Membrane recycling experimentsFor quantitative fluorescence microscopy, HUVEC monolayers were incubated for 1 h at37 8C with Fab fragments of P1.1, a monoclonal antibody against domain 5 of PECAMthat does not inhibit any known PECAM function10. During this time, the Fab bound toPECAM at the junction and was taken up into the subjunctional compartment. Controlexperiments showed that binding of Fab to PECAM was stable down to pH 3.0. We washedoff unbound antibody, chilled the monolayers on ice, and added rabbit antibody againstmouse IgG (RAM) for 1 h at 4 8C; these conditions were determined to saturably bind all ofthe available antibody that was present on PECAM molecules at the cell surface (that is,any monoclonal antibody bound to PECAM molecules that had not been sequestered, orany that had recycled back to the cell surface during the incubation). After this treatment,unbound RAM was washed away and FITC-labelled RAM was added in the cold for 1 h toallow ample time to penetrate along the endothelial cell borders. At time zero, themonolayers were rapidly warmed to 37 8C, allowing membrane trafficking to resume forvarious times before fixation. FITC–RAM can only bind to Fab fragments that were notpreviously in the junction, that is, Fab fragments on sequestered membrane that recycledto the junction or Fab fragments on membrane that now communicated with the junction.Newly synthesized PECAM molecules coming to the junction at this time would not belabelled by FITC–RAM, because they would not have had the opportunity to bind Fabfragments against PECAM.

Digitized images were made using a Leica DMIRB microscope equipped with aPrinceton Instruments cooled CCD (charge-coupled device) camera driven by Image-1/MetaMorph Imaging System software (Universal Imaging Corporation). Quantitativeanalysis of images was done with MetaMorph software (Universal Imaging Corporation).Briefly, after background subtraction, the total integrated intensity per field was measuredfor five random fields per time point. We plotted mean fluorescence intensities per field foreach time point.

For flow cytometry, HUVEC monolayers were treated as above, but FITC–RAM Fabwas used as the detecting agent. At specified times, cells were rapidly removed from theplates, given a brief (2-min) exposure to trypsin plus EDTA and quenched in ice-coldserum-containing medium. Pilot experiments had shown that this procedure does notaffect the surface levels of immunologically detectable PECAM. We analysed cells by flowcytometry on a FACScalibur cytometer using CellQuest software (Becton Dickenson).

Fluorescence quenching experimentsExternal fluorescence quenching experiments were carried out on confluent HUVECmonolayers grown on cover slip dishes. We incubated the monolayers with FITC-labelledP1.1 Fab for 1 h at 37 8C or 4 8C, and then washed the cultures and chilled them in ice-coldPBS. Digitized images of representative fields were captured using a Leica DMIRBmicroscope, as described above. Immediately after obtaining the image, the pH of thesurrounding buffer was dropped to 5.5 by flooding the dish with 20 mM 2-(N-morpholino)-ethanesulphonic acid (MES) buffer and a second image of the same field wascaptured within 2 s of the change in pH (ref. 9) using the same imaging settings. Controlexperiments in which HUVEC monolayers were allowed to endocytose FITC–dextran intoendosomal or lysosomal compartments showed that this treatment had no effect onintracellular pH.

Recycling during TEMMonolayers were incubated with P1.1 Fab as for the quantification of recyclingexperiments. After chilling and saturable binding of junctional Fab with F(ab

0)2 fragments

of RAM, freshly isolated human monocytes (or THP-1 monocyte line in one series ofcontrol experiments) were added, along with rhodamine-labelled F(ab

0)2 fragments of

RAM, at 4 8C. These monocytes had been incubated in a blocking rabbit antibody againstPECAM or a preimmune IgG, and then washed extensively before their addition. After themonocytes had settled on the monolayer, the cultures were quickly warmed to 37 8C toallow a rapid and synchronous wave of TEM. After 10–15 min, TEM was stopped by rapidwashing and fixation of the monolayers. The degree of TEM was analysed by a typical TEMassay5, and membrane recycling was detected by the rhodamine-conjugated antibodyagainst mouse IgG, which would only stain the P1.1 Fab on recycling PECAM. Monocyteswere identified by staining with OKM1 antibodies conjugated to Alexa-488 (MolecularProbes), according to the manufacturer’s directions. We examined samples by confocalmicroscopy using a Zeiss LSM 510 microscope and analysed images using MetaMorphsoftware.

Received 26 September; accepted 12 November 2002; doi:10.1038/nature01300.

1. Butcher, E. C. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and

diversity. Cell 67, 1033–1036 (1991).

2. Springer, T. A. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep

paradigm. Cell 76, 301–314 (1994).

3. Muller, W. A. & Randolph, G. J. Migration of leukocytes across endothelium and beyond: molecules

involved in the transmigration and fate of monocytes. J. Leukoc. Biol. 66, 698–704 (1999).

4. Muller, W. A. Leukocyte–endothelial cell interactions in the inflammatory response. Lab. Invest. 82,

521–534 (2002).

5. Muller, W. A., Weigl, S. A., Deng, X. & Phillips, D. M. PECAM-1 is required for transendothelial

migration of leukocytes. J. Exp. Med. 178, 449–460 (1993).

6. Berman, M. E., Xie, Y. & Muller, W. A. Roles of platelet/endothelial cell adhesion molecule-1

(PECAM-1, CD31) in natural killer cell transendothelial migration and b2 integrin activation.

J. Immunol. 156, 1515–1524 (1996).

7. Liao, F., Ali, J., Greene, T. & Muller, W. A. Soluble domain 1 of platelet-endothelial cell adhesion

molecule (PECAM) is sufficient to block transendothelial migration in vitro and in vivo. J. Exp. Med.

185, 1349–1357 (1997).

8. Schenkel, A. R., Mamdouh, Z., Chen, X., Liebman, R. M. & Muller, W. A. CD99 plays a major role in

the migration of monocytes through endothelial junctions. Nature Immunol. 3, 143–150 (2002).

9. Yamashiro, D. J. & Maxfield, F. R. Kinetics of endosome acidification in mutant and wild-type chinese

hamster ovary cells. J. Cell Biol. 105, 2713–2721 (1987).

10. Liao, F., Huynh, H. K., Eiroa, A., Greene, T., Polizzi, E. & Muller, W. A. Migration of monocytes across

endothelium and passage through extracellular matrix involve separate molecular domains of

PECAM-1. J. Exp. Med. 182, 1337–1343 (1995).

11. Feng, D., Nagy, J. A., Hipp, J., Dvorak, H. F. & Dvorak, A. M. Vesiculo-vacuolar organelles and the

regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and

serotonin. J. Exp. Med. 183, 1981–1986 (1996).

12. Vasile, E., Qu, H., Dvorak, H. F. & Dvorak, A. M. Caveolae and vesiculo-vacuolar organelles in bovine

capillary endothelial cells cultured with VPF/VEGF on floating Matrigel-collagen gels. J. Histochem.

Cytochem. 47, 159–167 (1999).

13. Schmidt, A., Hannah, M. J. & Huttner, W. B. Synaptic-like microvesicles of neuroendocrine cells

orginate from a novel compartment that is continuous with the plasma membrane and devoid of

transferrin receptor. J. Cell Biol. 137, 445–458 (1997).

14. Feng, D., Nagy, J. A., Pyne, K., Dvorak, H. F. & Dvorak, A. M. Neutrophils emigrate from venules by a

transendothelial cell pathway in response to fMLP. J. Exp. Med. 187, 903–915 (1998).

15. Bamforth, S., Lightman, S. & Greenwood, J. Ultrastructural analysis of interleukin-1b-induced

leukocyte recruitment to the rat retina. Investig. Ophtalmol. Vis. Sci. 38, 25–35 (1997).

16. Hammersen, F. & Hammersen, E. The ultrastructure of endothelial gap formation and leukocyte

emigration. Prog. Appl. Microcirc. 12, 1–34 (1987).

17. Allport, J. R., Muller, W. A. & Luscinskas, F. W. Monocytes induce reversible focal changes in vascular

endothelial cadherin complex during transendothelial migration under flow. J. Cell Biol. 148, 203–216

(2000).

18. Shaw, S. K., Bamba, P. S., Perkins, B. N. & Luscinskas, F. W. Real-time imaging of vascular endothelial-

cadherin during transmigration across endothelium. J. Immunol. 167, 2323–2330 (2001).

19. Muller, W. A., Ratti, C. M., McDonnell, S. L. & Cohn, Z. A. A human endothelial cell-restricted,

externally disposed plasmalemmal protein enriched in intercellular junctions. J. Exp. Med. 170,

399–414 (1989).

20. Muller, W. A. & Weigl, S. Monocyte-selective transendothelial migration: Dissection of the binding

and transmigration phases by an in vitro assay. J. Exp. Med. 176, 819–828 (1992).

21. Ali, J., Liao, F., Martens, E. & Muller, W. A. Vascular endothelial cadherin (VE-Cadherin): cloning and

role in endothelial cell–cell adhesion. Microcirculation 4, 267–277 (1997).

Supplementary Information accompanies the paper on Nature’s website

(ç http://www.nature.com/nature).

Acknowledgements We thank R. Liebman for technical assistance; P. Newman for the P1.1

antibody; and P. Brennwald, T. McGraw and T. Ryan for discussions and comments on the

manuscript. Supported by NIH grants (to W.A.M. and F.R.M.), a Charles H. Revson Foundation

Fellowship (to Z.M.), and an Atorvastatin Research Award from Pfizer/Parke Davis (to L.P.).

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to W.A.M.

(e-mail: wamuller@med.cornell.edu).

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

Cdc42 regulates GSK-3b andadenomatous polyposis colito control cell polaritySandrine Etienne-Manneville & Alan Hall

MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, CancerResearch UK Oncogene and Signal Transduction Group, and Department ofBiochemistry and Molecular Biology, University College London, Gower Street,London WC1E 6BT, UK.............................................................................................................................................................................

Cell polarity is a fundamental property of all cells. In highereukaryotes, the small GTPase Cdc42, acting through a Par6–atypical protein kinase C (aPKC) complex, is required to estab-lish cellular asymmetry during epithelial morphogenesis, asym-metric cell division and directed cell migration1–5. However, littleis known about what lies downstream of this complex. Here weshow, through the use of primary rat astrocytes in a cellmigration assay, that Par6–PKCz interacts directly with andregulates glycogen synthase kinase-3b (GSK-3b) to promotepolarization of the centrosome and to control the direction ofcell protrusion. Cdc42-dependent phosphorylation of GSK-3boccurs specifically at the leading edge of migrating cells, and

letters to nature

NATURE | VOL 421 | 13 FEBRUARY 2003 | www.nature.com/nature 753© 2003 Nature Publishing Group

induces the interaction of adenomatous polyposis coli (Apc)protein with the plus ends of microtubules. The association ofApc with microtubules is essential for cell polarization. Weconclude that Cdc42 regulates cell polarity through the spatialregulation of GSK-3b and Apc. This role for Apc may contributeto its tumour-suppressor activity.

Scratch-induced cell migration in astrocyte monolayers is associ-ated with the spatially restricted activation of Cdc42, which actsthrough a Par6–PKCz complex to control cell polarity. In a searchfor molecules that act downstream of Par6–PKCz, we examined thephosphorylation state of GSK-3b, a protein previously implicatedin the establishment of polarity6–10. The level of phosphorylation atserine 9 of GSK-3b increases soon after scratching a monolayer,reaches a maximum at 1 h, and lasts for at least 12 h (note thatwound closure is complete by 24 h; Fig. 1a). The total levels of

GSK-3b remain constant. Figure 1b shows that GSK-3b phos-phorylation occurs preferentially at the leading edge of migratingcells, where we previously reported co-localization of Cdc42, Par6and PKCz5. Ectopically expressed enhanced green fluorescent pro-tein (EGFP)–Cdc42 localizes together with phosphorylated GSK-3b(Fig. 1b). To determine whether GSK-3b physically associates withPKCz11, the proteins were immunoprecipitated and analysed onwestern blots. We found that the two proteins can be precipitatedtogether and exist in a complex, but after scratch-inducedmigration, significant dissociation occurs (Fig. 1c). PhosphorylatedGSK-3b cannot be detected in the PKCz precipitate (not shown),indicating that phosphorylation of GSK-3b leads to its dissociationfrom PKCz. Figure 1c shows that GSK-3b can be precipitatedwith an anti-Par6 antibody, suggesting a larger complex containingGSK-3b, Par6 and PKCz.

To confirm that GSK-3b phosphorylation is dependent on theCdc42–Par6–PKCz pathway, cells were pre-treated with toxinB10463 (inhibits Cdc42, Rac, Rho), toxin B1470 (inhibits Rac,Ral, Rap1, R-ras) and C3 transferase (inactivates Rho). Only thetoxin that inhibits Cdc42 prevents GSK-3b phosphorylation(Fig. 1d). Inhibition of all PKC isoforms with GF109203X(Fig. 1d) or RO 31-8220 (not shown), or inhibition of PKCz witha cell-permeable PKCz-specific pseudo-substrate (Fig. 1d), preventsGSK-3b phosphorylation, whereas depletion of conventional ornovel PKCs by 12-O-tetradecanoylphorbol-13-acetate (TPA) treat-ment does not (Fig. 1d). Furthermore, inhibition of phosphatidyl-inositol-3-OH kinase (PI(3)K) with wortmannin or LY 294002 hasno effect (not shown), indicating that Akt/PKB is not involved.Finally, transfection of COS cells with Par6, which activates PKCz,or with wild-type (but not kinase-dead) PKCz induces phosphoryl-ation of GSK-3b on serine 9 (Fig. 1e).

Phosphorylation of GSK-3 at serine 9 inhibits its catalyticactivity12,13. To explore the significance of this, GSK-3 S9A, a non-phosphorylatable, constitutively activated mutant was micro-injected into leading-edge cells. The establishment of polarity wasvisualized by the reorientation of the centrosome to face thedirection of migration, and by the formation of cell protrusionsperpendicular to the scratch5. GSK-3 S9A is almost as effective asdominant-negative Cdc42 (N17Cdc42) in blocking the reorienta-tion of the centrosome (Fig. 2a). Expression of a kinase-dead

Figure 2 Spatially localized inhibition of GSK-3 is required to establish cell polarity.

a, b, After scratching a monolayer, leading-edge cells were microinjected with the

indicated constructs (a), or incubated with inhibitors (b). Centrosome polarization was

determined 8 h later. c, Cells at the leading edge were microinjected, and the number of

expressing cells with protrusions was determined 8 h later. d, Cells were scratched,

incubated with LiCl (20 mM) or SB216763 (20 mM), and 16 h later they were fixed and

stained with an anti-tubulin antibody.

Figure 1 Glycogen synthase kinase-3b (GSK-3b) is phosphorylated downstream of

Cdc42 and protein kinase Cz (PKCz) during astrocyte migration. a, Astrocyte monolayers

were scratched and were incubated for the indicated times. Phosphorylated (Ser 9) and

total GSK-3b were analysed by western blotting (WB). b, Cell monolayers were scratched

and immediately microinjected with green fluorescent protein (GFP)–Cdc42. Cells were

fixed 0 or 4 h later and stained with anti-phospho-GSK-3 (Ser 9) antibody (left and middle

panels). GFP–Cdc42 (right panel) localizes with phospho-GSK-3 (Ser 9) at the leading

edge. Scale bar, 10 mm. c, Astrocytes were lysed before (2) or 1 h after (þ) scratching.

Endogenous GSK-3b or PKCz were immunoprecipitated (IP) and analysed on western

blots with anti-PKCz or anti-GSK-3b antibodies, respectively. Par6 was

immunoprecipitated and analysed with anti-Par6 or anti-GSK-3b antibodies.

d, Astrocytes were pre-treated with toxin B 1470 (3 h, 10 pg ml21), toxin B 10463 (3 h,

1 pg ml21), C3 toxin (overnight, 5 mg ml21), GF109203X (20 mM, 1 h), PKCz pseudo-

substrate (10 mM, 1 h) or 12-O-tetradecanoylphorbol-13-acetate (TPA) (160 nM,

overnight), scratched in the presence of the inhibitors, and incubated for 1 h. GSK-3b

phosphorylation was visualized as above. e, COS cells were transfected and lysed after

48 h, and phosphorylated (Ser 9) and total GSK-3b visualized. Each experiment was

repeated three times. FL, full length; KD, kinase-dead; WT, wild type.

letters to nature

NATURE | VOL 421 | 13 FEBRUARY 2003 | www.nature.com/nature754 © 2003 Nature Publishing Group

version of GSK-3b also inhibits centrosome reorientation (Fig. 2a).This is reminiscent of our previous observations that dominant-negative or constitutively activated Cdc42 inhibit polarity, andpoints to the importance of localization of signalling activities5.To visualize the effects of global GSK-3 inhibition, cells were pre-treated with two chemically distinct inhibitors, LiCl and SB216763,both of which block centrosome polarity (Fig. 2b). Close inspection(Fig. 2c, d) reveals that in the presence of GSK-3 inhibitors, cellprotrusions still form but are randomly oriented.

GSK-3 is a component of numerous signal transduction path-ways. In the Wnt pathway, GSK-3 is part of an axin–Apc–b-catenincomplex, and Wnt induces inactivation of GSK-3, stabilization ofb-catenin and induction of gene transcription14,15. The loss of APCactivity in human cancers also leads to b-catenin stabilization16. Todetermine the effects of localized GSK-3 inhibition in migratingcells, lysates from resting and scratched astrocyte monolayers wereanalysed on western blots. Thirty minutes after scratch-inducedmigration, stabilization of b-catenin is observed (Fig. 3a). Thispersists for at least 24 h and is dependent on Cdc42 and PKCz(Fig. 3a, c). Figure 3b reveals that b-catenin accumulates at theleading edge of migrating cells and is still visible at cell–cell contacts,which are maintained during the migration assay. There is nodetectable increase in nuclear b-catenin or inhibition of astrocytepolarization on treatment with cycloheximide or actinomycinD. Accumulation of b-catenin at the leading edge of cells isabolished by expression of N17Cdc42, kinase-dead PKCz or GSK-3b S9A (data not shown). It can also be disrupted with cytochalasinD (not shown), but, in this case, cell polarity is not affected5. Thedata indicate that the accumulation of b-catenin at the leading edgereflects the localized inhibition of GSK-3, but is not required for cellpolarity. It may, however, contribute other activities to the processof cell migration.

The tumour-suppressor gene product Apc is phosphorylatedby GSK-3 (refs 17, 18). Apc participates in the destabilization ofb-catenin, but can also associate with the plus ends of microtubulesand regulate microtubule dynamics19,20. Figure 4a, b shows that2–4 h after scratch-induced migration, Apc becomes associated withthe plus ends of microtubules specifically at the leading edge, andthis association is dependent on Cdc42, PKCz and phosphorylationof GSK-3 at serine 9. Through residues located in the carboxy-terminal region, Apc can associate with microtubules directly, or bymeans of the protein EB1 (refs 21, 22). EB1 also localizes at the plusends of microtubules, but unlike Apc, this is independent of Cdc42,PKCz (data not shown) and GSK-3 S9A (Fig. 4b). To determinewhether the association of Apc with microtubules is required forpolarization, Apc lacking the microtubule- and EB1-binding sites(Apc-DCt) was expressed in leading-edge cells. This inhibits centro-some reorientation, whereas full-length Apc or the amino-terminaldomain of Apc does not (Fig. 4c). None of the Apc constructsinhibits protrusion formation (data not shown).

We conclude that Cdc42 activates Par6–PKCz, leading to phos-phorylation and inactivation of GSK-3b at the leading edge ofmigrating astrocytes. Inactivation of GSK-3b may affect severalmicrotubule-associated proteins23,24, but a key consequence is thespatially restricted association of Apc with the plus ends of micro-

Figure 4 The association of Apc with microtubules is regulated by the Cdc42–PKCz–

GSK-3 pathway. a, Astrocyte monolayers were scratched and fixed 8 h later. Cells were

stained with anti-tubulin (green) and anti-Apc (red) antibody. Scale bar, 10 mm. Apc

localization at the plus ends of microtubules was confirmed by expression of a GFP-tagged

protein (data not shown), but no accumulation of GFP–Apc was visible in the nucleus,

indicating that the nuclear staining seen here may be an artefact. b, Astrocyte monolayers

were scratched and leading-edge cells were immediately microinjected with the indicated

constructs. Four hours later, cells were fixed and stained with anti-tubulin (green), anti-

Apc or anti-EB1 (lower-right panel) (red) antibody and an antibody to detect the expressed

proteins. Expressing cells are indicated with a white arrow; arrowheads indicate Apc

association with the microtubule plus-ends in the non-injected cells. Scale bar, 10 mm.

c, Astrocytes were microinjected immediately after scratching with the indicated

constructs (Xenopus Apc full length (FL), amino acids 1–2830; APC-Nt, amino acids

1–1035; APC-DCt, amino acids 1–1962). Centrosome polarization was determined 8 h

after wounding.

Figure 3 b-Catenin is stabilized and localized at the leading edge of migrating cells.

a, Scratched monolayers were incubated for different times, and b-catenin levels were

assessed by western blot. An anti-tubulin antibody was used as a loading control. b, Cells

were fixed and stained with anti-b-catenin antibody 4 h after scratching. Arrowheads

show b-catenin localized at the leading edge of migrating cells. Scale bar, 10 mm.

c, b-Catenin levels in astrocytes pre-treated with toxin B 10463 (3 h, 1 pg ml21), C3 toxin

(overnight, 5 mg ml21), GF109203X (20 mM, 1 h), LiCl (1 h, 20 mM) or SB216763 (1 h,

20 mM), scratched in the presence of the inhibitors, and incubated for 6 h before lysis.

letters to nature

NATURE | VOL 421 | 13 FEBRUARY 2003 | www.nature.com/nature 755© 2003 Nature Publishing Group

tubules, which is essential for establishing cell polarity. The role ofApc is unclear; it could directly affect microtubule dynamics25,26,or—perhaps through EB1—interact with the dynactin–dynein com-plex, which is required for centrosome reorientation5,27,28. Theseobservations link together two important signalling pathways thathave been independently implicated in controlling cell polarityduring the development of multicellular organisms: Cdc42–Parproteins on one hand2, and GSK-3–b-catenin–Apc on the other10.The essential role described here for Apc in regulating the polarity ofmigrating cells may be an important aspect of its functionalcontribution to human cancer. A

MethodsMaterialsWe used the following reagents: anti-a-tubulin (Sigma), anti-GSK-3b, anti-b-catenin,anti-EB1 (all Transduction Labs), anti-PKCz, anti-Cdc42 (both Santa CruzBiotechnology), anti-pericentrin (BabCO), anti-phospho-GSK-3b (Ser 9) (Biosource),anti-Apc (I. Nathke) and anti-Par6C (amino acids 2–16; P. Aspenstrom), GF109203X,RO 31-8220 and SB216763 (all Calbiochem), PKCz pseudo-substrate (Biosource), toxinsB10463 and B1470 (C. von Eichel-Streiber), GSK-3 constructs (V. M. Lee and R. Kypta)and Apc constructs (B. M. Gumbiner). GTPases, Par6 and PKCz constructs have beendescribed previously5.

Cell culture and scratch-induced migrationPrimary astrocyte monolayers and scratch assays have been described previously5.Individual wounds around 300 mm wide were made with a microinjection needle, andwound closure occurred 16–24 h later. For biochemical analysis, multiple wounds weremade with an eight-channel pipette (with 2-ml tips) scratched several times across a90-mm dish. Centrosomes were localized using anti-pericentrin antibody5, and thosewithin the quadrant facing the wound were scored as positive. We examined 300 cells foreach condition. To assess polarized morphology, astrocytes were microinjected withpEGFP as a control or with the indicated DNA constructs and biotin-dextran, and werefixed 8 h later. Cells expressing these constructs were scored as protruding when theirlength was at least four times their width.

ImmunoprecipitationCells were washed with ice-cold PBS containing 1 mM orthovanadate and lysed at 4 8C inbuffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM orthovanadate, 1% Nonidet-P40,2 mM phenylmethylsulphonyl fluoride, 5 mM EDTA, 20 mg ml21 aprotinin, 20 mg ml21

leupeptin). Nuclei were discarded after centrifugation at 10,000g for 10 min. Lysates wereincubated for 2 h at 4 8C with specific antibodies and protein G Sepharose, andimmunoprecipitates were collected by centrifugation and were analysed on 8%SDS–polyacrylamide gel electrophoresis gels.

Received 25 November 2002; accepted 14 January 2003; doi:10.1038/nature01423.

Published online 29 January 2003.

1. Etienne-Manneville, S. & Hall, S. Rho GTPases in cell biology. Nature 420, 629–635 (2002).

2. Ohno, S. Intercellular junctions and cellular polarity: the PAR–aPKC complex, a conserved core

cassette playing fundamental roles in cell polarity. Curr. Opin. Cell Biol. 13, 641–648 (2001).

3. Gotta, M., Abraham, M. C. & Ahringer, J. CDC-42 controls early cell polarity and spindle orientation

in C. elegans. Curr. Biol. 11, 482–488 (2001).

4. Kay, A. J. & Hunter, C. P. CDC-42 regulates PAR protein localization and function to control cellular

and embryonic polarity in C. elegans. Curr. Biol. 11, 474–481 (2001).

5. Etienne-Manneville, S. & Hall, A. Integrin-mediated Cdc42 activation controls cell polarity in

migrating astrocytes through PKCz. Cell 106, 489–498 (2001).

6. Dominguez, I., Itoh, K. & Sokol, S. Y. Role of glycogen synthase kinase 3b as a negative regulator of

dorsoventral axis formation in Xenopus embryos. Proc. Natl Acad. Sci. USA 92, 8498–8502 (1995).

7. Emily-Fenouil, F., Ghiglione, C., Lhomond, G., Lepage, T. & Gache, C. GSK3b/shaggy mediates

patterning along the animal-vegetal axis of the sea urchin embryo. Development 125, 2489–2498 (1998).

8. He, X., Saint-Jeannet, J.-P., Woodgett, J. R., Varmus, H. E. & Dawid, I. Glycogen synthase kinase-3 and

dorsoventral patterning in Xenopus embryos. Nature 374, 617–622 (1995).

9. Pierce, S. B. & Kimelman, D. Regulation of Spemann organizer formation by intracellular Xgsk-3.

Development 121, 755–765 (1995).

10. Ferkey, D. M. & Kimelman, D. GSK-3: new thoughts on an old enzyme. Dev. Biol. 225, 471–479 (2000).

11. Oriente, F. et al. Insulin receptor substrate-2 phosphorylation is necessary for protein kinase Cz

activation by insulin in L6hIR cells. J. Biol. Chem. 276, 37109–37119 (2001).

12. Harwood, J. A. Regulation of GSK-3: a cellular multiprocessor. Cell 105, 821–824 (2001).

13. Troussard, A. A., Tan, C., Yoganathan, T. N. & Dedhar, S. Cell–extracellular matrix interactions

stimulate the AP-1 transcription factor in an integrin-linked kinase- and glycogen synthase kinase 3-

dependent manner. Mol. Cell. Biol. 19, 7420–7427 (1999).

14. Li, L. et al. Axin and Frat1 interact with Dvl and GSK, bridging Dvl to GSK in the Wnt-mediated

regulation of LEF-1. EMBO J. 18, 4233–4240 (1999).

15. Moon, R. T., Bowerman, B., Boutros, M. & Perrimon, N. The promise and perils of Wnt signaling

through b-catenin. Science 296, 1644–1646 (2002).

16. Polakis, P. Wnt signaling and cancer. Genes Dev. 14, 1837–1851 (2000).

17. Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B. & Polakis, P. Regulation of intracellular b-catenin

levels by the adenomatous polyposis coli (APC) tumour-suppressor protein. Proc. Natl Acad. Sci. USA

92, 3046–3050 (1995).

18. Rubinfeld, B. et al. Binding of GSK3b to the APC-b-catenin complex and regulation of complex

assembly. Science 272, 1023–1026 (1996).

19. Bienz, M. The subcellular destinations of APC proteins. Nature Rev. Mol. Cell Biol. 3, 328–338 (2002).

20. Mogensen, M. M., Tucker, J. B., Mackie, J. B., Prescott, A. R. & Nathke, I. S. The adenomatous

polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in

establishing parrallel arrays of microtubule bundles in highly polarized epithelial cells. J. Cell Biol. 157,

1041–1048 (2002).

21. Su, L. K. et al. APC binds to the novel protein EB1. Cancer Res. 55, 2972–2977 (1995).

22. Barth, A. I. M., Siemers, K. A. & Nelson, W. J. Dissecting interactions between EB1, microtubules and

APC in cortical clusters at the plasma membrane. J. Cell Sci. 115, 1583–1590 (2002).

23. Wagner, U., Utton, M., Gallo, J.-M. & Miller, C. C. J. Cellular phosphorylation of Tau by GSK-3b

influences tau binding to microtubules and microtubule organisation. J. Cell Sci. 109, 1537–1543

(1996).

24. Lucas, F. R., Goold, R. G., Gordon-Weeks, P. R. & Salinas, P. C. Inhibition of GSK-3b leading to the loss

of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium.

J. Cell Sci. 111, 1351–1361 (1998).

25. Nakamura, M., Zhou, X. Z. & Lu, K. P. Critical role for the EB1 and APC interaction in the regulation

of microtubule polymerization. Curr. Biol. 11, 1062–1067 (2001).

26. Zumbrunn, J., Kinoshita, K., Hyman, A. A. & Nathke, I. S. Binding of the adenomatous polyposis coli

protein to microtubules increases microtubule stability and is regulated by GSK3b phosphorylation.

Curr. Biol. 11, 44–49 (2001).

27. Palazzo, A. F. et al. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-

regulated microtubule stabilization. Curr. Biol. 11, 1536–1541 (2001).

28. Berrueta, L., Tirnauer, J. S., Schuyler, S. C., Pellman, D. & Bierer, B. E. The APC-associated protein EB1

associates with components of the dynactin complex and cytoplasmic dynein intermediate chain.

Curr. Biol. 9, 425–428 (1999).

Acknowledgements This work was supported by a Cancer Research UK programme grant, the

Medical Research Council and by an EMBO Long-Term Fellowship (S.E.-M.). We thank S. Martin,

V. M. Lee, R. Kypta, B. M. Gumbiner, P. Aspenstrom, I. Nathke and C. von Eichel-Streiber for

plasmids and reagents.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to A.H.

(e-mail: alan.hall@ucl.ac.uk).

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

Structure of the extracellularregion of HER2 alone and incomplex with the Herceptin FabHyun-Soo Cho*†, Karen Mason‡, Kasra X. Ramyar*, Ann Marie Stanley*,Sandra B. Gabelli*, Dan W. Denney Jr‡ & Daniel J. Leahy*†

* Department of Biophysics and Biophysical Chemistry, and † Howard HughesMedical Institute, The Johns Hopkins University School of Medicine, 725 NorthWolfe Street, Baltimore, Maryland 21205, USA‡ Genitope Corporation, 525 Penobscot Drive, Redwood City, California 94063,USA.............................................................................................................................................................................

HER2 (also known as Neu, ErbB2) is a member of the epidermalgrowth factor receptor (EGFR; also known as ErbB) family ofreceptor tyrosine kinases, which in humans includes HER1(EGFR, ERBB1), HER2, HER3 (ERBB3) and HER4 (ERBB4)1.ErbB receptors are essential mediators of cell proliferation anddifferentiation in the developing embryo and in adult tissues2,and their inappropriate activation is associated with the devel-opment and severity of many cancers3. Overexpression of HER2is found in 20–30% of human breast cancers, and correlates withmore aggressive tumours and a poorer prognosis4. Anticancertherapies targeting ErbB receptors have shown promise, and amonoclonal antibody against HER2, Herceptin (also known astrastuzumab), is currently in use as a treatment for breastcancer5. Here we report crystal structures of the entire extra-cellular regions of rat HER2 at 2.4 A and human HER2 complexedwith the Herceptin antigen-binding fragment (Fab) at 2.5 A.These structures reveal a fixed conformation for HER2 thatresembles a ligand-activated state, and show HER2 poised tointeract with other ErbB receptors in the absence of direct ligand

letters to nature

NATURE | VOL 421 | 13 FEBRUARY 2003 | www.nature.com/nature756 © 2003 Nature Publishing Group