degradationofactivatedk-rasorthologueviak-ras-specific ... · prognosis and resistance to...

Degradation of Activated K-Ras Orthologue via K-Ras-specificLysine Residues Is Required for Cytokinesis*

Received for publication, October 30, 2013, and in revised form, December 7, 2013 Published, JBC Papers in Press, December 13, 2013, DOI 10.1074/jbc.M113.531178

Kazutaka Sumita‡1,2, Hirofumi Yoshino‡1, Mika Sasaki‡1, Nazanin Majd‡, Emily Rose Kahoud§, Hidenori Takahashi§,Koh Takeuchi¶, Taruho Kuroda�, Susan Lee**, Pascale G. Charest**, Kosuke Takeda**‡‡, John M. Asara§,Richard A. Firtel**, Dimitrios Anastasiou§§, and Atsuo T. Sasaki‡3

From the ‡Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute,Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, Collegeof Medicine, Cincinnati, Ohio 45267, the §Department of Medicine, Harvard Medical School, Division of Signal Transduction, BethIsrael Deaconess Medical Center, Boston, Massachusetts 02115, the ¶Biomedicinal Information Research Center, National Instituteof Advanced Industrial Science and Technology, 2-3-26 Aomi, Koto, Tokyo 135-0064, Japan, the �Department of Basic MedicalSciences, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan, the **Section of Cell andDevelopmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0380, the‡‡Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore,138667, Republic of Singapore, and the §§Division of Physiology and Metabolism, Medical Research Council National Institute forMedical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

Background: Targeting oncogenic K-Ras for cancer therapy has remained challenging.Results: Ubiquitination specifically occurs on the activated K-Ras orthologue in Dictyostelium via evolutionary conserved K-Raslysines, which promotes K-Ras protein degradation.Conclusion: Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium.Significance: This work reveals a novel negative feedback regulation for the K-Ras isoform, which is critical for cytokinesis inDictyostelium.

Mammalian cells encode three closely related Ras proteins,H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations fre-quently occur in human cancers, which lead to dysregulated cellproliferation and genomic instability. However, mechanisticrole of the Ras isoform regulation have remained largelyunknown. Furthermore, the dynamics and function of negativeregulation of GTP-loaded K-Ras have not been fully investi-gated. Here, we demonstrate RasG, the Dictyostelium ortho-logue of K-Ras, is targeted for degradation by polyubiquitina-tion. Both ubiquitination and degradation of RasG were strictlyassociated with RasG activity. High resolution tandem massspectrometry (LC-MS/MS) analysis indicated that RasG ubiq-uitination occurs at C-terminal lysines equivalent to lysinesfound in human K-Ras but not in H-Ras and N-Ras homologues.Substitution of these lysine residues with arginines (4KR-RasG)diminished RasG ubiquitination and increased RasG proteinstability. Cells expressing 4KR-RasG failed to undergo propercytokinesis and resulted in multinucleated cells. Ectopicallyexpressed human K-Ras undergoes polyubiquitin-mediateddegradation in Dictyostelium, whereas human H-Ras and a Dic-tyostelium H-Ras homologue (RasC) are refractory to ubiquiti-nation. Our results indicate the existence of GTP-loaded K-Ras

orthologue-specific degradation system in Dictyostelium, andfurther identification of the responsible E3-ligase may provide anovel therapeutic approach against K-Ras-mutated cancers.

Ras is a small GTPase that can cycle between a GTP-boundactive state and a GDP-bound inactive state. GTP-loaded Raselicits an array of downstream signaling such as Raf/MEK/ERKand PI3K/AKT pathways to promote cell survival and prolifer-ation (1–5).

Mammalian cells encode four closely related Ras proteins,H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B that are highly homol-ogous in the first 85% of the protein and share a C-terminalCAAX box that targets them for farnesylation. The C-terminalhypervariable region (HVR)4 renders distinctive roles of Rasisoforms. The HVR of K-Ras 4B (hereafter called K-Ras), adominant K-Ras isoform, contains a poly-basic, lysine-rich,cluster that is required for K-Ras plasma membrane localiza-tion. The HVRs of the other Ras isoforms, including K-Ras 4A,another minor K-Ras isoform, provide palmitoylation site(s) fortargeting to the plasma membrane (6 –10). Extensive researchwithin the past two decades has shown that Ras isoforms differin their ability to activate downstream effectors and promotetransformation (11–13). In mouse tumor models, only the con-stitutively active K-Ras mutant, but not H-Ras or N-Rasmutants, promotes colorectal carcinogenesis and hyperprolif-

* This work was supported in part by National Institutes of Health Grants2P01CA120964 (to J. M. A.) and 5P30CA006516 (to J. M. A.) and the Univer-sity of Cincinnati (to A. T. S.).

1 These authors contributed equally to this work.2 Supported in part by the American Association of Neurological Surgeons.3 To whom correspondence should be addressed: UC Cancer Inst., Dept. of

Internal Medicine, Div. of Hematology Oncology, UC Neuroscience Insti-tute, Dept. of Neurosurgery, Brain Tumor Center, University of Cincinnati,College of Medicine, Cincinnati, OH 45267. Tel.: 513-558-2160; Fax: 513-558-6703; E-mail: [email protected].

4 The abbreviations used are: HVR, hypervariable region; CHX, cycloheximide;7KR-RasG, seven lysine residues in the membrane-targeting region substi-tuted for arginine residues; 4KR-RasG, four lysines found to be ubiquiti-nated are substituted with arginines.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 7, pp. 3950 –3959, February 14, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

3950 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 7 • FEBRUARY 14, 2014

eration of endoderm progenitor cells (1, 3, 14, 15). In humancancers, K-Ras is the most frequently mutated Ras isoform.Furthermore, K-Ras mutations are associated with poor clinicalprognosis and resistance to chemotherapy (6, 8, 16 –18).Despite numerous attempts, a small molecule inhibitor forK-Ras is yet to be developed for clinical use. Identification ofKRas-specific regulatory mechanisms is likely to provideimportant insights into the development of novel therapeuticapplications for K-Ras-mutated tumors.

To date, many of the physiological roles of Ras have beenidentified through studies using model organisms. However,not all model organisms have both H-Ras and K-Ras isoforms.Saccharomyces cerevisiae, a unicellular organism, has H-Rashomologue genes Ras1 and Ras2, but it does not encode for aK-Ras orthologue. Caenorhabditis elegans and Drosophilamelanogaster have a single K-Ras orthologous gene but not anH-Ras orthologue. Dictyostelium discoideum, a social amoebathat has the ability to alternate between a unicellular and amulticellular form, has both K-Ras and H-Ras orthologues.Therefore, to investigate whether ubiquitination comprises anevolutionarily conserved mechanism for Ras regulation, we uti-lized the Dictyostelium system.

EXPERIMENTAL PROCEDURES

Materials—Anti-FLAG (M2) antibody and 3� FLAG pep-tide were from Sigma Aldrich; anti-Ras (Ab-3) antibody wasfrom EMD Millipore; and anti-ubiquitin antibody (P4D1) wasfrom Santa Cruz Biotechnology. FLAG-tagged Ras mutantswere generated by the standard PCR method and subclonedinto pEXP4(�). All constructs were sequenced. We producedGST-RBD in BL21 bacteria (Stratagene) and purified it on glu-tathione-Sepharose (Amersham Biosciences) per the manufac-turer’s instructions.

Cell Culture—All cell lines were cultured axenically in HL5medium at 22 °C. Transformants were maintained in 40 �g/mlG418.

Tandem Mass Spectrometry Analysis—FLAG-RasG express-ing RasG-null cells were grown exponentially in HL5 media.The a billion of FLAG-RasG/RasG cells were then collected andlysed with radioimmune precipitation assay buffer and sub-jected to anti-FLAG (M2)-agarose immunoprecipitation. Thebeads were washed with radioimmune precipitation assaybuffer and FLAG-RasG, and its ubiquitinated forms were elutedwith 3�FLAG peptide. Samples were prepared from SDS-PAGE gels, stained with Coomassie Blue, and excised for massspectrometry sequencing and ubiquitin site mapping. Gelpieces were reduced with DTT, and Cys residues were alkylatedwith iodoacetamide and digested overnight using modifiedtrypsin (Promega) at 37 °C at pH 8.3. Peptides were extractedand analyzed by data-dependent microcapillary tandem massspectrometry using both linear ion trap (Thermo Scientific, SanJose, CA) and a high resolution/mass accuracy hybrid linear iontrap LTQ-Orbitrap XL mass spectrometer (Thermo Scientific)coupled to an EASY-nLC nanoflow HPLC (Proxeon Biosys-tems). MS/MS spectra acquired via collision-induced dissocia-tion were searched against the reversed and concatenatedSwiss-Prot protein database (version 55.8, UniProt) with a fixedmodification for carbamidomethylation (�57.0293) and the

variable modifications for oxidation (�15.9949) and ubiquiti-nation (�114.10, GG tag) using the Sequest algorithm in Pro-teomics Browser software (Thermo Scientific). Peptides fromRasG were identified by database scoring, and peptides modi-fied by ubiquitin were validated manually to be sure that all b-and y- series ions were consistent with the modified residue andadditional validation was performed using GraphMod softwarein Proteomics Browser software. The peptide false discoveryrate was �1.5% based on reversed database hits.

RESULTS AND DISCUSSION

Dictyostelium RasG, K-Ras Homologue, Undergoes Ubi-quitination—Dictyostelium has five Ras isoforms (RasB, RasC,RasD, RasG, and RasS), among which RasG is the closest homo-logue to human K-Ras 4B (K-Ras). RasG contains the charac-teristic lysine stretch of K-Ras in its C-terminal HVR, and thepositions of the lysine residues in RasG are well aligned withthe lysines in the HVR of human K-Ras (Fig. 1A). In contrast,the HVR of RasG has poor sequence homology to that of humanH-Ras and human N-Ras. To investigate regulation of RasG, weused RasG knock-out Dictyostelium cells stably expressingFLAG-RasG (WT-RasG/�RasG) (11, 19) and measured proteinstability of RasG in response to chemoattractant (cAMP) stim-ulation. The FLAG-RasG/�RasG cells were placed in buffercontaining only Na/KPO4 and treated with the protein synthe-sis inhibitor, cycloheximide (CHX). The result showed thatCHX led to an �50% decrease in RasG protein levels in �30min (Fig. 1B). The half-life of the RasG protein was furthershortened by chemoattractant (cAMP) stimulation. Theseresults suggest that RasG protein stability can be dynamicallydecreased by extracellular stimuli.

To investigate whether ubiquitin-mediated degradation sys-tem could account for RasG destabilization, RasG was immu-noprecipitated with anti-FLAG resin from WT-RasG/�RasGcells. Immunoprecipitated RasG was detected with anti-FLAGantibody as an expected band (�25 kDa). The anti-FLAG anti-body also detected slower migrating bands above the 30-kDamolecular mass marker, which were not detected in the controlwild-type cells (Ax3). A similar band ladder pattern was alsodetected with anti-Ras antibody, indicating that ladder bandswere modified forms of RasG protein. The increment of RasGladder bands was �9 kDa, which is equivalent to the size ofubiquitin. Consistent with this hypothesis, the slow-migratingRasG bands were recognized with anti-ubiquitin antibody (Fig.1C). These results indicated that RasG has a ubiquitinated formand that an evolutionarily conserved mechanism of K-Ras reg-ulation by ubiquitination may exist in Dictyostelium.

RasG Undergoes Activation-dependent Ubiquitination—Next,we investigated whether RasG protein stability might be modu-lated by RasG activation and/or its downstream signaling. Weintroduced a point mutation (Thr-35 to Ala) in RasG that inter-feres with the GTP-mediated switch transition and thereforeabrogates effector binding of Ras (14, 15, 20, 21). To study themutant RasG stability, we used vegetative Dictyostelium cellsrather than developmentally competent cells because mutation ofRasG can affect Dictyostelium development (16–18, 22, 23). TheWT-RasG/�RasG cells or T35A-RasG/�RasG cells were grown toexponential phase and then treated with cycloheximide. Consis-

Negative Feedback Regulation of K-Ras by Ubiquitination

FEBRUARY 14, 2014 • VOLUME 289 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 3951

tent with the result seen in developed cells, CHX treatment led toa 50% decrease in wild-type RasG protein levels within 30 min (Fig.2A) in the vegetative growth condition. In contrast, the half-life ofT35A-RasG protein was significantly extended.

To investigate whether RasG ubiquitination is triggered bythe downstream effector pathway of RasG, ubiquitination levelsof wild-type RasG and T35A-RasG were assessed. Non-ubiq-uitinated T35A-RasG expression levels were �2 times morethan WT-RasG. However, the ubiquitination level of T35A-RasG was �3 times less than that of WT-RasG. Taking intoconsideration the increased protein stability of T35A-RasG,these results suggest that RasG induces its own ubiquitination

via downstream effector pathway for its degradation. Notably,mono-ubiquitinated T35A-RasG was still detectable, albeit atlower levels compared with wild-type RasG. This suggests theexistence of yet another RasG ubiquitination pathway that isindependent of RasG activity. However, such a pathway maynot be relevant for RasG degradation, as T35A-RasG is morestable compared with wild-type RasG (Fig. 2B).

Ubiquitination Sites of RasG Are Located in the GuanineNucleotide-binding Domain and C-terminal HypervariableRegion—To gain further insight into the role of RasG ubiquiti-nation, we determined ubiquitination sites in RasG. FLAG-RasG protein and its ubiquitinated forms were purified with

Homo

A

C

RasGK-Ras4AK-Ras4BH-Ras

Dictyostelium discoideum

Sapiens

N-Ras REIRQHKLRKLNPPDESGPGCMSCKCVLS

REIRK-DLKGDSKPEKGKKKRPLKACTLLREIRQYRLKKISKEEKTPGCVKIKKCIIMREIRKHKEKM-SKDGKKKKKKSKTKCVIM

REIRQYRMKKLNSSDDGTQGCMGLPCVVM

RasP-loop Switch I & II guanine ring-binding

G4 G5G3G2G1

Hyper-Variable Region

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IP: αFlagTCL

αFlag αFlag αRas αUb.

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CHX. (min)0 15 30 60 15 30 60

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FIGURE 1. RasG, Dictyostelium K-Ras homologue, undergoes polyubiquitination. A, schematic diagram of G-domain of Ras (upper panel). The C-terminalHVRs of RasG and human Ras isoforms are aligned (lower panel). B, the FLAG-tagged WT-RasG/�RasG cells were treated with 100 �g/ml CHX for indicated times,in the presence (�) or absence (�) of 10 �M of cAMP, and cell extracts were analyzed with anti-FLAG antibody. Relative protein levels of RasG were quantifiedand are shown on the right. These blots are representative of two separate experiments. C, RasG undergoes polyubiquitination (Poly-Ub.RasG). FLAG-taggedRasG was immunoprecipitated (IP) from total cell lysate (TCL) of FLAG-tagged RasG stably expressing RasG knock-out cells (FLAG-RasG/�RasG), and immuno-blotted with the indicated antibodies. Wild-type cells, Ax3 cell line, were used as control. Ubiquitinated Ras is indicated with arrowheads. These blots arerepresentative of three separate experiments.



anti-FLAG resin followed by FLAG peptide elution. The bandsmigrating at the positions for mono- and diubiquitinated formof RasG were digested by trypsin and subjected to LC-MS/MSanalysis. Major ubiquitination sites were found at Lys residues169, 173, 176, and Lys-178, and a minor fraction of Lys-117ubiquitination was found (Fig. 3 A–C)5. The four major ubiquiti-nation sites are located in the C-terminal HVR. HVR can besubdivided into two regions, the linker region and membrane-targeting region (19, 20, 24, 25). Interestingly, all four ubiquiti-nated lysine residues are evolutionarily conserved from Dictyo-stelium RasG to human K-Ras4A and K-Ras4B and are alllocated in the linker region (Fig. 3A).

RasG Receives Lys-48-linked Polyubiquitination—Linkage-specific ubiquitination often determine the fate of the ubiquiti-nated proteins. Potentially seven different types of ubiquitinlinkages can be formed, depending on whether the ubiquitinsare attached to Lys-6, Lys-11, Lys-27, Lys-29, Lys-33, Lys-48, orLys-63 on the ubiquitin (26 –28). To determine the ubiquitinlinkage, we undertook the LC-MS/MS analysis. LC-MS/MSdetected 90% of entire ubiquitin protein from the diubiquiti-nated RasG. Strikingly, we found that diubiquitinated RasGcontained Lys-48-linked ubiquitination in the diubiquitinatedform of RasG (Fig. 3D), whereas there was no ubiquitinatedfragment of other regions detected, including Lys-63.

To further verify the result, we immunoprecipitated RasG forWestern blot analysis and then blotted the membrane withlinkage specific ubiquitin antibodies. We observed a strong sig-nal by Lys-48-linkage-specific ubiquitin antibody. Next wetested whether polyubiquitinated form of RasG contain Lys-63-

linked ubiquitin. The anti-Lys-63-linked ubiquitin antibodydisplayed robust signal in the total cell lysate fraction, which aredistinctive pattern compared with the anti-Lys-48-linked ubiq-uitin antibody, consistent with previous reports (26 –28). How-ever we could not detect Lys-63-linked ubiquitin signal in theRasG-immunoprecipitated sample (Fig. 4A). These data areconsistent with the LC-MS/MS result (Fig. 3D).

To further verify the finding, we examined the effect ofproteasome inhibitors on ubiquitination of RasG. We testedfour proteasome inhibitors, which have been widely used, andincreased concentration to 5�10-fold the commonly used dose.Unfortunately, lactacystin, epoxomicin, and ALLN did notinduce any detectable changes of ubiquitination signal of cellu-lar proteins. This is in agreement with the previous studies thatDictyostelium cellular proteins were refractory to MG132,MG262, and lactacystin, presumably due to the restricted per-meability to and high rate of drug exclusion from the Dictyos-telium or both (29, 30). At 100 �M concentration, the treatmentof MG132 slightly induced accumulation of the polyubiquiti-nated cellular proteins (Fig. 4A). Under this condition, therewas further accumulation of the Lys-48-ubiquitinated RasG.This suggests that Lys-48-linked ubiquitination of RasG isdegraded, if not all, via the ubiquitin-proteasome system.

C-terminal Lysine Cluster Ubiquitination Leads to RasGDestabilization—To further investigate the function of ubiq-uitination in the HVR of RasG, we made a RasG mutant whereall four lysines found to be ubiquitinated are substituted witharginines (4KR-RasG). We also made a mutant that has theseven lysine residues in the membrane-targeting regionsubstituted for arginine residues (7KR-RasG) (Fig. 4, A and B).FLAG-tagged 4KR-RasG and 7KR-RasG mutants were stably5 J. M. Asara, unpublished observations.

A

(short expos.)

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FIGURE 2. Polyubiquitination and degradation of RasG requires RasG activation and its downstream effector. A, T35A-RasG mutant increased proteinstability. The WT- and T35A-RasG stably expressing cells were treated with 100 �g/ml CHX for indicated times, and cell extracts were analyzed with anti-FLAGantibody. Relative protein levels of RasG were quantified by ImageJ software and shown in the bottom panel. These blots are representative of two separateexperiments. B, T35A-RasG mutant decreased ubiquitination level. FLAG-tagged WT- and T35A- RasG were immunoprecipitated (IP) from total cell lysate (TCL)of FLAG-tagged RasG stably expressing RasG knock-out cells (FLAG-RasG/�RasG) and immunoblotted (IB) with the indicated antibodies. Wild-type cells, Ax3cell line, were used as control. Ubiquitinated Ras is indicated with arrowheads. short expos., short exposure; Poly-Ub., polyubiquitination.



REIRK-DLAGDSAPEAGAKKRPLKACTLL

RasGK-Ras4AK-Ras4BH-Ras


HomoSapiens

N-Ras REIRQHKLRKLNPPDESGPGCMSCKCVLS

REIRK-DLKGDSKPEKGKKKRPLKACTLLREIRQYRLKKISKEEKTPGCVKIKKCIIMREIRKHKEKM-SKDGKKKKKKSKTKCVIM

REIRQYRMKKLNSSDDGTQGCMGLPCVVM

WT-RasG REIRK-DLKGDSKPEKGKKKRPLKACTLL4KR-RasG

REIRK-DLAGDSAPEAGAAARPLAACTLL7KR-RasG

Ub. Ub. Ub.Ub.

Ub. Ub. Ub.Ub.

Ras G4 G5G3G2G1

Ub. Ub. Ub.Ub.

Ubiquitination sites of mono-Ub.-RasG

Ubiquitination sites of di-Ub.-RasG

Major ubiquitination sites of RasG A

B

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ubiquitination

ubiquitination

Ubiquitination sites of ubibuiqin from di-Ub.RasG q q

Lys-48

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Protein coverage

Legend

Peptide spectra

M*:+15.99490 NQ#:+0.98402 K@:+114.04293

Linker Membrane-targeting



expressed into RasG-null cells, and their ubiquitination levelswere assessed. Fig. 4B shows that the ubiquitination level of7KR-RaG was 9 times lower than that of wild-type RasG. Theresults of Figs. 1B and 2A suggest that activated-RasG receives

polyubiquitination. Importantly, we found that polyubiquitina-tion levels were increased �2 times in G12V-RasG and Q61L-RasG constitutively active mutants, which are locked in GTP-bound state, compared with that of wild-type RasG. Consistent

FIGURE 3. Ubiquitination of RasG occurred on K-Ras specific lysine residues. A, ubiquitination (Ub.) sites in RasG was identified by tandem mass spectrom-etry and shown in the red boxed lysine residues. The sequence of the RasG ubiquitination sites and corresponding region of human Ras isoforms are aligned(lower panel). The identified ubiquitination sites were mutated to arginine residues in 4KR and 7KR RasG mutants as shown in the lower panel. B–D, diagram ofLC/MS/MS identified in vivo ubiquitination sites in RasG and ubiquitin. FLAG-tagged RasG was stable expressed and immunoprecipitated with anti-FLAGantibody. The IP was run out on SDS-PAGE, stained with Coomassie, and the band corresponding to monoubiquitinated RasG (B), diubiquitinated (di-Ub.) RasG(C and D) was cut out, and isolated and subjected to tryptic digest and LC/MS/MS analysis. Amino acid coverage map showing the tryptic peptides sequencedby LC-MS/MS are in dark green and the detected ubiquitination sites are highlighted in magenta. Light green and blue highlight oxidations of Asn/Gln and Met,which are most likely an in vitro processing artifact.

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FIGURE 4. Lys-48-linked ubiquitination of RasG regulates RasG stability and cytokinesis. A, WT-RasG receives Lys-48-linked polyubiquitination. FLAG-tagged WT- and 7KR-RasG mutants cells were treated with the indicated proteasome inhibitors for 40 min. WT- and 7KR-RasG were immunoprecipitated (IP)from total cell lysate (TCL) and immunoblotted (IB) with the indicated antibodies. Only MG132 displayed inhibition of proteasome inhibition, leading accumu-lation of polyubiquitinated proteins. B, 4KR- and 7KR-RasG mutants decreased ubiquitination level. Ubiquitination level of the indicated RasG mutants wasassessed as in Fig. 1A. These blots are representative of two separate experiments. Relative polyubiquitinated RasG levels were assessed by quantification ofpoly- and non ubiquitinated (Ub.) RasG signal intensity by ImageJ software. Polyubiquitination signal was divided by non-ubiquitinated RasG signal to estimatethe relative ubiquitination level of WT- and RasG mutants. C, 4KR- and 7KR-RasG mutants increased protein stability. Protein stability of the indicated RasGmutants was assessed as in Fig. 2. These blots are representative of two separate experiments. D, 4KR-RasG mutant increased number of nuclei in suspensionculture. WT and 4KR mutant expressing RasG -null cells were shaken for 3 days, plated, and stained with DAPI. Random fields were photographed, and fractionsof cells containing the designated number of nuclei were determined. Averages of two experiments comprising �200 cells for each line are shown. These blotsare representative of two separate experiments. DMSO, dimethyl sulfoxide.



with the results of Figs. 1 and 2, this suggests the activated formof RasG receives polyubiquitination. To further verify this, weintroduce mutation of 4KR into Q61L-RasG rather than WT-RasG because 4KR mutation may reduce RasG activation bynon-ubiquitin-related pathway. Strikingly, 4KR-Q61L-RasGreduced ubiquitination level, 17 times less than Q61L-RasG(Fig. 4B). The results indicated that these four lysine residuesfound by mass spectrometry analysis were bona fide ubiquitina-tion sites. However, both 7KR- and 4KR-Q61L mutants stillreceive a small fraction of polyubiquitination, suggesting that thereis another site(s) for polyubiquitination. It is also possible that themutation of the C-terminal lysine may lead to ubiquitination ofanother site(s), which is otherwise not ubiquitinated.

We have further investigated the role of the HVR ubiquitina-tion on RasG protein stability. As shown in Fig. 4C, wild-typeRasG protein level was quickly decreased after CHX treatment,while 4KR- and 7KR-RasG mutants significantly increasedprotein stability. Consistent with the elevated Lys-48-linkedpolyubiquitination level (Fig. 4, A and B), the degradation of G12V-and Q61L-RasG was faster than that of wild-type RasG. Impor-tantly, Q61L-RasG destabilization was completely reversed bythe four-lysine residue mutation. These results indicate thatubiquitination of HVR in RasG regulates RasG protein stability.

RasG Ubiquitination Pathway Is Critical for Cytokinesis—RasG is critical for cytokinesis (20, 21, 31). Recently, it hasbeen shown that Cdc42, a Rho family GTPase essential forcytokinesis, is down-regulated during mitotic exit, which isrequired for proper cytokinesis (22, 23, 32). To assess thefunctional significance of RasG ubiquitination, we assessedcytokinesis of wild-type- and 4KR-RasG-expressing RasG-null cells using suspension culture where cytokinesis-defi-cient mutants become multinucleated (20, 24, 25, 33, 34).Consistent with previous reports, RasG-null cells becamemultinucleated after 3 days of shaking culture in suspension.The expression of wild-type RasG prevented the emergenceof multinucleated cells (Fig. 4D). However, the expression of4KR-RasG mutant failed to reverse the cytokinesis defect ofRasG-null cells.

Next, to test whether KR-mutant retains activity, we firstperformed a biochemical assay. The GST-Ras-binding domainpulldown assay reveals that 7KR mutant has high basal activity,which is more than Q61L mutant. Thus, the 7KR mutantundergoes GTP loading and binds to its effector molecules (Fig.5A). However, the 7KR mutant may no longer be the same asWT-RasG because the mutant protein level is higher than theWT, similar to its basal activation levels.

ΔRasG

WT-RasG/ΔRasG

7KR-RasG/ΔRasG

High density low density

WT 7KR Q61L

Input InputInput Boundto RBD

Boundto RBD

Boundto RBD

αFlag

GTP-Ras

total GTP-Ras

total GTP-Ras

total

IB:

non-specificband

RasG

A

B

FIGURE 5. Effect of ubiquitination on the basal RasG activity and developmental activity. A, 7KR-RasG increases basal activity. RasG wild-type (WT) ormutants were stably expressed in �RasG cells, and their activation level was analyzed by a GST-RBD pulldown assay of Ras as described in Ref. 51. Q61L-RasGhas much higher basal activity than WT-RasG. 7KR mutant increases basal activity, presumably because the 7KR-RasG could evade from the activation-depen-dent RasG degradation. B, 7KR-RasG cells retain developmental activity, similar to WT-RasG cells. RasG wild-type (WT) or 7KR mutant were stably expressed in�RasG cells and plated on non-nutrient agar to induce the developmental process as described previously (52, 53). Although the impeding mutation of GTPaseactivity of RasG has shown to attenuate the developmental process (35), the 7KR-RasG cells displayed highly comparable developmental progression toWT-RasG cells. IB, immunoblot.



Therefore, we next examined whether 7KR-RasG has a rolein developmental process of Dictyostelium, which requiresproper regulation of chemotaxis and gene expression. The cellswere seeded on the development plate at various cell densities,and their developmental processes were monitored for thedevelopmental process for 48 h. Interestingly, although consti-tutive RasG activation has been shown to lead to developmentaldefects in Dictyostelium (35), the 7KR-RasG mutant cells havenormal developmental functioning, indistinguishable to that ofWT-RasG cells (Fig. 5B). The biochemical and developmentalanalyses suggest that ubiquitin-mediated regulation of RasG isnot involved in the normal developmental process even thoughthe 7KR-mutant has higher basal Ras activity. It should benoted that cells under the developmental assay condition,which does not have nutrients, do not proliferate. Furthermore,cytokinesis defect of RasG cells are only prominent when theyare cultured in suspension (20, 24, 25). Thus, the cytokinesisdefect of RasG mutant cells was suppressed in the developmen-tal assay where cells were plated on agar.

Cytokinesis failure is one of the well described events thatinduce aneuploidy and genomic instability (36 –38). Further-more, it has been shown that Ras is activated at the cellularpoles during cytokinesis in Dictyostelium (14, 31, 39, 40) andthat K-Ras is shown to have an important role in cytokinesisand genome stability in mammalian cells (32, 41, 42). The find-ings in Dictyostelium RasG potentially reflect the circumstanceof tumorigenesis driven by hyper-activation of K-Ras: theexpression of mutated K-Ras not only activates downstreamsignaling but also induces aneuploidy and promotes tumori-genesis (33, 34, 43, 44).

H-Ras Homologue, RasC, Does Not Undergo Ubiquitination—Next, we investigated whether other Ras isoforms in Dictyoste-lium are ubiquitinated similarly to RasG. To test this possibility,we focused on Dictyostelium RasC, a functional orthologue ofhuman H-Ras, which is a critical mediator of chemoattractant-induced PI3K and AKT activation (36, 38). RasC does not havelysine residues in its linker region, and RasC shares several res-idues in the linker region with mammalian H-Ras (Fig. 6A).FLAG-tagged wild-type RasC was stably expressed into RasC-null mutant cells (WT-RasC/�RasC). The expression of RasCcomplemented the developmental defect of RasC-null cellsindicating that the exogenously expressed RasC protein wasfunctional. Unlike RasG, we did not detect ubiquitination ofRasC (Fig. 6B). We also expressed G12V-RasC, a mutant that isconstitutively bound to GTP and found that G12V-RasC didnot undergo ubiquitination either. Consistent with this finding,both wild-type and G12V-RasC proteins are very stable (Fig.6C). These results reveal that RasC is refractory to ubiquitina-tion-mediated degradation.

Ubiquitination Specifically Occurs on K-Ras Orthologue, butNot on H-Ras Orthologue, in Dictyostelium—Sequence align-ment revealed that the identified ubiquitination sites of RasGwere conserved in mammalian K-Ras 4A and 4B, but not inH-Ras (Figs. 1A and 6A). Therefore, we next examinedwhether human K-Ras protein could be recognized for ubiq-uitination in Dictyostelium cells. Human G12V-K-Ras4B wasstably expressed into RasG-null cells and immunoprecipitatedwith anti-FLAG agarose. Western blotting with anti-ubiquitinantibody revealed that G12V-K-Ras was highly ubiquitinated inDictyostelium at levels similar to RasG ubiquitination (Fig. 6D).

IB:αUb.

IB:αFlag

IP:αFlag

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CG

12V

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+cAMP (100µM)

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B C-

poly-basic regionRasG


Homo SapiensRasCH-Ras

REIKR--WNQQNPQNEEMLPPKKRGCIILREIRQHKLRKLNPPDESGPGCMSCKCVLS

REIRK-DLKGDSKPEKGKKKRPLKACTLL

Ub. Ub. Ub.Ub.

Linker Membrane-targeting

cAMP (min)0 2/32 3Ax3

0 2/32 3K-Ras4BG12V

0 2/32 3RasG

0 2/32 3RasGG12V

IB:αUb.

IB:αFlag

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H-R

as

H-R

asG

12V

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GG

12V

K-Ras4B

H-Ras

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CHX. 0 20 40 60 (min) HVR

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E

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lati

ve

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sC

le

ve

l

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0CHX. (min)0 15 30 60 15 30 60

+cAMP-

CHX. 6040200 (min)

Rela

tive R

as level

100

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0

K-Ras4B

KRas/H

HRas/KH-Ras

FIGURE 6. Ubiquitination specifically occurs on K-Ras, but not H-Ras. A, the sequence of the RasG ubiquitination (Ub.) sites and corresponding region ofDictyostelium RasC and human H-Ras are aligned. B, Dictyostelium RasC does not undergo ubiquitination. FLAG-tagged WT- and G12V-RasC were stablyexpressed in RasC-null cells. Ubiquitination level of RasC and RasG was assessed as in Fig. 1. These blots are representative of two separate experiments. C, RasCis a highly stable protein. Protein stability of RasC was assessed as in Fig. 2. These blots are representative of two separate experiments. D, human K-Ras undergopolyubiquitination, whereas human H-Ras is refractory to ubiquitination in Dictyostelium. FLAG-tagged K-Ras and H-Ras were stably expressed in �RasG cells.The ubiquitination level of K-Ras and H-Ras were assessed as in Fig. 1. These blots are representative of two separate experiments. E, C-terminal HVR determinedthe protein stability of K-Ras and H-Ras in Dictyostelium. A schematic diagram of K-Ras and H-Ras chimeric mutants (left). Human K-Ras, H-Ras, and chimericmutants were stably expressed in �RasG cells, and their protein stability was assessed as in Fig. 2. These blots are representative of two separate experiments.IB, immunoblot.



To further examine whether human Ras proteins undergoubiquitination in Dictyostelium in an isoform-specific manner,human wild-type and G12V-H-Ras were stably expressed inDictyostelium. Fig. 6D, right panel, shows that in Dictyostelium,ubiquitination of wild-type H-Ras and G12V-H-Ras was notobserved in contrast to the ubiquitination of K-Ras. The mostsignificant difference of K-Ras and H-Ras is their C-terminalHVR region. To further examine the importance of K-Ras HVRfor ubiquitination, we have generated a chimeric H-Ras proteinthat harbors the HVR of K-Ras (H-Ras/K) and a chimeric K-Rasthat harbors the HVR of H-Ras (K-Ras/H). Fig. 6E shows that ahalf-life of K-Ras/H protein prolonged about six times morethan that of wild-type K-Ras, a half-life of H-Ras/K mutant wasabout three times less than wild-type H-Ras.

Using biochemical and genetic approaches, we have foundthat RasG, a Dictyostelium K-Ras orthologue, undergoes ubiq-uitination and that the extent of ubiquitination is dependent ondownstream effector signaling. Ubiquitination specificallyoccurred on lysine residues that are conserved in K-Ras HVRlinker domain. RasG ubiquitination regulates RasG protein lev-els and is required for proper cytokinesis. Ectopically expressedhuman K-Ras can be polyubiquitinated in Dictyostelium. How-ever, human H-Ras and Dictyostelium RasC, a H-Ras ortho-logue, are refractory to ubiquitination. Thus, Dictyostelium hasa ubiquitination system that is specific for activated RasG thatcan also target human K-Ras.

Recently, three types of ubiquitin-mediated Ras regulatorypathways have been discovered: 1) ubiquitination of Ras iso-forms at Lys-117 or Lys-147 leads to RasGEF-independent Rasactivation (14, 39, 40, 45); 2) ubiquitination of H-Ras by theE3-ubiquitin ligase RabEX5 results in H-Ras endomembranetranslocation (41, 42); 3) ubiquitination of phosphorylated Rasisoforms by �TrCP, another E3 ligase, results in Ras protea-some-mediated degradation (43, 44). So far, K-Ras-specificdegradation has not been described in any system. Thus, this isthe first report that demonstrates a negative feedback regula-tion of a K-Ras orthologue. Furthermore, it is conceivable thatubiquitination at the K-Ras-specific lysines may alternate sub-cellular localization of RasG because of its proximity to theHVR.

There are �500 E3 ligases in the human genome, whereas theDictyostelium genome has �100 potential E3-ligases. In ourdatabase search, there was no putative E3 ligase that harbors aRas binding domain. Because E3 ligase often consists of severalsubunits, we assume that the activated RasG/K-Ras-specific E3ligase may form a protein complex that contains subunit bind-ing to activated-Ras. We also speculate that RasG/K-Ras E3ligase may recognize GTP-RasG/KRas, which is in the bindingstatus with its effector, to specifically regulate effector pathway.This could be analogous mechanism to the ubiquitin-mediateeffector selection of Ras, which we have reported recently (14,39, 45). We expect that active RasG-specific E3 ligase has a role incytokinesis, based on our observation. Thus, Dictyosteliummutants that have a cytokinesis defect may contain the RasG E3ligase. Future biochemical purification and/or genetic analysisshould reveal the responsible machinery for RasG ubiquitination.

Currently, it is unclear whether mammalian cells have a sim-ilar mechanism of K-Ras regulation. Several studies in human

cancers revealed that increased Ras protein levels in tumors canbe attributed to suppressing microRNAs against Ras and/orgenomic amplification of the Ras locus. It is possible that aK-Ras-specific degradation system may act as tumor suppres-sor and thus suppressed in cancers. Consistent with this notion,it has been shown that expression of proapoptotic Ras effectorssuch as Nore1 and RASSFs (Ras association domain family) arefrequently down-regulated by promoter methylation or chro-mosomal rearrangement in various cancers (46 –50). Theseobservations support the possibility that K-Ras degradationpathway might be conserved from Dictyostelium to human,which could be down-regulated in cancer cells. Future studiesto identify an E3 ligase specific to RasG and K-Ras are requiredand may provide a novel approach to target K-Ras-mutatedcancers.

Acknowledgments—We gratefully acknowledge the members of theUniversity of Cincinnati Cancer Institute, the UC Brain Tumor Cen-ter and the Izayoi Meeting for stimulating discussions and helpfulsuggestions, Dr. Melanie Cushion, Dr. Antonis Koromilas, Mary Kem-per, Dillon Chen, and Janice Connelly for help in critical reading andpreparing this manuscript. We sincerely thank Dr. Lewis Cantley forthe critical comments, support, and encouragement. We also thankDrs. Ronald Warnick, Sarah Kozma, Carol Mercer, and GeorgeThomas for generous support and feedback. We thank Xuemei Yangand Min Yuan for help with mass spectrometry experiments.

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