von hippel-lindau gene product directs cytokinesis: a new tumor … · 2011. 6. 24. · pvhl...

Research Article

IntroductionVon Hippel Lindau (VHL) syndrome is a multisystem neoplasticdisorder characterized by central nervous system and retinalhemangioblastomas, renal cell carcinoma (RCC), andpheochromocytomas. About 20–30% of VHL patients have largeor partial germline deletions, 30–38% have missense mutations,and 23–27% have nonsense or frameshift mutations (Maher andKaelin, 1997; Stolle et al., 1998). As part of the E3 ubiquitin ligasecomplex, the Von Hippel Lindau tumor suppressor protein (pVHL)targets the -subunits (1, 2, and 3) of hypoxia-inducible factor (HIF-) for degradation (Cockman et al., 2000; Maxwell et al.,1999), a function that has been associated with the highly vascularnature of pVHL-associated neoplasms. Other functions of pVHLin preventing RCC tumorigenesis include a role in the assembly ofextracellular matrix (He et al., 2004; Ohh et al., 1998) and instabilization of both P53 and JADE-1 molecules (Roe et al., 2006;Zhou et al., 2004; Zhou et al., 2002). pVHL has also been shownto associate with and control microtubule stability and dynamics atthe cell periphery (Hergovich et al., 2003; Lolkema et al., 2004).Most recently, Thoma et al. showed that pVHL inactivation leadsto spindle misorientation, deregulated spindle checkpoint, andincreased aneuploidy (Thoma et al., 2009). However, sufficientknowledge to establish the direct link between aneuploidy andtumorigenesis in pVHL-null patients is inadequate. In this study,we examined the novel role of pVHL in the regulation ofcytokinesis and linked the chromosomal instabilities associatedwith defective cytokinesis to tumorigenesis.

Cytokinesis is the process by which cells physically separateafter the duplication and segregation of genetic material.Constriction of the cell membrane by actin and myosin results information of the cleavage furrow and, finally, the midbody structure.The final stage of cell division requires cleavage of the midbodyinto two daughter cells. Endobrevin, a member of v-SNAREmembrane fusion machinery, has been reported to localize to themidbody and be required for the terminal stage of cytokinesis(Low et al., 2003). Polo-like kinase 1 (Plk1)-dependentphosphorylation of centrosome protein 55 (Cep55) is also requiredfor midbody localization and cytokinesis (Fabbro et al., 2005).Other investigators showed that Alix, a protein associated with theESCRT-1 subunit Tsg101 (Strack et al., 2003; von Schwedler et al.,2003), is recruited to the midbody during cytokinesis by aninteraction with Cep55 and is essential for final midbody abscission(Carlton and Martin-Serrano, 2007). Although the roles of proteinslike Endobrevin, Plk1, Cep55, Alix and Tsg101 are welldocumented in midbody abscission, how they are regulated duringmidbody abscission is still under studied.

pVHL-deficient RCC 786-O cells are hypertriploid, andreintroduction of wild-type pVHL partially rescues the DNAcontent to a near-diploid chromosome complement (Giles andVoest, 2005). Our study shows that wild-type pVHL localizes tothe cytokinetic bridge during cytokinesis and partially rescuespolyploidy in these cells, whereas localization of pVHL-K171G(lysine 171 mutated to glycine) is substantially decreased at thecytokinetic bridge and fails to alter the prevalence of polyploidy.

SummaryOne of the mechanisms of tumorigenesis is that the failure of cell division results in genetically unstable, multinucleated cells. Herewe show that pVHL, a tumor suppressor protein that has been implicated in the pathogenesis of renal cell carcinoma (RCC), plays animportant role in regulation of cytokinesis. We found that pVHL-deficient RCC 786-O cells were multinucleated and polyploid.Reintroduction of wild-type pVHL into these cells rescued the diploid cell population, whereas the mutant pVHL-K171G failed to doso. We demonstrate that lysine 171 of pVHL is important for the final step of cytokinesis: the midbody abscission. The pVHL-K171Gcaused failure to localize the ESCRT-1 interacting protein Alix and the v-SNARE complex component Endobrevin to the midbody in786-O cells, leading to defective cytokinesis. Moreover, SUMOylation of pVHL at lysine 171 might modulate its function as acytokinesis regulator. pVHL tumor suppressor function was also disrupted by the K171G mutation, as evidenced by the xenografttumor formation when 786-O clones expressing pVHL-K171G were injected into mice. Most RCC cell lines show a polyploidchromosome complement and consistent heterogeneity in chromosome number. Thus, this study offers a way to explain the chromosomeinstability in RCC and reveals a new direction for the tumor suppressor function of pVHL, which is independent of its E3 ubiquitinligase activity.

Key words: pVHL, CIN, SUMOylation, Cytokinesis, Alix, Endobrevin

Accepted 22 February 2011Journal of Cell Science 124, 2132-2142 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.087122

Von Hippel-Lindau gene product directs cytokinesis: anew tumor suppressor functionSutapa Sinha1, Gourish Mondal2, Eun Ju Hwang3, Da Woon Han3, Shamit K. Dutta1, Seethalakshmi Iyer1, S. Ananth Karumanchi4, Keun Il Kim3, Fergus J. Couch2 and Debabrata Mukhopadhyay1,*1Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, MN 55905, USA2Department of Pathology, Mayo Clinic Foundation, Rochester, MN 55905, USA3Department of Biological Sciences, Sookmyung Women’s University, Seoul 140-742, Korea4Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA*Author for correspondence ([email protected])

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We demonstrate that pVHL-deficient 786-O cells and 786-O-VHL-K171G cells are defective in localization of Alix andEndobrevin to the midbody. pVHL-null RCC A498 cells werealso defective in localization of Alix to the midbody, whereas thecells expressing wild-type pVHL, Caki-1 (a human renalcarcinoma cell line), were not. Post-translational modification orSUMOylation of pVHL at lysine 171 might be pivotal for itsfunction as a cytokinesis regulator. Further, we report thatpolyploid cells expressing pVHL-K171G injected subcutaneouslyinto mice can lead to tumor formation. Therefore, the lysine 171residue in the -domain of pVHL appears to be important for theregulation of cytokinesis, one of the central regulatorymechanisms of cell biology, and thus in the initiation of tumorformation.

2133SUMOylated pVHL potentiates cytokinesis

ResultsDepletion of pVHL leads to chromosome instabilityMissense and truncating mutations of the VHL gene are oftenreported in human cancer, indicating the tumor suppressor role ofpVHL (Crossey et al., 1994; Kim and Kaelin, 2004; Richards etal., 1994; Richards et al., 1993; Zbar et al., 1996). To delineate afunctional link between chromosome instability (CIN) and pVHLfunction, we used isogenic RCC lines that are either deficient inpVHL (786-O-empty) or have stable expression of wild-type pVHL[786-O-VHL(wt)] or mutated pVHL (e.g. 786-O-VHL-K171G).Immunofluorescent staining of 786-O-empty cells with anti--tubulin antibody revealed a significantly increased frequency ofmultinucleated cells (P0.003) in the absence of pVHL expression(Fig. 1A,B). Cells expressing pVHL-K171G showed a similarly

Fig. 1. Frequency of multinucleated anddiploid cells. (A)Frequency ofmultinucleated cells. RCC 786-O-empty,786-O-VHL(wt) and 786-O-VHL-K171G(clone C3) cells were stained with anti--tubulin (red) antibody and DAPI (blue).Immunofluorescence staining shows 786-O-empty and 786-O-VHL-K171G cells withmultiple nuclei and unresolved cytokineticbridges compared with 786-O-VHL(wt) cells.(B)Bars represent the percentage of cellswith multiple nuclei (MuNu) and withunresolved cytokinetic bridge (Cyt Br)structure. (C)Bars represent the effect ofwild-type pVHL and pVHL-K171G on thepercentage of cells with multiple nuclei andwith unresolved cytokinetic bridge structurein HeLa and Caki-1 cells; control siRNA(consi), VHL siRNA (VHLsi). (D)Frequencyof diploid cell population. Metaphase spreadsof colcemid-treated 786-O-empty, 786-O-VHL(wt) and 786-O-VHL-K171G cells weregenerated and analyzed. Chromosomenumber (Ch no.) is indicated. (E)Bars showthe percentage of diploid cells in the presenceor absence of wild-type pVHL or pVHL-K171G mutant (n30). Figures representthree separate experiments with similarresults.

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increased occurrence of multinucleation following anti--tubulinstaining (Fig. 1A,B) but not pVHL-N90G cells (asparagine 90 toglycine mutants) (Fig. 1B). pVHL mutation at the asparagine 90site, one of the reported pVHL missence mutation sites (Stebbinset al., 1999), and N90I (asparagine 90 to isoleucine) mutation havebeen shown to be defective in HIF- ubiquitylation (Cockman etal., 2000). Here, N90G mutation of pVHL was used as a negativecontrol for cytokinetic function. Notably, unresolved cytokineticbridge structures were more frequently observed (Fig. 1B) inpVHL-K171G-expressing and pVHL-deficient 786-O cells than in786-O cells expressing wild-type pVHL or pVHL-N90G duringthe terminal stage of mitosis. Moreover, depletion of pVHL withsiRNA in the cell lines expressing wild-type pVHL, HeLa andCaki-1, resulted in disruption of cytokinesis and increasedfrequencies of multinucleated cells (P0.03 and 0.02, respectively)(Fig. 1C). Consistent with that, overexpression of the pVHL-K171G mutant, but not wild-type pVHL, in HeLa cells also led tosimilar defects (Fig. 1C). We assume that overexpression of pVHL-K171G in HeLa cells expressing wild-type pVHL acted as adominant-negative and over-road the wild-type pVHL function.

A twofold increase in aneuploidy due to reduced spindle assemblycheckpoint activity and an absence of tetraploid cells has beenreported in pVHL-deficient primary mouse embryonic fibroblasts(MEFs) (Thoma et al., 2009). However, enumeration ofchromosomes in metaphase spreads of pVHL-deficient 786-O cellsrevealed substantial numerical chromosome aberrations (>80%)consisting mostly of polyploidy (Fig. 1D,E). Furthermore,reintroduction of wild-type pVHL, but not pVHL-K171G, rescuedthe population of diploid cells (P0.002) (Fig. 1D,E). Thus,cytogenetic analysis revealed that, unlike the absence of polyploidcells in pVHL-deficient MEFs reported by Thoma and associates(Thoma et al., 2009), the RCC lines contain hypertriploid andtetraploid cells. When considered together, these data suggest thatmassive disruption of the mitotic function of these cells and potentialdisruption of cytokinesis leads to unresolved cytokinetic bridges andmultinucleated cells in the absence of normal pVHL function.

pVHL localizes to the cytokinetic bridge and regulatescytokinesisThe involvement of pVHL in the regulation of microtubule stabilityand mitotic spindle orientation has been reported previously(Hergovich et al., 2003; Thoma et al., 2009). To further investigatethe potential link between pVHL function and mitotic progression,we first studied the cellular localization of pVHL throughoutmitosis. A reconstituted RCC line, 786-O, that stably expresseswild-type pVHL was subjected to immunofluorescence stainingwith anti-pVHL antibody followed by confocal microscopy. pVHLco-localized with -tubulin to the spindle midzone during lateanaphase (Fig. 2A) and to the cytokinetic bridge during cytokinesis(Fig. 2A,C). Similar staining of endogenous pVHL using anti-pVHL antibody in RCC Caki-1 cells indicated normal localizationof pVHL at the cytokinetic bridge during cytokinesis (Fig. 2B).These observations corroborate the findings of Thoma andcolleagues, who have also shown the co-localization of pVHL with -tubulin at the spindle midzone and to the cytokinetic bridgeduring mitosis (Thoma et al., 2009). Immunofluorescence stainingof the isogenic pVHL-null 786-O cell line did not detect any suchsignal either in interphase or telophase (Fig. 2A), confirming thespecificity of the immunofluorescence staining. Consistent withthat, treatment of HeLa cells with pVHL-specific siRNA abolishedthe anti-pVHL antibody signal as compared with cells treated with

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control siRNA (supplementary material Fig. S1A), and furtherverified the specificity of the pVHL antibody. To evaluate the exactlocation of pVHL at the cytokinetic bridge, wild-type pVHL-expressing 786-O or HeLa cells were co-stained for pVHL andeither the midbody component mitotic kinesin-like protein (MKLP-1) or septin-6. The results showed that pVHL co-localized withMKLP-1 (Fig. 2D) at the central spindle region during anaphasein both wild-type pVHL-expressing 786-O and HeLa cells. Three-dimensional analysis (Fig. 2E) of the confocal images for thepVHL and MKLP-1 co-staining in wild-type pVHL-expressing786-O cells further confirmed the co-localization of pVHL withMKLP-1 during anaphase. However, pVHL was not detected atthe tip of the midbody or at the Fleming body with MKLP-1 orseptin-6 during the terminal step of cytokinesis (Fig. 2D,F).Moreover, we observed that MKLP-1 was properly localized at themidbody of pVHL-null 786-O cells (data not shown), ruling outany role of pVHL for the midbody localization of MKLP-1.

Finally, we checked various pVHL point mutants for theirlocalization ability using immunofluorescence staining.Interestingly, the same K171G mutant of pVHL that showedelevated multinucleation also showed significantly reducedlocalization of pVHL to the spindle midzone during late anaphaseand at the cytokinetic bridge during cytokinesis (Fig. 2A,C). Bycontrast, other reported pVHL mutants (N90G, Y112H and L188V)did not show a significant reduction of their localization at thecytokinetic bridge (Fig. 2A,C and data not shown). We alsoobserved reduced nuclear localization of pVHL with K171Gmutation (Fig. 2A) during interphase.

pVHL is required for localization of microtubule-associated proteins at the terminal midbodyThe final stage of cytokinesis requires cleavage of the midbody,which consists of dense spindle microtubules, by membrane fusion.Normal localization of wild-type pVHL to the cytokinetic bridgeand the increased frequency of multinucleation in cells lackingpVHL at their cytokinetic bridge suggested an involvement ofpVHL in cytokinesis. To explore this potential role, we analyzedthe localization of a number of known cytokinesis regulatoryproteins in pVHL-deficient 786-O and isogenic wild-type pVHLreconstituted cells. These cells were stained with antibodies againstcytokinesis regulatory kinases Plk1 and Aurora B, proteinsassociated with the midbody components Cep55, Alix, Tsg101 andthe v-SNARE complex component Endobrevin. Depletion of pVHLdid not affect the localization of either of the mitotic kinases (Plk1or Aurora B) (Fig. 3A,B) or the microtubule-associated midbodycomponents Cep55 (Fig. 3G) and Tsg101 (Fig. 3H). By contrast,the Cep55-interacting protein Alix, which is recruited by Cep55 atthe terminal stage of cytokinesis, was absent from the midbody(Fig. 3C,D). Similar to Alix, Endobrevin was also absent from themidbody (Fig. 3E,F). We also verified the localization of the abovemarkers in the RCC cells A498 (pVHL-null) and Caki-1 (wild-type pVHL). Absence of pVHL did not affect the localization ofPlk1, AuroraB, Cep55 and Tsg101 (Fig. 3I) in A498 cells. However,as expected, Alix was absent from the midbody (Fig. 3I) in thesecells. Furthermore, we found that all cytokinesis regulatory markerstested in wild-type pVHL-expressing Caki-1 cells were properlylocalized to the midbody (Fig. 3I).

Next, to verify the specificity of these cytokinesis-associateddefects in pVHL-deficient cells, we expressed various point mutantsof pVHL in 786-O cells and performed immunofluorescenceanalysis of the midbody localization of all the midbody markers

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described above. Supplementary material Table S1 summarizes theeffect of pVHL mutation on the localization of all the markerstested. Interestingly, only the K171G mutation of pVHL, whichleads to displacement of pVHL from the cytokinetic bridge, alsoexcluded Alix and Endobrevin from the midbody (Fig. 3C–F andsupplementary material Table S1). However, western blot analysis


showed no significant difference in the expression of any of theseproteins in pVHL-deficient cells compared with wild-type pVHL-expressing cells (supplementary material Fig. S1B). We, therefore,suggest that the multinucleated phenotype and CIN in pVHL-deficient or pVHL-K171G mutated cells resulted from theimpairment of midbody cleavage.

Fig. 2. Localization of pVHL during mitosis. (A)Cells were co-stained with anti-pVHL (green) and anti--tubulin (red) antibodies. Magnified images of selectedregions are included. Association of pVHL with the spindle microtubule throughout mitosis was observed (arrows and bracket). Nuclear localization of pVHL wasindicated during interphase (arrow, arrowhead). (B)In Caki-1 cells, co-staining with anti-pVHL (green) and anti--tubulin (red) denoted the localization of pVHLat the cytokinetic bridge (arrows). (C)ImageJ quantitation data showed the relative pVHL intensities at the cytokenetic bridge. (D)Cells were co-stained with anti-MKLP-1 (red) and anti-pVHL (green) antibodies and show the co-localization of pVHL with MKLP-1 at the spindle midzone but not at the center of the midbodyor Fleming body. (E)Three-dimensional analysis was done using Zeiss LSM 510 confocal microscope. (F)786-O-VHL(wt) cells were co-stained with pVHL(green) and septin6 (red) antibodies, septin6 localization at the midbody is indicated by an arrow. Figures represent three separate experiments with similar results.

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Fig. 3. Localization of the midbody markers. (A–C,E,G) RCC cell lines were co-stained with antibodies against -tubulin (red) and Plk1, AuroraB, Alix,Endobrevin and Cep55 (green). Images of midbodies are shown. Cells expressing pVHL-K171G show a defect in localization of Alix and Endobrevin but not withPlk1, Cep55 and AuroraB at the midbody. (D,F) Frequency of Alix (D) and Endobrevin (F) localization at the midbody. Bars represent the percentage of cells withprominent, weak or absent Alix and Endobrevin localization at the midbody. (H)786-O-empty and 786-O cells expressing wild-type pVHL were co-stained withantibodies against -tubulin (green) and Tsg101 (red). (I)RCC A498 and Caki-1 cells were co-stained with antibodies against -tubulin (green) and Plk1, Cep55,AuroraB, Alix and Tsg101 (red). Images of midbodies are shown, arrows indicate midbody localization at different midbody markers. Figures represent threeseparate experiments with similar results.

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pVHL associates with cytokinesis regulatory midbodycomponentsTo investigate how pVHL directs the localization of Alix orEndobrevin to the midbody of dividing cells, the presence of pVHLin Alix and Endobrevin immunoprecipitates from mitotic 293T


cells expressing 3�FLAG-HA–VHL(wt) and 3�FLAG-HA–VHL-K171G (see Materials and Methods) were evaluated by westernblot. 293T cells were synchronized at the G1–S phase with adouble thymidine block and followed by release for 8.5 hours toenrich mitotic populations of cells. We observed that wild-type

Fig. 4. Association of pVHL with midbody markers.(A)During first release after thymidine block, 293T cellswere transfected with 3�FLAG-HA–VHL(wt) and3�FLAG-HA–VHL-K171G. Immunoprecipitations weredone with antibody against Alix and immunoblottedagainst pVHL and Alix. (B)Western blot analysis withpVHL, Alix and Endobrevin antibodies afterimmunoprecipitation from 293T cells expressing wild-type pVHL or mutant pVHL with Endobrevin antibody.(C)Immunoprecipitations were done with 293T celllysates using Cep55 antibody, and immunoblotted withantibodies against pVHL, Alix and Cep55.(D)Immunoprecipitations were done using synchronized293T cells with Tsg101 antibody, and immunoblotted forpVHL, Alix and Tsg101. (E)Immunoprecipitation withCep55 antibody and western blot with Tsg101 and Cep55antibodies in 293T cells. Middle panels are whole cellextracts from the indicated cell lines. NIH ImageJquantitation data for the interactions of Alix, pVHL,Endobrevin, Cep55 and Tsg101 are shown with bargraphs. Error bars indicate s.d. Non-specific antibodieswere used as controls. Figures represent three separateexperiments with similar results.

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pVHL is associated with both Alix and Endobrevin (Fig. 4A,B),and that the K171G mutation decreased the association of bothAlix and Endobrevin with pVHL (Fig. 4A,B). Immunoprecipitationdata also revealed that Alix and Endobrevin were in the samecomplex (Fig. 4B), and that the K171G mutation of pVHL impairedthe interaction of Alix with Endobrevin in 293T cells. We made asimilar observation when lysates from synchronized 786-O cellsexpressing wild-type pVHL or pVHL-K171G wereimmunoprecipitated with Endobrevin antibody and immunoblottedwith antibody against Alix (supplementary material Fig. S1C).

Because interactions between Alix, Cep55 and Tsg101 areimportant for efficient cytokinesis (Morita et al., 2007), we alsoassessed the influence of the pVHL mutant on these complexes. Asexpected, we observed decreased interaction of Alix with Cep55 in293T or RCC 786-O cells expressing pVHL-K171G comparedwith cells expressing wild-type pVHL (Fig. 4C and supplementarymaterial Fig. S1D). Additionally, we found that the associationbetween Alix and Tsg101 increased in the presence of wild-typepVHL compared with the pVHL-K171G mutant in 293T cells(Fig. 4D). Although we have observed the reduced staining ofpVHL-K171G to the cytokinetic bridge during cytokinesis (Fig.2A), we could not detect any change in association of pVHL withCep55 or Tsg101 (Fig. 4C,D, respectively) in 293T whole celllysates. Interestingly, we found decreased interaction of Tsg101with Cep55 (Fig. 4E).


Thus, K171G mutation of pVHL appears to alter the interactionsof pVHL with Alix and thereby inhibits the association of Alixwith proteins involved in midbody abscission, i.e. Cep55, Tsg101,and Endobrevin by exerting a dominant-negative effect. The dataalso suggest that the lysine 171 site of pVHL is important for theinteraction of pVHL with Endobrevin.

SUMOylation of pVHL enables cytokinetic regulatoryfunctionExistence of lysine 171 within a common SUMOylation motifyKxE (Rodriguez et al., 2001; Sampson et al., 2001) (Fig. 5A)indicated a possible modification of pVHL by SUMOylation.Interestingly, co-transfection of pVHL together with SUMO-1 orSUMO-2 and Ubc9 constructs in HeLa cells yielded clear, slowlymigrating bands corresponding to the estimated size specific toSUMOylated pVHL (Fig. 5B). Both SUMO-1 and SUMO-2 wereconjugated to pVHL to a similar extent in the in vivo SUMOylationassays (Fig. 5C). Slow-migrating bands detected by anti-FLAGimmunoblotting disappeared when the SUMOylation assay wasperformed with a SUMOylation-defective form of SUMOs(SUMO-1DGG or SUMO-2DGG), suggesting that the bands areSUMOylated pVHL (supplementary material Fig. S2A). Theidentity of SUMOylated pVHL was further confirmed by Ni-NTA-agarose pull-down assay against His-tagged SUMO-1 or SUMO-2, followed by immunoblotting with anti-FLAG antibody

Fig. 5. Detection of SUMOylation on pVHL.(A)Putative SUMOylation site containing conservedyKxE signature in pVHL is presented. (B)Plasmidconstructs for pVHL (p3�FLAG-CMV10-HA–VHL) andUbc9 (pFLAG-CMV10-Ubc9) were transfected to HeLacells in the presence of SUMO-1 (pcDNA4-His-Max-SUMO-1) or SUMO-2 (pcDNA4-His-Max-SUMO-2), orabsence of SUMO proteins. Cells were harvested 36 hoursafter transfection and analyzed by western blotting.(C)Increasing concentrations of SUMO expressionplasmids were added in the same transfection experimentsas described in B. (D)Wild-type pVHL (Wt) and threemutant forms of pVHL (K159R, K171R and K196R) weresubjected to transfection-based SUMOylation assays asdescribed in Materials and Methods to confirm theSUMOylation site in pVHL. (E)Stable clones of 786-O-empty, 786-O-VHL(wt) and 786-O-VHL-K171G (C3 andC3.1) were transfected with pcDNA4-His-Max-SUMO-1or pcDNA4-His-Max-SUMO-2, and pFLAG-CMV2-Ubc9and were subjected to in vivo SUMOylation assay.Experiments were repeated at least three times.

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(supplementary material Fig. S2B). pVHL was clearly detected inpull-down samples as a SUMO-conjugated protein (supplementarymaterial Fig. S2B).

Additionally, we found that among three lysine residues ofpVHL, only K171R abrogated both SUMO-1 and SUMO-2modifications of pVHL but not K159R or K196R (Fig. 5D). 786-O cells stably expressing wild-type pVHL or the pVHL-K171Gmutant were transfected with SUMO-1 or SUMO-2 and UbC9-expressing plasmids. SUMOylated pVHL bands were abolished bypVHL-K171G mutation in 786-O cells (Fig. 5E). Moreover, theK171G mutation also abrogated the SUMO modification of pVHLin HeLa cells (supplementary material Fig. S2C). We did not findany change in ubiquitylation or neddylation of pVHL-K171Gcompared with wild-type pVHL (data not shown).

pVHL-K171G mutation fails to suppress tumor formationin nude mouse xenograft modelThe pVHL-K171G mutation leads to a cytokinesis defect thatultimately develops a phenotype of multinucleation and polyploidy.Reintroduction of wild-type pVHL into RCC has been reported toblock the ability of these cells to form tumors in nude mice(Iliopoulos et al., 1995; Schoenfeld et al., 1998). Therefore, westudied whether the K171G mutation compromised the tumorsuppressor function of pVHL.

pVHL is a component of the E3 ubiquitin ligase complex thattargets the -subunit of HIF for degradation (Maxwell et al., 1999).Tumor cells devoid of functional pVHL produce inordinate amountsof HIF-regulated hypoxia-inducible genes such as VEGF (Gnarra


et al., 1996; Levy et al., 1996; Siemeister et al., 1996). Theexpression of VEGF mainly accounts for the vascular phenotypeof pVHL-associated tumors. Glucose transporter-1 (Glut-1)expression is also increased in pVHL-defective RCC (Iliopoulos etal., 1996; Ozcan et al., 2007). Using western blot analysis, wefound a significant reduction of HIF-2 expression in mutantstable cell lines compared with 786-O-empty cells, with amagnitude of reduction similar to that observed in 786-O-VHL(wt)cells (Fig. 6A). However, pVHL-null, wild-type and mutant 786-O cells showed similar levels of HIF-2 mRNA expression (Fig.6B). Thus, it seems that the K171G mutation does not impair theHIF-2 degradation function of pVHL. However, we detectedmore significant upregulation of both VEGF-A and Glut-1expression in 786-O-VHL-K171G stable clones than in 786-O-VHL(wt) cells using real-time polymerase chain reaction (PCR)(Fig. 6C), suggesting that non-HIF-2 pathways account forupregulation of these proteins.

Next, we subcutaneously injected the following cells into nudemice: 786-O-empty, 786-O clones expressing wild-type pVHL,and two 786-O clones (C3 and C3.1) expressing the pVHL-K171Gmutant. Tumor take and size were monitored and measured weekly(Fig. 6D and supplementary material Table S2). As expected, 786-O-empty cells formed tumors (9 of 10), whereas mice injectedwith 786-O-VHL(wt) were tumor-free (0 of 10) (Fig. 6D andsupplementary material Table S2). Notably, mice injected with C3and C3.1 clones of 786-O-VHL-K171G also developed tumors(Fig. 6D and supplementary material Table S2), but the averagetumor weight and tumor-take time were lower for 786-O-VHL-

Fig. 6. Effect of pVHLmutation on tumor suppressorfunction. (A)Effect of pVHL-K171G on HIF-2expression. HIF-2 expression was determined in 786-O-empty cells, 786-O-VHL(wt) and two clones of 786-O-VHL-K171G using western blot. K171G mutation didnot show any effect on the pVHL-mediated HIF-2degradation. (B)HIF-2 mRNA expression. RelativeHIF-2 mRNA expressions are shown for 786-O-empty,786-O-VHL(wt) and 786-O-VHL-K171G (C3) cells.(C)Relative expressions of VEGF-A and glucosetransferase-1 (Glut-1). Increased expression of VEGF-Aand Glut-1 mRNAs were found in both clones C3 andC3.1 of the 786-O-VHL-K171G stable cell line. Figuresrepresent three separate experiments with similar results.(D)pVHL-K171G fails to suppress tumor formation in anude mice xenograft model. The graph shows tumorgrowth over time in nude mice injected with the stableclones 786-O-empty, 786-O-VHL(wt) and 786-O-VHL-K171G (C3 and C3.1).

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K171G than for 786-O-empty tumors. The results demonstrate thatthe K171G mutation of pVHL affects the proper cytokinesis of thedividing cells, leads to polyploidy, and impairs pVHL tumorsuppressor activity.

Furthermore, we performed western blot analysis for pVHL andHIF-2 on lysates from 786-O-empty and 786-O-VHL-K171Gtumors and assessed HIF-2 expression in tumor sections byimmunohistochemistry with anti-HIF-2 antibody. Interestingly,we observed a significant induction of HIF-2 expression by bothwestern blot and immunostaining (supplementary material Fig.S3A,B). Given the cell line data, this HIF-2 upregulation intumors of 786-O-VHL-K171G–injected mice is probably asecondary effect, possibly due to the development of a hypoxicenvironment in 786-O-VHL-K171G tumors.

DiscussionLoss of pVHL can activate cellular pathways that are stronglyassociated with both tumor initiation and progression. Becauseupregulation of HIFs is thought to be an important pathway thatmediates tumor progression in pVHL-deficient states, the pathwaysmediating tumor initiation are not clearly defined. Althoughaneuploidy, a form of CIN in pVHL-deficient MEFs, has beenlinked to a deregulated spindle assembly checkpoint (Thoma et al.,2009), the source of increased multinucleation and polyploidy inpVHL-deficient RCC remains unexplained. In this study, weobserved that pVHL-deficient 786-O cells show increasedmultinucleation and the frequent occurrence of unresolvedcytokinetic bridge structures. Moreover, cytogenetic analysisrevealed that pVHL-deficient 786-O cells are polyploid in nature.Consistent with that, an increase in multinucleation by knockingdown pVHL or overexpressing pVHL-K171G in HeLa or Caki-1cells further supports our hypothesis.

We also report that pVHL co-localizes with MKLP-1 at thespindle midzone during late anaphase but becomes separated at theterminal midbody. More specifically, MKLP-1 localizes to theFlemming body, whereas pVHL localizes to the stem region ofcytokinetic bridge during cell cleavage. Because polyploidy can beattributed to failed cytokinesis (Chi and Jeang, 2007), a number ofkey cytokinesis regulatory proteins, which are required for thefinal abscission step, have been analyzed. We observed that pVHL-null 786-O cells and 786-O cells expressing pVHL-K171G mutantare defective in localization to the midbody of Alix and Endobrevin,but not of upstream effectors Aurora B, Plk1 and Cep55 ordownstream effecter Tsg101. The lysine 171 residue in pVHL isunique because we did not find any effect on the localization ofAlix or Endobrevin by other reported mutants of pVHL, such asN90G, Y112H and L188V. We found that pVHL-deficient-A498cells were also defective in Alix localization at the midbody butnot wild-type pVHL Caki-1 cells. Recent studies have establishedthat the Cep55, Alix and Tsg101 axis functions at the terminalmidbody and regulates the final cleavage (Morita et al., 2007).Alix and Tsg101 concentrate at the centrosome and are thenrecruited to the midbody through direct interaction with thecentrosome-associated protein Cep55 (Morita et al., 2007). Whenwe checked the association of pVHL with Alix, Cep55 and Tsg101,we found a decreased association of pVHL-K171G compared withwild-type pVHL with Alix but not with Cep55 or Tsg101. We alsoobserved decreased interaction of Alix with both Cep55 and Tsg101in cells overexpressing pVHL-K171G. pVHL-K171G also affectedthe interaction between Cep55 and Tsg101. Therefore, we proposethat pVHL is a part of the Cep55–Alix–Tsg101 complex, and that


pVHL interaction with Alix is important for the recruitment of Alixto the Cep55–Tsg101 complex at the midbody. Endobrevin, amember of SNARE membrane fusion machinery, localizes to themidbody and is required for the final abscission step (Low et al.,2003) during cytokinesis. Our data also suggest that Endobrevin isa component of the pVHL–Alix–Cep55–Tsg101 complex, and thatEndobrevin interaction with pVHL is crucial for the localization ofEndobrevin at the midbody.

The involvement of lysine 171 of pVHL in the regulation ofmidbody abscission intrigued us to explore the functionalimportance of the site. We identified the tumor suppressor pVHLas a target for SUMOylation. We observed reduced interaction ofpVHL-K171G with Alix and Endobrevin in absence ofSUMOylation. Thus, we suggest that SUMO modification of pVHLis necessary for the complex formation with Alix and Endobrevin.However, the complex formation of pVHL with Cep55 or Tsg101is independent of pVHL SUMOylation. We also observedsignificantly reduced nuclear localization of pVHL-K171G in theabsence of SUMOylation. In-depth studies are underway todelineate the mechanism of how SUMOylation alters the functionof pVHL as a cytokinesis regulator. This study reveals a previouslyunexplored role of the pVHL.

pVHLD95-123 and, more specifically, type 2A pVHL mutantsY98H and Y112H have been shown to disrupt the microtubule-destabilizing function of pVHL (Hergovich et al., 2003). Recently,Thoma and associates showed that pVHL Y98H and Y112H alsofailed to correct the spindle misorientation phenotype of 786-Ocells but were not associated with the level of the mitotic arrestdeficient protein 2 (Mad-2) (Thoma et al., 2009). However, we didnot find a change in pVHL-directed localization of Alix orEndobrevin with reported pVHL mutants such as Y112H, N90G,or L188V (type 2C). Therefore, spindle assembly checkpoint failureand defective cytokinesis are different pVHL-regulated events.

Inactivation of a number of tumor suppressor genes has beenimplicated in cytokinesis failure and CIN (Caldwell et al., 2007;Daniels et al., 2004; Fujiwara et al., 2005). We report that pVHL-null and pVHL-K171G mutant cells are polyploid because ofcytokinesis failure and are able to form tumors in mice. AlthoughK171G is not a reported mutation, pVHL truncating mutations thatexclude this site are very common in pVHL-associated disease.Loss of heterozygosity (LOH) has been frequently observed inRCC patient samples (Gnarra et al., 1994). Almost 30% of patientshave a deletion of one or more exons of the pVHL gene;approximately half of them (15%) include the entire gene(Hoebeeck et al., 2005).

Therefore, these data provide the evidence that, in pVHL-deficient cells, CIN may result from the combined failure of spindleassembly checkpoint (Thoma et al., 2009) and cytokinesis. Thecapacity for pVHL to regulate cytokinesis appears to be independentof its ability to regulate HIFs. Finally, our data show a new avenueof pVHL’s tumor suppressor activity, which is regulated bySUMOylation.

Materials and MethodsCell culture and transfectionHuman RCC 786-O cells were cultured as described by Sinha et al. (Sinha et al.,2008). 786-O cells were stably transfected with pcDNA3.1hygro (786-O-empty),pRC-HA–VHL [786-O-VHL(wt)], pcDNA3.1hygro-HA–VHL-K171G (786-O-VHL-K171G), or pcDNA3.1hygro-HA–VHL-N90G (786-O-VHL-N90G) and maintainedin DMEM medium with 10% FBS supplemented with G418 (1 mg/ml) or hygromycin(150 mg/ml). 293T cells were grown in DMEM medium and transfected with 2 mgof pBabe-puro-HA–VHL-L188V or pBabe-puro-HA–VHL-Y112H DNA using theEffectene transfection reagent (Qiagen, Hilden, Germany) according to the

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manufacturer’s protocol. The retrovirus was isolated 48 hours after transfection.Retrovirus solution (1ml, >2�107 plaque-forming units/ml) and 5 ml of freshmedium were added to the 786-O cells with 10 mg/ml polybrene, and positive cloneswere selected using puromycin (2 mg/ml). The human RCC cell lines (A498; ATCCHTB-44, and Caki-1; ATCC HTB46) were maintained in MEM and McCoy’s 5A(Hyclone Laboratories, Logan, UT) medium, respectively, containing 10% FBS(Fisher Scientific, Pittsburgh, PA) and 1% penicillin–streptomycin (Invitrogen,Carlsbad, CA). HeLa cells were cultured in DMEM medium.

HeLa or 293T cells were seeded at a density of 6�106/100-mm plate 24 hoursbefore transfection. 3�FLAGCMV10-HA–VHL(wt) and 3�FLAGCMV10-HA–VHL-K171G DNA transfections were carried out with the Effectene transfectionreagent (Qiagen) following the manufacturer’s protocol. The pVHL expression waschecked 48 hours after transfection.

AntibodiesAnti-pVHL and anti-b-actin antibodies were from BD Biosciences (San Diego, CA).Anti-HA and Alix antibodies were purchased from Cell Signaling. Anti-Cep55 andanti-FLAG antibodies were from Sigma-Aldrich (St Louis, MO). Septin-6 andEndobrevin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,CA). Anti--tubulin, Aurora B, and Tsg101 antibodies were from Abcam (Cambridge,MA). Anti-HIF-2 and MKLP-1 antibodies were from Novus Biologicals (Littleton,CO). Anti-Plk1 antibody was from Upstate Biologicals (Millipore, Lake Placid,NY). Anti-Xpress antibody was purchased from Invitrogen.

RNA interferenceCaki-1 and HeLa cells were transfected with siRNA against VHL (Qiagen). ControlsiRNA from Dharmacon (Lafayette, CO) was used. HeLa and Caki-1 cells wereseeded at 70% confluence in DMEM and McCoy’s 5A media, respectively, 24 hoursprior to transfection. Before the transfection, fresh antibiotic-free medium was addedto cells. DharmaFECT 1 (Dharmacon, Lafayette, CO) was used for the transfectionwith 100 nM of each siRNA according to the manufacturer’s instructions.

Immunofluorescence and confocal microscopyImmunofluorescence and confocal microscopy were carried out as describedpreviously (Wu et al., 2009).

Chromosome enumerationMetaphase spreads of colcemid-treated cells were generated and analyzed as describedby Baker and associates (Baker et al., 2006). To prepare metaphase spreads, 2�106

cells were treated with 0.05 mg/ml colcemid (GIBCO BRL) for 8 hours at 37°C,suspended in 5 ml 0.075 M KCl, and incubated at room temperature for 30 minutes.Cells were then fixed in Carnoy’s solution (75% methanol, 25% acetic acid), washed,and finally re-suspended in approximately 0.5 ml fixative. Aliquots were droppedonto glass slides, stained with Giemsa solution, and analyzed on an Olympus AX70microscope using a 100� objective.

Cell synchronization293T cells were synchronized with double thymidine block, performed with a firstblock for 16 hours, an 8 hour release, and a second block for 16 hours. Cells werethen released for 8.5 hours to enrich the mitotic population. The final concentrationof thymidine used in the block medium was 2 mM. RCC cell lines were alsosynchronized with double thymidine (2 mM) block and released for 12 hours toenrich the mitotic population.

Immunoprecipitation and western blot analysisWhole-cell lysates in NP40 buffer supplemented with protease inhibitor cocktailwere prepared. Collected lysates were subjected to immunoprecipitation followed byimmunoblot analysis (Sinha et al., 2009b).

Mutagenesis for the replacement of lysine residuesMutagenesis of lysine (K) residues 159, 171 and 196 in pVH to arginine (R) wasperformed using QuickChange XL Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA). Mutated constructs were confirmed by sequencing and subcloned intop3xFlagCMV10 vector.

In vivo SUMOylation assayHeLa cells were grown on six-well plates and transfected with p3�FLAG-CMV10-HA–VHL(wt) or p3�FLAG-CMV10-HA–VHL-K159R or p3�FLAG-CMV10-HA–VHL-K171R or p3�FLAG-CMV10-HA–VHL-K196R, pcDNA4-His-Max-SUMO-1 or pcDNA4-His-Max-SUMO-2, and pFLAG-CMV2-Ubc9 using PolyFectreagent (Qiagen). After transfections (36 hours), the cells were lysed, sonicatedbriefly, and then centrifuged. Then proteins were separated by SDS–PAGE, and theSUMOylated-pVHL was detected by western blot analysis.

786-O cells stably transfected with pcDNA3.1hygro (786-O-empty), pRC-HA–VHL [786-O-VHL(wt)], and pcDNA3.1hygro-HA–VHL-K171G (786-O-VHL-K171G) were again transfected with pcDNA4-His-Max-SUMO-1 orpcDNA4-His-Max-SUMO-2, and pFLAG-CMV2-Ubc9 using PolyFect reagent.SUMOylation assay was carried out as described above.

Mutant SUMO constructs with deletion of C-terminal di-glycine residues weregenerated by adding a stop codon on the site for the first glycine using PCR andsubcloned in p3�FLAG-CMV10 vector (Sigma-Aldrich). SUMOylation assay wascarried out as described above.

HeLa cells were also transfected with pRC-HA–VHL(wt) and pcDNA3.1hygro-HA–VHL-K171G using PolyFect reagent. SUMOylation assay was carried out asdescribed above.

Ni-NTA-pulldown assayHeLa cells expressing pVHL and SUMOylation system were lysed in phosphatebuffed saline (PBS) containing 1% SDS and then diluted 10 times with PBScontaining 10 mM imidazole. Cell extract (500 mg) was mixed with Ni-NTA-agarose(Qiagen) for 4 hours. After incubation, beads were washed with the same bufferthree times, boiled in SDS-PAGE loading buffer, and subjected to SDS-PAGEfollowed by western blotting.

RNA preparation and quantitative real time PCRRNA preparation and real-time PCR (RT-PCR) was carried out as describedpreviously (Sinha et al., 2009a). VEGF-A, Glut-1, HIF-2 and ACTB primers werepurchased from SABiosciences (Frederick, MD). Relative expression was calculatedusing the comparative Ct method (Pichiorri et al., 2008).

Tumor model and in vivo anti-tumor activityDetails of the RCC subcutaneous tumor model are described in supplementarymaterial Table S2. Female, 6-week-old, nude mice were obtained from the NationalInstitutes of Health and housed in the institutional animal facilities. All animal workwas performed under protocols approved by the Mayo Clinic Institutional AnimalCare and Use Committee. To establish tumor growth in mice, we injected 5�106

786-O-empty, 786-O-VHL(wt), and two clones (C3 and C3.1) of 786-O-VHL-K171G cells, resuspended in 100 ml of PBS, subcutaneously into the right flank.Tumors were allowed to grow for 4 weeks. Beginning at week 4 after tumor cellinjection, tumors were measured every week, and primary tumor volumes werecalculated by the formula: volume 1/2a � b2, where a is the longest tumor axisand b is the shortest tumor axis. At week 9, all mice injected with 786-O-empty cellswere sacrificed by asphyxiation with CO2. At week 13, 786-O-VHL-K171G tumor-bearing mice were sacrificed; tumors were removed, measured and prepared forimmunohistochemistry and western blot.

Histological studyTumors were removed and fixed in neutral buffered 10% formalin at roomtemperature for 24 hours prior to embedding in paraffin and sectioning. Sectionswere deparaffinized and then subjected to hematoxylin-eosin and HIF-2immunochemistry staining according to the manufacturer’s instructions. Stablediaminobenzidine was used as a chromogen substrate, and the HIF-2 sections werecounterstained with a hematoxylin solution. Photographs of the entire cross-sectionwere digitized using an Olympus camera (DP70).

Statistical analysisStatistical analysis was performed with statistical SPSS software (version 11.5;Chicago, IL). The independent-samples t-test was used to test the probability ofsignificant differences between groups. Statistical significance was defined as P<0.05;statistically high significance was defined as P<0.01. Error bars were given on thebasis of standard deviation values calculated.

This work is partly supported by NIH grants CA78383 and a giftfrom Atwater Foundation to D.M.; CA116167 and CA122340 to F.J.C.pBabe-puro-HA–VHL-L188V and pBabe-puro-HA–VHL-Y112Hretroviral backbone constructs were a generous gift from William G.Kaelin Jr (Dana-Faber Cancer Institute, Boston, MA). We thank Janvan Deursen and Asish Ghosh, Mayo Clinic, for discussions. We alsoacknowledge Jim Tarara, Mayo Clinic, for helping with confocalmicroscopy. Deposited in PMC for release after 12 months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/13/2132/DC1

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