chromosome 7. Cancer Res. 51, 4097–4101 (1991).

19. Zondag, G. C. M. et al. Oncogenic Ras downregulates Rac activity, which leads to increased Rho

activity and epithelial-mesenchymal transition. J. Cell Biol. 149, 775–781 (2000).

20. Kemp, C. J., Donehower, L. A., Bradley, A. & Balmain, A. Reduction of p53 gene dosage does not

increase initiation or promotion but enhances malignant progression of chemically induced skin

tumors. Cell 74, 813–822 (1993).

21. Joneson, T. & Bar-Sagi, D. Suppression of Ras-induced apoptosis by the Rac GTPase. Mol. Cell Biol. 19,

5892–5901 (1999).

22. Robles, A. I. et al. Reduced skin tumour development in cyclin D1-deficient mice highlights the

oncogenic ras pathway in vivo. Genes Dev. 12, 2469–2474 (1998).

23. Sander, E. E. et al. Matrix-dependent Tiam1/Rac signalling in epithelial cells promotes either cell-cell

adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J. Cell Biol. 143,

1385–1398 (1998).

24. Sander, E. E., ten Klooster, J. P., van Delft, S., Van der Kammen, R. A. & Collard, J. G. Rac

downregulates Rho activity: reciprocal balance between both GTPases determines cellular

morphology and migratory behaviour. J. Cell Biol. 147, 1009–1022 (1999).

25. Braga, V. M. M., Machesky, L. M., Hall, A. & Hotchin, N. A. The small GTPases rho and rac are

required for the establishment of cadherin-dependent cell-cell contacts. J. Cell Biol. 137, 1421–1431

(1997).

26. Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H. & Takai, Y. Regulation of cell-cell adhesion by Rac and

Rho small G proteins in MDCK cells. J. Cell Biol. 139, 1047–1059 (1997).

27. Bracke, M. E., Van Roy, F. M. & Mareel, M. M. The e-cadherin/catenin complex in invasion and

metastasis. Curr. Top. Microbiol. Immunol. 213, 123–161 (1996).

28. Perl, A. K., Wilgenbus, P., Dahl, U., Semb, H. & Christofori, G. A causal role for E-cadherin in the

transition from adenoma to carcinoma. Nature 392, 190–193 (1998).

29. de Rooij, J. & Bos, J. L. Minimal Ras-binding domain of Raf1 can be used as an activation-specific

probe for Ras. Oncogene 14, 623–625 (1997).

Supplementary Information accompanies the paper on Nature’s website

(http://www.nature.com/nature).

AcknowledgementsWe thank J.-Y. Song and the Animal Pathology department of the Netherlands CancerInstitute for histological analysis; D. Stuart, P. Demant and A. Hurlstone for advice;P. Krimpenfort and T. Schrauwers for experimental support; V. Zoumbourlis andA. Balmain for cell lines; and the animal facility of the Netherlands Cancer Institute forproviding animal care. Part of this work was supported by the Dutch Cancer Society, theEuropean Community, and the Association for International Cancer Research.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to J.G.C.

(e-mail: j.collard@nki.nl).

..............................................................

Efp targets 14-3-3j for proteolysisand promotes breast tumour growthTomohiko Urano*†, Tomoyuki Saito*†‡, Tohru Tsukui§, Masayo Fujita*,Takayuki Hosoi*, Masami Muramatsu§, Yasuyoshi Ouchi*& Satoshi Inoue*§k

* Department of Geriatric Medicine, Graduate School of Medicine, The Universityof Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan§ Research Center for Genomic Medicine and Department of Molecular Biology,Saitama Medical School, 1397-1 Yamane, Hidaka-shi, Saitama 350-1241, JapankCREST, Japan Science and Technology Corporation, Kawaguchi, Saitama,332-0012, Japan† These authors contributed equally to this work.............................................................................................................................................................................

Oestrogen exerts its influence on target organs through activat-ing oestrogen receptors (ERs) and regulating downstream genesby means of their oestrogen-responsive elements. Efp, a targetgene product of ERa1–3, is a member of the RING-finger B-boxcoiled-coil (RBCC) motif family4. Efp is predominantly expressed

in various female organs2 as well as in breast cancers5, and isthought to be essential for oestrogen-dependent cell proliferationand organ development—Efp-disrupted mice display underdeve-loped uteri and reduced oestrogen responsiveness6. Here we showthat Efp is a RING-finger-dependent ubiquitin ligase (E3) thattargets proteolysis of 14-3-3j, a negative cell cycle regulator thatcauses G2 arrest7. We demonstrate that tumour growth of breastcancer MCF7 cells implanted in female athymic mice is reducedby treatment with antisense Efp oligonucleotide. Efp-overexpres-sing MCF7 cells in ovariectomized athymic mice generate tumoursin the absence of oestrogen. Loss of Efp function in mouseembryonic fibroblasts results in an accumulation of 14-3-3j,which is responsible for reduced cell growth. These data providean insight into the cell-cycle machinery and tumorigenesis ofbreast cancer by identifying 14-3-3j as a target for proteolysis byEfp, leading to cell proliferation.

We have used MCF7 cells as a model system to explore thecontribution of Efp to growth of breast tumours. MCF7 cells wereimplanted into intact female athymic mice followed by ovariectomyor administration of sense/antisense Efp oligonucleotides (Fig. 1a–d). Levels of Efp were markedly reduced in tumours obtained fromovariectomized or antisense Efp-treated mice compared with con-trols, suggesting that both treatments impaired Efp expression (Fig.1a). Tumour volume did not increase in ovariectomized mice,consistent with the oestrogen-dependent nature of MCF7 cells(Fig. 1d). Tumour growth was observed in control mice (Fig. 1c,d); however, a dose-dependent inhibition of tumour growth wasobserved in mice treated with antisense Efp (Fig. 1b–d). We inferthat tumour growth is modulated by Efp expression, implicatingEfp as an oncogenic factor in breast cancers.

To test this hypothesis, we generated MCF7 cells that stablyexpressed Efp (Efp-MCF7 cells) and investigated whether Efpmodulates progression of the cell cycle. Endogenous levels ofp21Cip1 and 14-3-3j were reduced in Efp-MCF7 cells as comparedwith control MCF7 cells (vector-MCF7 cells) (Fig. 2a). Theseproteins are negative regulators of cell cycle progression and areimportant for G1 and G2 arrest after DNA damage7,8, suggestingthat Efp may regulate cell cycle checkpoints (Fig. 2a). This wasconfirmed by cell cycle analysis using flow cytometry, as a highpercentage of Efp-MCF7 cells were in the proliferating stage,particularly in S phase, whereas vector-MCF7 cells were primarilyin the G1 phase (Fig. 2b). To determine whether Efp overexpressioninfluences tumour growth, we inoculated transfected MCF7 cellsinto intact female athymic mice. After two months, both vector-MCF7 and Efp-MCF7 cells showed tumour formation; however, thetumours were larger in Efp-MCF7 mice (data not shown). The roleof Efp as a primary regulator of oestrogen-dependent signalling wasverified by implanting transfected MCF7 cells into ovariectomizedmice. Tumours did not grow in vector-MCF7 mice, whereasprominent tumour growth was observed in ovariectomized miceinoculated with Efp-MCF7 cells (Fig. 2c). To further confirm therole of Efp in tumour growth, transfected MCF7 cells were inocu-lated into intact athymic mice, followed by ovariectomy when thetumour volume reached 180 mm3 (Fig. 2d). Tumour volume invector-MCF7 animals did not increase further, whereas tumours inEfp-MCF7 animals continued to grow in the absence of gonadaloestrogens. This indicates that elevated levels of Efp promote cellgrowth, indicating that Efp might directly regulate the cell cyclemachinery.

As a first step towards understanding the role of Efp in cell cycleprogression, we used yeast two-hybrid screening to identify Efp-interacting proteins. Notably, a single interacting clone coding for14-3-3j was identified by screening 3 £ 106 yeast clones trans-formed with a mouse embryo complementary DNA library. 14-3-3j was originally identified as an epithelial-specific marker, HME1,which is downregulated in a few breast and colon cancer cell lines7,9.Although 14-3-3j seems to be an important negative cell cycle

‡ Present address: Pharmacological Research Department, Teikoku Hormone MFG Co. Ltd, 1604 Shimo-

sakunobe, Takatsu-ku, Kawasaki, Japan.

letters to nature

NATURE | VOL 417 | 20 JUNE 2002 | www.nature.com/nature 871© 2002 Nature Publishing Group

regulator, a regulatory mechanism for 14-3-3j activity was pre-viously unknown.

Next, we tested whether Efp can specifically interact with andregulate the activity of 14-3-3j in mammalian cells. COS7 cells weretransiently transfected with Efp and 14-3-3j. Both proteins loca-lized in the cytoplasm (Fig. 3a) and immunoprecipitation con-firmed that Efp and 14-3-3j formed a complex in the cells (Fig. 3b).

In contrast, we did not detect interaction between Efp and other cellcycle checkpoints such as p21Cip1, p27Kip1 and p57Kip2 (data notshown). To define 14-3-3j-binding domains in Efp, we constructeda series of Efp deletion mutants and analysed their binding to full-length 14-3-3j by immunoprecipitation (Fig. 3c). Neither amino-terminal Efp (residues 1–122) containing the RING finger domainnor carboxy-terminal Efp (residues 440–630) containing the SPRY

Figure 1 Inhibition of Efp expression suppresses tumour growth by MCF7 cells. Athymic

female mice were inoculated with MCF7 cells then treated with an ovariectomy (OVX), or

administration of vehicle (saline) or sense/antisense Efp oligonucleotides (sense-L and

antisense-L, 20 mg per week; sense-H/sense and antisense-H/antisense, 100 mg per

week). a, Efp expression in tumours obtained from mice inoculated with MCF7 cells.

b, Weight of tumours caused by MCF7 cells in nude mice. c, Reduced tumour size in mice

6 weeks after inoculation with antisense Efp or with OVX. d, Reduced tumour volume in

mice treated with antisense Efp or with OVX. Asterisk, P , 0.05; double asterisk,

P , 0.01; triple asterisk, P , 0.001; n ¼ 5.

Figure 2 Efp promotes cell cycle progression and tumour growth by MCF7 cells in an

oestrogen-independent manner. a, Efp overexpression downregulates levels of 14-3-3j

or p21Cip1 in MCF7 cells. Efp-MCF7 cells stably express Efp. b, Efp overexpression

increases the percentage of cells in the proliferation stage of the cell cycle. c, Hormone-

independent tumour growth is observed in ovariectomized nude mice bearing Efp-MCF7

cells. Mice were inoculated with MCF7 cells and observed after 2 months. d, Tumour

volume of mice bearing MCF7 transfectants after ovariectomy. Mice were ovariectomized

at a tumour volume of 180 mm3. Triple asterisk, P , 0.001.

letters to nature

NATURE | VOL 417 | 20 JUNE 2002 | www.nature.com/nature872 © 2002 Nature Publishing Group

domain could bind to 14-3-3j. However, Efp (81–450) containingthe B-box coiled-coil domain was able to interact with 14-3-3j. TheB-box coiled-coil domain often associates with a RING finger toform a larger conserved motif, RBCC 4, and it is this motif in Efp thatspecifically interacts with 14-3-3j. It is notable that the RBCC motiffamily includes several oncogenic proteins such as PML10 and TIF1(ref. 11). We next investigated whether Efp overexpression affects

the expression of 14-3-3j. Co-transfection of Efp and 14-3-3j intoCOS7 cells resulted in lower levels of 14-3-3j protein comparedwith cells transfected with 14-3-3j alone (Fig. 3d). Similar resultswere obtained in HEK293 cells (data not shown). To explorewhether Efp overexpression can modulate 14-3-3j turnover, weperformed pulse and chase experiments in transfected COS7 cells(Fig. 3e). In the vector-transfected cells, 35S-labelled 14-3-3jimmunoprecipitated by anti-haemagglutinin (HA) remains atmore than 50% after 3 h, whereas the degradation rate of 14-3-3jwas explicitly enhanced in Efp-overexpressed cells (12.2% 14-3-3jremaining after 3 h in Efp-overexpressed cells compared with 58.4%in control cells). The reduced levels of 14-3-3j in MCF7 cells causedby adenoviral Efp transduction were recovered by MG132, apotent inhibitor of proteasome function12 (Fig. 3f). The findingthat MG132 increases the amount of 14-3-3j binding to Efp wasfurther confirmed by immunoprecipitation (Fig. 3g). These datasuggest that Efp directly downregulates 14-3-3j levels through aproteasome-dependent mechanism.

We next investigated how Efp downregulates expression of 14-3-3j. One possible mechanism is that Efp functions as an E3 ubiquitinligase similar to other RING finger proteins such as Rbx1 (refs

Figure 3 Efp conjugates to 14-3-3j and promotes 14-3-3j proteolysis in a proteasome-

dependent manner. a, Immunoreactivities of Efp (fluorescein isothiocyanate (FITC), left

panel) and 14-3-3j (rhodamin, right panel) expressed in COS7 cells were co-localized in

the cytoplasm. b, Interaction of Efp with 14-3-3j. Lysates of COS7 cells transfected with

indicated plasmids were analysed by immunoblotting using anti-Efp antibody (WB: Efp),

combined without (input) or with (IP: HA) immunoprecipitation using anti-HA. c, Specific

interaction of the B-box coiled-coil domain in Efp with 14-3-3j. Lysates of transfected

COS7 cells were immunoprecipitated with anti-Myc and analysed by immunoblotting

using anti-HA (middle panel: IP: Myc, WB: HA). The amounts of Myc-Efp mutants (WB:

Myc) are shown in the bottom panel. d, Efp overexpression downregulates exogenous 14-

3-3j. Lysates of transfected COS7 cells were analysed by anti-Efp (top panel) or anti-HA

(bottom panel). e, 14-3-3j degradation is accelerated by Efp expression. COS7 cells

transiently transfected with HA-14-3-3j and vector/Myc-Efp were pulse-labelled with

[35S]methionine for 30 min, and chased for the indicated time. f, Adenoviral Efp

expression downregulates endogenous 14-3-3j in a proteasome-dependent manner.

MCF7 cells transduced with b-galactosidase (Adv-Efp 2) or Efp (Adv-Efp þ)

adenoviruses were incubated with either vehicle (MG132 2) or MG132 (10 mM) for 8 h.

Lysates were analysed by anti-Efp (top panel) or anti-14-3-3j (bottom panel). g, MG132

enhanced in vivo interaction between adenovirally expressing Efp and endogenous 14-3-

3j. Lysates of MCF7 cells were immunoprecipitated with anti-14-3-3j and analysed by

anti-Efp.

Figure 4 Efp is a RING-finger-dependent ubiquitin ligase that targets 14-3-3j. a, 14-3-

3j downregulation is mediated through the RING finger in Efp. COS7 cells were co-

transfected with HA-14-3-3j and the indicated Myc-Efp mutants. Top panel, 14-3-3j

levels; bottom panel, expression of Efp with or without Myc-tagging. b, Association of Efp

with UbcH8. COS7 cells were co-transfected with Myc-Efp and Flag-tagged UbcH7/

UbcH8. c, The RING finger in Efp is required for interaction with UbcH8. COS7 cells were

co-transfected with Myc-Efp (residues 1–122) and Flag-tagged UbcH7/UbcH8. d, Efp and

UbcH8 promote 14-3-3j ubiquitination. Anti-ubiquitin was used to detect ubiquitinated

forms in 14-3-3j immunoprecipitates obtained from transfected COS7 cells. e, The RING

finger in Efp is required for 14-3-3j ubiquitination. Anti-HA was used to detect HA-14-3-

3j and its ubiquitinated forms (Ubn) in 14-3-3j precipitates obtained from transfected

COS7 cells (top panel). The bottom panel shows expression of Efp with or without Myc-

tagging.

letters to nature

NATURE | VOL 417 | 20 JUNE 2002 | www.nature.com/nature 873© 2002 Nature Publishing Group

13, 14), BRCA1 (ref. 15), Cbl (ref. 16), Mdm2 (ref. 17), parkin (ref.18) and MID1 (ref. 19). The ubiquitination-dependent proteolysisis related to cell cycle progression and carcinogenesis20–23. Weconstructed a RING-finger-deleted Efp mutant that lacked the 31N-terminal amino acids (DRING-Efp), and tested its function inCOS7 cells. The DRING-Efp mutant failed to downregulate levels of14-3-3j, indicating that the RING finger in Efp is essential forregulation of 14-3-3j (Fig. 4a). Furthermore, Efp preferentiallybound to ubiquitin-conjugating enzyme UbcH8 rather than UbcH7(Fig. 4b). This interaction is RING-finger-dependent becauseEfp (1–122) was immunoprecipitated with UbcH8 (Fig. 4c). Wenext examined whether Efp can ubiquitinate 14-3-3j in a UbcH8-dependent manner. COS7 cells were transfected with Efp, 14-3-3j,ubiquitin and UbcH8/UbcH7. Large amounts of ubiquitinated14-3-3j were observed in cells transfected with UbcH8 but notwith UbcH7 (Fig. 4d). Cells transfected with the DRING-Efpmutant failed to ubiquitinate 14-3-3j, confirming that ubiquitina-tion requires the Efp RING finger (Fig. 4e). We conclude that Efpdownregulates 14-3-3j through ubiquitin-mediated degradation inwhich Efp functions as an E3 that selectively requires UbcH8.

We investigated further the effects of Efp abrogation on cellgrowth using mouse embryonic fibroblasts (MEFs) from Efpþ/þ

and Efp2/2 mice6 as models. The growth of Efpþ/þ MEFs wasnormal, whereas that of Efp2/2 MEFs was severely reduced (Fig. 5a).14-3-3j levels were markedly elevated in Efp 2/2 MEFs (Fig. 5b).The levels of Cdk2 and Cdc2 bound to p21Cip1 or 14-3-3j were

elevated in Efp 2/2 MEFs (Fig. 5c), although the levels of Cdk2 andCdc2 in both MEFs were not significantly different (Fig. 5c). Thissuggests that increased amounts of 14-3-3j and p21Cip1 in Efp 2/2

MEFs tend to entrap mitotic factors, leading to reduced growth. Weexamined the effects of 14-3-3j antisense/sense oligonucleotides onMEF cell growth by collecting cells 24 or 48 h after initial incubationwith oligonucleotides (Fig. 5d). The reduced cell growth in Efp2/2

MEFs was rescued by the antisense oligonucletide in a dose-dependent manner, suggesting that 14-3-3j is the principal check-point in Efp2/2 MEFs. To explore whether the accumulation of14-3-3j in Efp 2/2 MEFs is due to stabilization of the protein ratherthan activation of transcription, we performed pulse and chaseexperiments (Fig. 5e). The half-life (t1/2) of 14-3-3j was 4 h inEfp 2/2 MEFs (95% confidence interval, 6.7–2.9 h), which wassignificantly longer than t 1/2 in Efpþ/þMEFs (2.3 h, 95% confidenceinterval, 2.8–1.8 h, P , 0.001). The accumulation of 14-3-3j inEfp 2/2 MEFs was reversed dose-dependently by adenoviral Efptransduction (Fig. 5f). The reduced levels of 14-3-3j in Efp 2/2

MEFs adenovirally expressing Efp was rescued by MG132, which isconsistent with the results indicating that Efp degrades 14-3-3jthrough a ubiquitin-dependent pathway.

Our findings provide insight into the cell cycle machinery andgrowth of oestrogen-dependent tumours. In the classical scheme,DNA damage induces elevation of p53 levels and p53 promotestranscription of Cdk inhibitors, which recruit cyclin–Cdk com-plexes leading to cell cycle arrest and DNA repair8,24–27. Here we

Figure 5 Loss of Efp function causes an accumulation of 14-3-3j and inhibits cell

proliferation. a, The growth of Efp 2/2 MEFs is reduced compared with Efp þ/þ MEFs.

b, Accumulation of 14-3-3j in Efp 2/2 MEFs. c, High amounts of Cdk2 (top panel) or

Cdc2 (middle panel) associated with p21Cip1 or 14-3-3j are detected in Efp 2/2 MEFs.

The amounts of precipitated Cdk2 and Cdc2 are shown in the bottom panel. d, Reduced

growth in Efp 2/2 MEFs was rescued by antisense 14-3-3j oligonucleotide. MEFs were

incubated with antisense/sense oligonucleotides (antisense-L/sense-L, 100 nM;

antisense-H/sense-H, 400 nM), or with vehicle. The graph shows the number of cells

collected 24 or 48 h after initial oligonucleotide transfection. The bottom panel shows

14-3-3j levels. Lanes 1–5 and 6–10 show lysates from indicated cells collected 24 and

48 h after initial transfection, respectively. Asterisk, P , 0.05 compared with vehicle at

24 h; double asterisk, P , 0.01 compared with vehicle at 48 h; triple asterisk,

P , 0.001 compared with vehicle at 48 h. e, 14-3-3j turnover is more stabilized in

Efp 2/2 MEFs than in Efp þ/þ MEFs. Lysates were immunoprecipitated by anti-14-3-3j.

f, 14-3-3j accumulation in Efp 2/2 MEFs is reduced by adenoviral Efp expression in a

proteasome-dependent manner. MEFs adenovirally expressing b-galactosidase (lane 1)

or Efp at a low dose (40 MOI) (lane 2) or at a high dose (100 MOI) (lanes 3 and 4) were

subjected to immunoblotting using anti-14-3-3j. Cells in lane 4 were treated with MG132

(10 mM).

letters to nature

NATURE | VOL 417 | 20 JUNE 2002 | www.nature.com/nature874 © 2002 Nature Publishing Group

demonstrate a mechanism in which the function of 14-3-3j ismodulated by Efp-mediated proteolysis. The degradation of 14-3-3j is subsequently followed by dissociation of the protein fromcyclin–Cdk complexes, leading to cell cycle progression and tumourgrowth. Indeed, it has been shown recently that downregulation of14-3-3j alone is sufficient to immortalize primary keratinocytes28.The present data suggest that Efp may be an essential oncogenicfactor in breast cancers and perhaps other hormone-related malig-nancies. Our findings have important clinical relevance becausetargeting the Efp-mediated breakdown of 14-3-3j could provide anew therapy to block breast tumour proliferation. A

MethodsMCF7 tumour growth in nude miceMCF7 cells suspended in Matrigel (5 £ 106 cells per 0.1 ml) were injected subcutaneouslyinto female athymic mice (4-week-old BALB/c nu/nu). We calculated tumour volumesweekly by the tumour radii. When the tumour volume reached 300 mm3, mice wereovariectomized or treated with Efp sense/antisense oligonucleotides (phosphorothioate-modified). Oligonucleotide sequences are: antisense, 5 0 -AGGGGGCACAGCTCTGCCAT-30; sense, 5

0-ATGGCAGAGCTGTGCCCCCT-3

0. Six weeks later, animals were killed and

tumours were analysed by immunoblotting using anti-Efp antibody1. In experiments forEfp-expressing MCF7 cells, cells were transfected with pEF29 or pEF containing Efp. Twoclones for each construct containing either pEF alone (vector-MCF7 number 1 and 3) orpEF-Efp (Efp-MCF7 number 2 and 4) were selected by G418. Each cell suspension(5 £ 106 cells per 0.1 ml) was injected into five ovariectomized nude mice respectively. Inother experiments, five intact female athymic mice injected with MCF7 cells wereovariectomized at a tumour volume of 180 mm3.

Cell cycle analysisTransfected MCF7 cells were incubated with phenol red free DMEM containing 0.2%charcoal-stripped fetal bovine serum (FBS) 24 h before analysis. Trypsinized cells wereresuspended in hypotonic propidium iodide solution (50 mg ml21) containing 0.1%sodium citrate and 0.1% Triton X-100, and analysed on a FACScan flow cytometer(Becton Dickinson).

Yeast two-hybrid screenSaccharomyces cerevisiae strain EGY48 was transformed with pEG202-NLS-human Efp,and further transformed with a mouse embryo cDNA library (OriGene). Selected positiveclones according to the manufacturer’s instructions were sequenced.

Immunoprecipitation assaysIn experiments with COS7 cells, cells were lysed in Nonidet P-40 lysis buffer30 48 h afterplasmid transfection. Lysates were immunoprecipitated with anti-Myc (Santa Cruz), anti-Flag (Sigma), or anti-14-3-3j (Santa Cruz) antibodies. In experiments with MEFs, lysateswere immunoprecipitated with anti-p21Cip1 (Santa Cruz), anti-14-3-3j, or goatimmunoglobulin-g (IgG), and immunoblotted by anti-Cdk2 (Santa Cruz) and anti-Cdc2(Santa Cruz).

Pulse and chase experimentsMEFs were pulse-labelled with [35S]methionine (200 mCi ml21) in methionine-freemedium containing 10% dialysed FBS for 2 h, washed twice with pre-warmed PBS, andchased by culturing in DMEM containing 10% FBS for 10 h. Lysates from pulse-labelledcells were immunoprecipitated with anti-14-3-3j, resolved by SDS–polyacrylamide gelelectrophoresis, and analysed with a PhosphorImager (Molecular Dynamics). Inexperiments with COS7 cells, cells were pulse-labelled for 30 min and chased for 3 h.Lysates were immunoprecipitated with anti-HA (Santa Cruz).

Generation of recombinant adenovirusesRecombinant adenoviruses were generated by Adenovirus Expression Vector Kit (TaKaRa)and used at a multiplicity of infection (MOI) of 40–100 according to the manufacture’sprotocol.

In vivo ubiquitination assaysCOS7 cells were transfected with plasmids including Myc-Efp, HA-14-3-3j, ubiquitin,and Flag-tagged UbcH8 or UbcH7. Forty hours later, cells were incubated with MG132(10 mM, Peptide Institute) for 8 h, then immunoprecipitated with anti-14-3-3j.Precipitates were analysed by anti-ubiquitin (Santa Cruz) or anti-HA.

Preparation of Efp1/1 and Efp2/2 MEFsWe generated Efp2/2 mice as described6. MEFs were obtained from wild-type or Efp 2/2

13.5-day-old embryos and used at third to sixth generations. For growth rate analysis, cellswere synchronized for 72 h in DMEM containing 0.1% FBS, then trypsinized and plated ata density of 1 £ 105 cells per 35-mm dish in DMEM containing 10% FBS. In experimentsfor antisense oligonucleotide transfection, MEFs maintained for 24 h in DMEMcontaining 10% FBS were incubated with oligofectamine (Invitrogen) and eitherantisense/sense 14-3-3j oligonucleotides (phosphorothioate-modified) or vehicle alonefor 4 h according to the manufacture’s protocol. At the end of incubation, 10% FBS wassupplemented and maintained for the next 24 or 48 h. Antisense, 5 0 -ATCAGACTGGC

TCTCTCCAT-3 0 ; sense, 50-ATGGAGAGAGCCAGTCTGAT-3

0. In other experiments,

cells were seeded and incubated with medium containing 10% FBS for 48 h, then lysed inNonidet P-40 lysis buffer30.

Received 6 February; accepted 19 April 2002; doi:10.1038/nature00826.

1. Inoue, S. et al. Genomic binding-site cloning reveals an estrogen-respomsive gene that encodes a

RING finger protein. Proc. Natl Acad. Sci. USA 90, 11117–11121 (1993).

2. Orimo, A., Inoue, S., Ikeda, K., Noji, S. & Muramatsu, M. Molecular cloning, structure, and

expression of mouse estrogen-responsive finger protein Efp. Co-localization with estrogen receptor

mRNA in target organs. J. Biol. Chem. 270, 24406–24413 (1995).

3. Muramatsu, M. & Inoue, S. Estrogen receptors: how do they control reproductive and non-

reproductive functions. Biochem. Biophys. Res. Commun. 270, 1–10 (2000).

4. Freemont, P. S. RING for destruction? Curr. Biol. 10, R84–R87 (2000).

5. Ikeda, K., Orimo, A., Higashi, Y., Muramatsu, M. & Inoue, S. Efp as a primary estrogen-responsive

gene in human breast cancer. FEBS Lett. 472, 9–13 (2000).

6. Orimo, A. et al. Underdeveloped uterus and reduced estrogen responsiveness in mice with disruption

of the estrogen-responsive finger protein gene, which is a direct target of estrogen receptor. Proc. Natl

Acad. Sci. USA 96, 12027–12032 (1999).

7. Hermeking, H. et al. 14-3-3 j is a p53-regulated inhibitor of G2/M progression. Mol. Cell 1, 3–11 (1997).

8. Bunz, F. et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282,

1497–1501 (1998).

9. Prasad, G. L., Valverius, E. M., McDuffie, E. & Cooper, H. L. Complementary DNA cloning of a novel

epithelial cell marker protein, HME1, that may be down-regulated in neoplastic mammary cells. Cell

Growth Differ. 3, 507–513 (1992).

10. Jensen, K., Shiels, C. & Freemont, P. S. PML protein isoforms and the RBCC/TRIM motif. Oncogene

20, 7223–7233 (2001).

11. Le Douarin, B. et al. The N-terminal part of TIF1, a putative mediator of the ligand-dependent

activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO

J. 14, 2020–2033 (1995).

12. Traenckner, E. B., Wilk, S. & Baeuerle, P. A. A proteasome inhibitor prevents activation of NF-kB and

stabilizes a newly phosphorylated form of IkB-a that is still bound to NF-kB. EMBO J. 13, 5433–5441

(1994).

13. Skowyra, D. et al. Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and

Rbx1. Science 284, 662–665 (1999).

14. Kamura, T. et al. Rbx1, a component of the VHL tumour suppressor complex and SCF ubiquitin

ligase. Science 284, 657–661 (1999).

15. Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T. & Verma, I. M. Cancer-predisposing mutations

within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from

radiation hypersensitivity. Proc. Natl Acad. Sci. USA 98, 5134–5139 (2001).

16. Joazeiro, C. A. et al. The tyrosine kinase negative regulator c-Cbl as a RING-type. E2-dependent

ubiquitin-protein ligase. Science 286, 309–312 (1999).

17. Honda, R. & Yasuda, H. Association of p19 (ARF) with Mdm2 inhibits ubiquitin ligase activity of

Mdm2 for tumour suppressor p53. EMBO J. 18, 22–27 (1999).

18. Shimura, H. et al. Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.

Nature Genet. 25, 302–305 (2000).

19. Trockenbacher, A. et al. MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets

phosphatase 2A for degradation. Nature Genet. 29, 287–294 (2001).

20. Tyers, M. & Willems, A. R. One ring to rule a superfamily of E3 ubiquitin ligases. Science 284, 601–604

(1999).

21. Joazeiro, C. A. P. & Weissman, A. M. RING finger proteins: mediators of ubiquitin ligase activity. Cell

102, 549–552 (2000).

22. Pagano, M. et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-

dependent kinase inhibitor p27. Science 269, 682–685 (1995).

23. Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

24. Chan, T. A., Hermeking, H., Lengauer, C., Kinzler, K. W. & Vogelstein, B. 14-3-3j is required to

prevent mitotic catastrophe after DNA damage. Nature 401, 616–620 (1999).

25. Laronga, C., Yang, H. Y., Neal, C. & Lee, M. H. Association of the cyclin- dependent kinases and

14-3-3j negatively regulates cell cycle progression. J. Biol. Chem. 275, 23106–23112 (2000).

26. Kastan, M. B. et al. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective

in ataxia-telangiectasia. Cell 71, 587–597 (1992).

27. Cox, L. S. & Lane, D. P. Tumour suppressors, kinases and clamps: how p53 regulates the cell cycle in

response to DNA damage. Bioessays 17, 501–508 (1995).

28. Dellambra, E. et al. Downregulation of 14-3-3j prevents clonal evolution and leads to

immortalization of primary human keratinocytes. J. Cell Biol. 149, 1117–1129 (2000).

29. Kim, D. W., Uetsuki, T., Kaziro, Y., Yamaguchi, N. & Sugano, S. Use of the human elongation factor 1 a

promoter as a versatile and efficient expression system. Gene 91, 217–223 (1990).

30. Urano, T. et al. p57(Kip2) is degraded through the proteasome in osteoblasts stimulated to

proliferation by transforming growth factor-b1. J. Biol. Chem. 274, 12197–12200 (1999).

AcknowledgementsWe thank H. Yashiroda, A. Takada, K. Okamoto, M. Kabayashi, S. Inada and T. Hishinumafor technical assistance, and B. Blumberg and K. Horie for critical reading and commentson the manuscript. This work was supported in part by grants-in-aid from the Ministry ofHealth, Labor and Welfare, from the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to S.I.

(e-mail: INOUE-GER@h.u-tokyo.ac.jp).

letters to nature

NATURE | VOL 417 | 20 JUNE 2002 | www.nature.com/nature 875© 2002 Nature Publishing Group