–6. tagmin 1 is markedly increased by a specific interaction of its C2B domain with 49. Lippincott-Schwartz J, Cole NB, Donaldson JG (1998) Building a secretory apparatus: Role phosphatidylinositol 4,5-bisphosphate. J Biol Chem 284(38):25749–25760. of ARF1/COPI in Golgi biogenesis and maintenance. Histochem Cell Biol 109(5–6):449–462. 23. Torrecillas A, Laynez J, Menéndez M, Corbalán-García S, Gómez-Fernández JC (2004) 50. Antonny B, et al. (2005) Membrane curvature and the control of GTP hydrolysis in Calorimetric study of the interaction of the C2 domains of classical protein kinase C Arf1 during COPI vesicle formation. Biochem Soc Trans 33(Pt 4):619–622. isoenzymes with Ca2+ and phospholipids. Biochemistry 43(37):11727–11739. 51. Bigay J, Casella JF, Drin G, Mesmin B, Antonny B (2005) ArfGAP1 responds to membrane 24. van den Bogaart G, Meyenberg K, Diederichsen U, Jahn R (2012) Phosphatidylinositol curvature through the folding of a lipid packing sensor motif. EMBO J 24(13):2244–2253. 4,5-bisphosphate increases Ca2+ affinity of synaptotagmin-1 by 40-fold. J Biol Chem 52. Tucker J, et al. (1986) Expression of p21 proteins in Escherichia coli and stereochem- 287(20):16447–16453. istry of the nucleotide-binding site. EMBO J 5(6):1351–1358. 25. Lemmon MA (2008) Membrane recognition by phospholipid-binding domains. Nat 53. Scrima A, Thomas C, Deaconescu D, Wittinghofer A (2008) The Rap-RapGAP complex: GTP Rev Mol Cell Biol 9(2):99–111. hydrolysis without catalytic glutamine and arginine residues. EMBO J 27(7):1145–1153. 26. Martens S, Kozlov MM, McMahon HT (2007) How synaptotagmin promotes mem- 54. Ohi M, Li Y, Cheng Y, Walz T (2004) Negative staining and image classification - brane fusion. Science 316(5828):1205–1208. powerful tools in modern electron microscopy. Biol Proced Online 6:23–34.

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