p120 catenin roles in adhesion and signaling · 2001. 5. 3. · p120 catenin roles in adhesion and...

INTRODUCTION

p120 (p120ctn, hereafter p120) was originally identified as aprominent substrate of the Src oncoprotein (Reynolds et al.,1989). cDNA cloning revealed that it contains an Armadillo-repeat domain (Arm domain) that shares 22% identity with thatof the catenin Armadillo/β-catenin (Reynolds et al., 1992;Peifer et al., 1994) and led to experiments that demonstratedits direct interaction with cadherins (Reynolds et al., 1994;Daniel and Reynolds, 1995; Staddon et al., 1995; Shibamotoet al., 1995; see Fig. 1). Cadherins comprise a superfamily oftransmembrane cell-cell adhesion receptors that link adjacentcells via calcium-dependent homophillic interactions betweenthe cadherin extracellular domains. They regulate a variety ofbiological processes, including development, morphogenesis,and tumor metastasis (for review see Takeichi, 1995; Yap,1998). During tumor progression to malignancy, the aberrantloss of expression of epithelial cadherin (E-cadherin), themajor cell-cell adhesion molecule in epithelial cells, is widelybelieved to mediate the transition to metastasis (Perl et al.,1998; reviewed by Yap, 1998). Cadherin function is modulatedby a group of cytoplasmic proteins called catenins (i.e. α-catenin, β-catenin, plakoglobin and p120), which interactwith the cadherin intracellular domain. Defects in cateninexpression or function have also been linked to metastasis. Amajor role of catenins is to anchor the cadherin complex to theactin cytoskeleton. β-Catenin and plakoglobin act as bridgesconnecting E-cadherin to α-catenin, which in turn associateswith actin filaments either directly (Herrenknecht et al., 1991;

Nagafuchi et al., 1991; Rimm et al., 1995) or indirectly(Knudsen et al., 1995; Nieset et al., 1997). β-Catenin also hassignaling roles in the cytoplasm and nucleus that are importantin development and cancer. For example, β-catenin/armadillois a key player in the Wnt/Wg signaling pathway, directlymediating downstream events through transactivation oftranscription factors of the Lef1/TCF family (Molenaar et al.,1996; Behrens et al., 1996; van de Wetering et al., 1997). Inaddition, β-catenin interacts directly with the tumor suppressoradenomatous polyposis coli (APC; Su et al., 1993; Rubinfeldet al., 1993). Inactivating mutations in APC, or activatingmutations in β-catenin, cause the accumulation of β-catenin inthe cytoplasm and nucleus, leading to constitutive signalingthrough interaction with Lef1/TCF (Munemitsu et al., 1995;Korinek et al., 1997; Rubinfeld et al., 1997). Collectively,defects in APC or β-catenin function are thought to accountfor initiation of the majority of human colon cancer (Powell etal., 1992; Morin et al., 1997) and a smaller percentage ofmelanoma (Rubinfeld et al., 1997). The well-characterizeddual roles of β-catenin in adhesion and signaling establish animportant paradigm for other Arm proteins, many of which areknown to function as adhesion molecules (e.g. p120).

Unlike β-catenin, p120 does not interact with α-catenin orwith APC (Daniel and Reynolds, 1995), which implies thatp120 has a novel function in cadherin complexes. p120apparently has both positive and negative affects on cadherin-mediated adhesion, depending on signaling cues that remainunspecified. Tyrosine and serine/threonine kinases are re-emerging as important candidates for the regulaton of p120

1319Journal of Cell Science 113, 1319-1334 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS0834

p120 catenin (p120) is the prototypic member of a growingsubfamily of Armadillo-domain proteins found at cell-cell junctions and in nuclei. In contrast to the functionsof the classical catenins (α-catenin, β-catenin, and γ-catenin/plakoglobin), which have been studied extensively,the first clues to p120’s biological function have onlyrecently emerged, and its role remains controversial.Nonetheless, it is now clear that p120 affects cell-celladhesion through its interaction with the highly conservedjuxtamembrane domain of classical cadherins, and is likelyto have additional roles in the nucleus. Here, we summarize

the data on the potential involvement of p120 both inpromotion of and in prevension of adhesion, and proposemodels that attempt to reconcile some of the disparities inthe literature. We also discuss the structural relationshipsand functions of several known p120 family members, aswell as the potential roles of p120 in signaling and cancer.

Key words: Cell adhesion, p120ctn, ARVCF,δ-catenin/NPRAP/neurojungin, p0071, Plakophilin, Cadherin,Clustering, RhoA, Kaiso, Presenilin, Armadillo/β-catenin

SUMMARY

COMMENTARY

The p120 catenin family: complex roles in adhesion, signaling and cancer

Panos Z. Anastasiadis and Albert B. Reynolds*

Department of Cell Biology, Vanderbilt University, MCN #C-2310, 1161 21st Ave. South, Nashville, TN 37232-2175, USA*Author for correspondence (e-mail: [email protected])

Published on WWW 21 March 2000

1320

activity, which suggests that the story will eventually come fullcircle back to Src. In addition, given the β-catenin paradigm, itis perhaps not surprising that p120 also enters the nucleus (VanHengel et al., 1999, and, in this issue, Mariner et al., 2000),where it interacts with a novel transcription factor, Kaiso(Daniel and Reynolds, 1999; Fig. 1). In fact, p120 is theprototypic member of a growing gene family, whose proteinproducts localize both at junctions and in the nucleus.Elucidation of the potential signaling pathways implied by theseobservations promises to reveal important new informationabout cell-cell communication, differentiation and cancer.

p120 ISOFORMS AND NOMENCLATURE

Initially designated p120CAS (for cadherin-associated Srcsubstrate), p120 was renamed p120ctn (catenin; Reynolds andDaniel, 1997) to avoid confusion with a different Src substrate,p130CAS (Crk-associated substrate; Sakai et al., 1994). Inaddition, it is now evident that most cell types express multiple

isoforms of p120, which are derived by alternative splicing ofa single gene (Reynolds et al., 1994; Staddon et al., 1995; Moand Reynolds, 1996; Keirsebilck et al., 1998). Thus, thenomenclature has been further refined to account for thesemultiple forms (Fig. 2). N-terminal splicing events lead to theuse of four different ATGs (Keirsebilck et al., 1998), resultingin the expression of p120 isoform type 1, 2, 3 or 4, accordingto the respective ATG used as the translation start site.Furthermore, alternative splicing of the C-terminal end leadsto use of exon A, exon B, both exons A and B, or none of them.On rare occasions, p120 contains a sequence encoded by anadditional exon (exon C), which is inserted within Arm repeat6. Various combinations of these N- and C-terminal exonsgenerate the different p120 bands observed in various celltypes. The current, and definitive nomenclature, is shown inFig. 2 (for comparison with older ‘CAS’ nomenclature, seeReynolds and Daniel, 1997).

The existence of cell-type-specific expression patternsimplies functional differences between the p120 isoforms. Forexample, macrophages and fibroblasts make N-cadherin and

P. Z. Anastasiadis and A. B. Reynolds

Fig. 1. The role of p120 incell-cell adhesion andsignaling. The catenins(p120ctn, β-catenin,plakoglobin and α-catenin)bind to the cytoplasmic tailsof classical cadherins. β-catenin and plakoglobincompete for binding to the so-called catenin-bindingdomain (CBD) and mediatethe attachment of cadherins tothe actin cytoskeleton via α-catenin. In contrast, p120(including isoforms andprobably other p120subfamily members)associates with the cadherinjuxtamembrane domain(JMD) and does not bind toα-catenin. p120 may act as aswitch, either promoting orinhibiting cadherin-mediatedadhesion in response tounspecified signaling cues.p120, like β-catenin, exhibitsdual localization both at themembrane and in the nucleus.Wnt/Wg signaling duringdevelopment, or APCmutations in human cancer,stabilize a normally transientcytoplasmic pool of β-cateninthat translocates to thenucleus, where it participatesin transcriptional regulationthrough interactions withtranscription factors of theLef1/TCF family. Kaiso is anovel transcription factor that interacts with p120. Although the biological significance of the p120-Kaiso interaction is not known, p120 mayaffect nuclear signaling by transactivating Kaiso, which is postulated to act as a transcriptional repressor. In contrast to β-catenin signaling,there is no mechanism to promote the degradation of p120 in metastatic cells that have downregulated cadherins. Thus, cadherin binding maybe an in important factor in regulating the putative role of p120 in transcription through its sequestration at the cell membrane.

Ca++E-cadherin

p120ctn orfamily members

β-catenin orplakoglobin

α-cateninα-actinin

F-actin

Wnt

Fz

Kaiso

LEF/TCF

Cytoplasm

Nucleus

p120ctn

DSH

?

GSK3β

APC

Axin

?vinculin

β-catenin

Extracellular

(?)

CBD JMDPlasma Membrane

1321p120 catenin roles in adhesion and signaling

preferentially express p120ctn1A isoforms, whereas epithelialcells make E-cadherin and preferentially express smallerisoforms, such as p120ctn3A. Because alternative splicingusually affects only the N- and C-terminal domains, the Armdomain is left intact and free to interact with cadherins.Different isoforms may affect cadherin function by recruitingdistinct binding partners to the cadherin complex. Othermembers of the p120 family are also expressed as multipleisoforms, which for the most part have not been characterized.

The regulated expression of multiple p120 family members andtheir isoforms, might provide an important mechanism for fine-tuning the activities of the various cadherins in different cells.

THE p120 FAMILY

Two groups with different functionsMembers of the p120 family share a characteristic structural

1 2 3 4ATGs:

A B

p120ctn1ABC

C

p120ctn2ABC

p120ctn3ABC

p120ctn4ABC

Fig. 2. p120 nomenclature and alternativesplicing. p120 contains a central Armadillodomain that has ten Arm repeats (depictedby gray boxes) interrupted by short loopsin repeats 4, 6 and 9. Cell-type-specificalternative splicing events result inmultiple isoforms. Four N-terminal ATGstart sites generate p120 isoforms 1, 2, 3,and 4. p120 isoform 1 contains a putativecoiled-coil domain (yellow box), which isabsent from isoforms initiating at internalATGs 2-4 and is conserved among p120’sclose relatives (see Fig. 4). Additionalalternative splicing results in alternativeusage of exons in the C-terminal end.Exons A and B are often used, whereasexon C is used rarely. The splicingcomplicates the nomenclature. Isoformsare designated p120ctn 1-4, depending onthe N-terminal start site. The A, B, and/orC designations are included if the exon ispresent (e.g. p120ctn1A, p120ctn1BC). Theletter N (for none) is used if it is knownthat none of the C-terminal exons ispresent (e.g. p120ctn1N).

Fig. 3. Sequence alignment andorganization of the p120 Armrepeats. The recently elucidatedcrystal structure of the β-cateninArm repeats (Huber et al., 1997)makes it possible to realign thep120 Arm domain in the contextof structural data, and isimportant for mutationalanalysis. According to thisalignment, p120 has tencontiguous Arm repeats, each ofwhich consists of three helices(H1, H2 and H3). Repeats 4, 6and 9 contain looped-outstructures located betweenhelices H2 and H3. The basicmotif (KKGKGKK) is thusembedded within repeat 6.Conserved residues arehighlighted in red. In theconsensus motif (bottom), +denotes basic residues (H,K,R),yellow boxes denote small hydrophobic residues (A,C,P,V,T), green boxes denote large hydrophobic residues (F,I,L,M,W), whereas orangeboxes denote general hydrophobic residues (A,C,F,I,L,M,P,T,V, or W).

R1 (352-394) SLRKGMP PPSNWRQ PELPEVIAMLGF RL DAVKSNAAAYLQHLC

R2 (395-436) YRNDKV KTDVRKL KGIPILVGLLDH PK KEVHLGACGALKNIS

R3 (437-480) FGRDQDN KIAIKNC DGVPALVRLLRK AR DMDLTEVITGTLWNLS

R4 (481-538) SHDSIK MEIVD HALHALTDEVII PH SGWEREPNEDCKPRHIE WESVLTNTAGCLRNVS

R5 (539-587) SERSEAR RKLRECD GLVDALIFIVQA EIGQKDS DSKLVENCVCLLRNLS

R6 (588-645) YQVH REIPQA ERYQEALPTVANS TGPH AASCFGAKKGKGKKP TEDPANDTVDFPKRTS

R7 (646-686) PARG YELLFQP EVVRIYISLLKE SK TPAILEASAGAIQNLC

R8 (687-732) AGRWTYGRYI RSALRQE KALSAIAELLTS EH ERVVKAASGALRNLA

R9 (733-780) VDARN KELIGK HAIPNLVKNLPG GQ QNSSWNF SEDTVVSILNTINEVI

R10 (781-824) AENLEAA KKLRET QGIEKLVLINKS GN RSEKEVRAAALVLQTIW

H1H1 H2H2 H3H3

Consensus +. ... .G ..LV. L.. .. .. ..A . L+.LS A T A

1322 P. Z. Anastasiadis and A. B. Reynolds

p120-h1ABCARVCFNPRAP-D-catHSP0071plakophilin 1plakophilin 2aplakophilin 3

1 20 1 20 1 59 1 45 1 0 1 9 1 5

M D D S E V E S T A - S I L A S V K E Q E M E D C N V H S A A - S I L A S V K E Q E

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21 68 21 53 60 115 46 103 1 0

10 49 6 40

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69 108 54 90

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109 150 91 133

173 224 164 223 13 54 91 132 85 130

V E T Y T E E - D P E G A M S V V S V E T S - - - - - - - - D D G T T R R T E T T V K K V V K T V T T - - - - - - - - -E E T V T V E E D P G T P T S H V S I V T S - - - - - - - - E D G T T R R T E T K V T K T V K T V T T - - - - - - - - -H S N Q T L A L G E T T P S Q L P A R G T - - - - - - Q A R A T G Q S F S Q G T T S - R A G H L A G P E P A P P P P P -H N S Q N V S K A D N R Q Q H S F I G S T N N H V V R N S R A E G Q T L V Q P S V A N R A M R R V S S V P S R A Q S P SE C F Q D Q D N S T L A L P S D Q K M K T G - - - - - - - - T S G R Q R V Q E Q V M - M T V K R Q K S - - - - - - - - -H L V E N D F V G G R S P V P K T Y D M L - - - - - - - - - K A G T T A T Y E G R W G R G T A Q Y S S - - - - - - - - -H T M Q T G F S S R S Q G M S G D K T S T F R - - - - - - P I A K P A Y S P A S W S S R S A V D L T C S - - - - - - - -


151 197 134 174 225 275 224 280 55 95

133 175 131 171

- - - - - - - - - - R T V Q P V A M G P D - G L P V D A S S - - V S N N Y I Q T L G R D F R K N G N G G P G P Y V G Q A- - - - - - - - - - R T V R Q V P V G P D - G L P L L D G G P P L G P F A D G A L D R H F L L R G - G G P - - - - - - -- - - - - - - - P P R E P F A P S L G S A F H L P D A P P A A A A A A L Y Y S S S T L P A P P R G - G S P L A A P Q G GY V I S T G V S P S R G S L R T S L G S G F G S P S V T D P R P L N P S A Y S S T T L P A A - R A - A S P Y S Q - R P A- - - - - - - - - - K S S Q S S T L S H S - N R G S M Y D G - L A D N Y N Y G T T S R S S Y Y S K F Q A G - - - - - - -- - - - - - - - - Q K S V E E R S L R H P L R R L E I S P D S S P E R A H Y T H S D Y Q Y S Q R S - Q A G - - - - - - -- - - - - - - - - - R R L S S A H N G G S - A F G A V G Y G G T Q P T P P M P T R P V S F H E R G - G A A - - - - - - -


198 245 175 219 276 335 281 329 96 131

176 215 172 210

G T A T L P R N F H Y P P D G Y S R H Y E D G Y P G G S D - N Y G S L S R - - - - - - V T R I E E - - - - - R Y R P S M- V A T L S R A Y L S S G G G F P - - - E G P E P R D S P - S Y G S L S R G - - - - - L G M R P P - - - - - R A G P L GS P T K L Q R G G S A P E G A T Y A A P R G S S P K Q S P S R L A K S Y S T S S P I N I V V S S A G L S P I R V T S P PS P T A I R R I G S V T S R Q T S N - P N G - - P - - T P - Q Y Q T T A R V G S P L T L T D A Q T - - - - - R V A S P S- - N G S - W G Y P I Y N G T L K - - - R E - - P - D N R - R F S S Y S Q - - - - - - - - - M E N - - - - - W S R H Y P- H T L H H Q E S R R A A L L V P - - - - - - - P R Y A R S E I V G V S R - - - - - - - A G T T S - - - - - R Q R H F D- S R A D Y D T L S L P S L R L G - - - - - - - P G G L D D R Y S V V S E - - - - - - - - Q L E P - - - - - A A A S T Y


246 291 220 270 336 394 330 385 132 166 216 255 211 242

E G - - - - - Y R A P S R Q D V Y - - - - - - G P Q P Q V R V G G S S V D L H R F H P E P Y G L E D D Q R - - S M G - YP G P G D G C F T L P G H R E A F P - - - - V G P E P - G P P G G R S L P - E R F Q A E P Y G L E D D T R - - S L A - AT V Q S - T I S S S P I H Q L S S T I G T Y A T L S P T K R L V H A S E Q Y S K H S Q E L Y A T A T L Q R P G S L A A GQ G Q - - V G S S S P K R S G M T A V P Q H L G P S - L Q R T V H D M E Q F G Q Q Q Y D I Y E R M V P P R P D S L T - GR G - - - S C N T T G A G S D I C - - - - - - - - - - - - - - F M Q K I K A S R S E P D L Y C D - - - P R - - - - G - TT Y - - - - - H R Q Y Q H G S V S - - - - - - - - - - - - D T V F D S I P - A N P A L L T Y P R P G T S R - - S M G N LR A - - - - - - Y A Y K R Q A S S - - - - - - - - - - - - - - - G S S R A G G L D W P E A T E G P - P S R - - - - - - T


292 336 271 322 395 454 386 444 167 215 256 311 243 287

D D L D Y G - M M S D Y G T A R R T G - T P S D P R - - - R R L R S Y E D M I G - - - - - - - - E E V P S D Q - - Y Y WD D E G G P E L E P D Y G T A T R R - - R P E C G R G - - L H T R A Y E D T A D D G G E L A - - D E R P A F P - - M V TS R A S Y S S Q H G H L G P E L R A L Q S P E H H I D P I Y E D R V Y Q K P P M R S L S Q S Q G D P L P P A H T G T Y RL R S S Y A S Q H S Q L G Q D L R S A V S P D L H I T P I Y E G R T Y Y S P V Y R S P N H G - T V E L Q G S Q T A L Y RL R K G T L G S K G Q K T T Q N R Y S F Y S T C S G - - - - Q K A I K K C P V R P - P S - - - - C A S K Q D P - - V Y IL E K E N Y L T A G L T V G Q V R P - L V P L Q P V T - - - Q N R A S R S S W H Q S S F H S T R T L R E A G P S V A V DI R A P A M R T L Q R F Q S S H R S - - R G G T G S - - - - V S G A G L E P V A R - - - - - - - - - A P S V R S L S L S


337 349 323 335 455 514 445 492 216 228 312 329 288 300

A P L A - - - - - - - - - - - - - - - - Q H - - - - - - - - - - E R G S L A S - - - - - - - - - - - - - - - - - - - - -A P L A - - - - - - - - - - - - - - - - Q P - - - - - - - - - - E R G S M G S - - - - - - - - - - - - - - - - - - - - -T S T A P S S P G V D S V P L Q R T G S Q H G P Q N A A A A T F Q R A S Y A A G P A S N Y A D P Y R Q L Q Y C P S V E ST G V S - - - - G I G - - N L Q R T S S Q R - - - - - S T L T Y Q R N N Y A L N T T A T Y A E P Y R P I Q Y R - V Q E CP P I S - - - - - - - - - - - - - - - - C N - - - - - - - - - - K D L S F G H - - - - - - - - - - - - - - - - - - - - -S S G R - - - - - - - - - - - - - - - R A H - - - - - - L T V G Q A A A G G S - - - - - - - - - - - - - - - - - - - - -L A D S - - - - - - - - - - - - - - - - G H - - - - - - - - - - L P D V R G L - - - - - - - - - - - - - - - - - - - - -


350 388 336 380 515 572 493 550 229 267 330 375 301 341

- - - - - - - - - - L D S L R K G G P - - - - - - P P P N - - - W R Q P E L P E V I A M L G - - F R L D A V K S N A A A- - - - - - - - - - L D R L V R R S P S V D S A R K E P R - - - W R D P E L P E V L A M L R - - H P V D P V K A N A A AP Y S K S G P A L P P E G T L A R S P S I D S I Q K D P R E F G W R D P E L P E V I Q M L Q - - H Q F P S V Q S N A A AN Y N R L Q H A V P A D D G T T R S P S I D S I Q K D P R E F A W R D P E L P E V I H M L E - - H Q F P S V Q A N A A A- - - - - - - - - - - S R A S S K I C S - - - - - E D I E - - - C S G L T I P K A V Q Y L S - - S Q D E K Y Q A I G A Y- - - - - - - - - - G N L L T E R S T F T D S Q L G N A D - - - - M E M T L E R A V S M L E A D H M P P S R I S A A A T- - - - - - - - - - D S Y T G H R T L Q - - - - R L S S G - - - F D D I D L P S A V K Y L M - - A S D P N L Q V L G A A


389 447 381 439 573 632 551 610 268 325 376 433 342 399

Y L Q H L C Y R N D K V K T D V R K L K G I P V L V G L L D H P K K E V H L G A C G A L K N I S F G R D Q D - N K I A IY L Q H L C F E N E G V K R R V R Q L R G L P L L V A L L D H P R A E V R R R A C G A L R N L S Y G R D T D - N K A A IY L Q H L C F G D N K I K A E I R R Q G G I Q L L V D L L D H R M T E V H R S A C G A L R N L V Y G K A N D D N K I A LY L Q H L C F G D N K V K M E V C R L G G I K H L V D L L D H R V L E V Q K N A C G A L R N L V F G K S T D E N K I A MY I Q H T C F Q D E S A K Q Q V Y Q L G G I C K L V D L L R S P N Q N V Q Q A A A G A L R N L V F - R S T T - N K L E TF I Q H E C F Q K S E A R K R V N Q L R G I L K L L Q L L K V Q N E D V Q R A V C G A L R N L V F - E D N D - N K L E VY I Q H R C Y S D A A A K K Q A R S L Q A V P R L V K L F N H A N Q E V Q R H A T G A M R N L I Y - D N V D - N K L A L

N-term coiled-coil region

Arm domain

Fig. 4. For legend see p. 1324.



448 507 440 499 633 692 611 670 326 385 434 493 400 458

K N C D G V P A L V R L L R K A R D M D L T E V I T G T L W N L S S H D S I K M E I V D H A L H A L T D E V I I P H S GR D C G G V P A L V R L L R A A R D N E V R E L V T G T L W N L S S Y E P L K M V I I D H G L Q T L T H E V I V P H S GK N C G G I P A L V R L L R K T T D L E I R E L V T G V L W N L S S C D A L K M P I I Q D A L A V L T N A V I I P H S GK N V G G I P A L L R L L R K S I D A E V R E L V T G V L W N L S S C D A V K M T I I R D A L S T L T N T V I V P H S GR R Q N G I R E A V S L L R R T G N A E I Q K Q L T G L L W N L S S T D E L K E E L I A D A L P V L A D R V I I P F S GA E L N G V P R L L Q V L K Q T R D L E T K K Q I T G L L W N L S S N D K L K N L M I T E A L L T L T E N I I I P F S GV E E N G I F E L L R T L R E Q - D D E L R K N V T G I L W N L S S S D H L K D R L A R D T L E Q L T D L V L S P L S G


508 567 500 559 693 752 671 730 386 442 494 551 459 515

W E R E P N E D C K P R H I E W E S V L T N T A G C L R N V S S E R S E A R R K L R E C D G L V D A L I F I V Q A E I GW E R E P N E D S K P R D A E W T T V F K N T S G C L R N V S S D G A E A R R R L R E C E G L V D A L L H A L Q S A V GW E N S P L Q D D R K I Q L H S S Q V L R N A T G C L R N V S S A G E E A R R R M R E C D G L T D A L L Y V I Q S A L GW N N S S F D D D H K I K F Q T S L V L R N T T G C L R N L T S A G E E A R K Q M R S C E G L V D S L L Y V I H T C V NW C D - G N S N - M S R E V V D P E V F F N A T G C L R N L S S A - D A G R Q T M R N Y S G L I D S L M A Y V Q N C V AW P E - - G D Y P K A N G L L D F D I F Y N V T G C L R N M S S A G A D G R K A M R R C D G L I D S L V H Y V R G T I AP G G - - - P P L I Q Q N A S E A E I F Y N A T G F L R N L S S A S Q A T R Q K M R E C H G L V D A L V T Y I N H A L D


568 621 560 619 753 811 731 788 443 495 552 606 516 572

Q K D S D S K L V E N C V C L L R N L S Y Q V H R E I P Q A E R Y Q E A A P N - V A N N T G P - - - - - H A A S C F G AR K D T D N K S V E N C V C I M R N L S Y H V H K E V P G A D R Y Q E A E P G P L G S A V G S Q R R R R D D A S C F G GS S E I D S K T V E N C V C I L R N L S Y R L A A E T S Q G Q H M G T D E L D G L L C G E A N - G K D A E S S G C W G KT S D Y D S K T V E N C V C T L R N L S Y R L E L E V P Q A R L L G L N E L D D L L G K E S P - S K D S E P S - C W G KA S R C D D K S V E N C M C V L H N L S Y R L D A E V P T - - R Y R Q L E Y N - A R N A Y T E - - - - K S S T G C F S ND Y Q P D D K A T E N C V C I L H N L S Y Q L E A E L P E - - K Y S Q N I Y I Q N R N I Q T D - - - N N K S I G C F G SV G K C E D K S V E N A V C V L R N L S Y R L Y D E M P P S - A L Q R L E G R G R R D M A G A P - - P G E M V G C F T P


622 679 620 678 812 861 789 841 496 542 607 653 573 623

K K G K D E W F S R G K K P - I E D P A N D T V D F P K R T S P A R G Y E L L F Q P E V V R I Y I S L L K E S K T P - AK K A K E E W F H Q G K K D G E M D R N F D T L D L P K R T E A A K G F E L L Y Q P E V V R L Y L S L L T E S R N F - NK K K K K - - K - - - - - - - S Q D Q W D G V G P L P D C A E P P K G I Q M L W H P S I V K P Y L T L L S E C S N P - DK K K K K - - K R - - - T P - Q E D Q W D G V G P I P G L S K S P K G V E M L W H P S V V K P Y L T L L A E S S N P - AK S D K M - - - - - - - - - - M N N - - N Y D C P L P E E E T N P K G S G W L Y H S D A I R T Y L N L M G K S K K D - AR S R K V - - - - - - - - - - K E Q - - Y Q D V P M P E E K S N P K G V E W L W H S I V I R M Y L S L I A K S V R N - YQ S R R L R - - - - - - - - - E L P L T A D A L T F A E V S K D P K G L E W L W S P Q I V G L Y N R L L Q R C E L N R H


680 738 679 737 862 920 842 900 543 602 654 713 624 683

I L E A S A G A I Q N L C A G R W T Y G R Y I R S A L - R Q E K A L S A I A D L L T N E H E R V V K A A S G A L R N L AT L E A A A G A L Q N L S A G N W M W A T Y I R A T V - R K E R G L P V L V E L L Q S E T D K V V R A V A I A L R N L ST L E G A A G A L Q N L A A G S W K W S V Y I R A A V - R K E K G L P I L V E L L R I D N D R V V C A V A T A L R N M AT L E G S A G S L Q N L S A S N W K F A A Y I R G G R - P K R K G L P I L V E L L R M D N D R V V S S G A T A L R N M AT L E A C A G A L Q N L T A S K G L M S S G M S Q L I G L K E K G L P Q I A R L L Q S G N S D V V R S G A S L L S N M ST Q E A S L G A L Q N L T A G S G P M P T S V A Q T V V Q K E S G L Q H T R K M L H V G D P S V K K T A I S L L R N L ST T E A A A G A L Q N I T A G D R R W A G V L S R L A L E Q E R I L N P L L D R V R T A D H N Q L R S L T G L I R N L S


739 795 738 796 921 980 901 955 603 657 714 768 684 738

V D A R N K E L I G K H A I P N L V K N L P G G Q Q - - - N S S W N F S E D T V I S I L N T I N E V I A E N L E A A K KL D R R N K D L I G S Y A M A E L V R N V R N A Q A P P - R P G A C L E E D T V V A V L N T I H E I V S D S L D N A R SL D V R N K E L I G K Y A M R D L V H R L P G G N N S N N T A S K A M S D D T V T A V C C T L H E V I T K N M E N A K AL D V R N K E L I G K Y A M R D L V N R L P G G N G P S - - - - - V L S D E T M A A I C C A L H E V T S K N M E N A K AR H P L L H R V M G N Q V F P E V T R L L T S H T G - - - - - N T S N S E D I L S S A C Y T V R N L M A S Q P Q L A K QR N L S L Q N E I A K E T L P D L V S I I P D T V P - - - - - S T D L L I E T T A S A C Y T L N N I I Q N S Y Q N A R DR N A R N K D E M S T K V V S H L I E K L P G S V G - - - - - E K C P P A E V L V N I I A V L N N L V V A S P I A A R D


796 853 797 853 981 1040 956 1015 658 714 769 827 739 797

L R E T Q G I E K L V L I N K S G - - N R S E K E V R A A A L V L Q T I W G Y K E L R K P L E K E G W K K S D F Q V N LL L Q A R G V P A L V A L V A S - - - S Q S V R E A K A A S H V L Q T V W S Y K E L R G T L Q K D G W T K A R F Q S A AL R D A G G I E K L V G I S K S K G D K H S P K V V K A A S Q V L N S M W Q Y R D L R S L Y K K D G W S Q Y H F V A S SL A D S G G I E K L V N I T K G R G D R S S L K V V K A A A Q V L N T L W Q Y R D L R S I Y K K D G W N Q N H F I T P VY F S S S M L N N I I N L C R S - - - S A S P K A A E A A R L L L S D M W S S K E L Q G V L R Q Q G F D R N M L G T L AL L N T G G I Q K I M A I S A G D - A Y A S N K A S K A A S V L L Y S L W A H T E L H H A Y K K A Q F K K T D F V N S RL L Y F D G L R K L V F I K K K R D S P D S E K S S R A A S S L L A N L W Q Y S K L H R D F R A K G Y R K E D F L G P


854 875 854 878

1041 1100 1016 1072 715 726 828 837 798 797

N N A S - - R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S Q S S H S - Y D D S T L P L I D RA T A K G P K G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A L S P G G - F D D S T L P L V D KS T I E R D R Q R P Y S S S R T P S I S P V R V S P N N R S A S A P A S P R E M I S L K E R K T D Y E C T G S N A T Y HS T L E R D R - - - F K S H P S L S T T N Q Q M S P I I Q S V G S T S S S P A L L G I R D P R S E Y D R T Q P P M Q Y YG A N S - L R N - - F T S R F T A K A Y H S - - - L K D


876 929 879 926

1101 1157 1073 1129 727 726 838 837 798 797

N Q K S D K K P D R E E I Q M S N M G S N T K S L D N N Y S T P N - E R G D H N R T L D R - - S G D L G - - - D M E P LS L E G E K T G S R D V I P M D A L G P - - - - - D G - Y S T V D - R R E R R P R G A S S - - A G E A S - - - E K E P LG A K G E H T S R K D A M T A Q N T G I S T - L Y R N S Y G A P A - E D I K H N Q V S A Q P V P Q E P S - R K D Y E T YN S Q G D A T H K G - - L Y P G S S K P S P - I Y I S S Y S S P A R E Q N R R L Q H Q Q L Y Y S Q D D S N R K N F D A Y


930 962 927 956

1158 1217 1130 1184 727 726 838 837 798 797

K G T T P L M Q D E G Q E S L E E E L D V - - - - - - - - - - - - - - - - - L V L D D E G G Q V S - - - - - - - - - - YK L D - P S R K - - A P P P G P S R P A V - - - - - - - - - - - - - - - - - R L V D A V G D A K P - - - - - - - - - - QQ P F Q N S T R N Y D E S F F E D Q V H H R P P A S E Y T M H L G L K S T G N Y V D F Y S A A R P Y S E L N Y E T S H YR L Y L Q S P H S Y E D P Y F D D R V H F - P A S T D Y S T Q Y G L K S T T N Y V D F Y S T K R P - - - - S Y R A E Q Y


963 968 957 962

1218 1225 1185 1211 727 726 838 837 798 797

P - - S M Q K I P - - V D S W V P A S P D S W V P G S P D S W V Y D Q D A Q Q R N S F F L T L F R L R

PDZ-binding motif

Arm domain

Fig. 4. For legend see p. 1324.

1324

organization of the Arm-repeat domain (see Figs 2, 3), whichsuggests an ancient evolutionary relationship. However, twogroups have emerged that differ both in the degree of similaritythat they share with p120, and in their subcellular localizations.The first group includes ARVCF (Armadillo repeat genedeleted in Velo-Cardio-Facial syndrome; Sirotkin et al., 1997),δ-catenin/NPRAP/neurojungin (neural plakophilin-relatedArmadillo protein; Paffenholz and Franke, 1997; Zhou et al.,1997) and p0071 (Hatzfeld and Nachtsheim, 1996). Theseproteins share >45% identity with p120 in their Arm domainsand, with the possible exception of p0071 (Hatzfeld andNachtsheim, 1996), bind to and colocalize with classicalcadherins at adherens junctions (Lu et al., 1999; Paffenholz etal., 1999; Mariner et al., 2000). The intron-exon boundaries ofp120 and ARVCF are nearly identical (Keirsebilck et al.,1998), further highlighting the similarity between theseproteins. The second group, the plakophilins, have ~30%identity to p120 in their Arm domains, which are otherwiseorganized similarly to that of p120. Unlike the first group,which interact with cadherins through their Arm domains,plakophilins localize to desmosomes through interactions

mediated by their head (N-terminal) domain, and coordinateintermediate filaments (Hatzfeld et al., 1994; Heid et al., 1994;Mertens et al., 1996; Schmidt et al., 1999; Bonne et al., 1999;Kowalczyk et al., 1999; Schmidt et al., 1997; Smith and Fuchs,1998). They exhibit significantly higher similarity to oneanother than to p120 (Bonne et al., 1999). Because of thesignificant differences between these subgroups, we willhereafter refer to them separately as the p120 subfamily (whichincludes ARVCF, δ-catenin/NPRAP and p0071), and theplakophilin subfamily (which includes plakophilins 1, 2 and 3).All of these proteins also localize to the nucleus (Bonne et al.,1999; Schmidt et al., 1997; Mertens et al., 1996; Van Hengelet al., 1999; Mariner et al., 2000); this interesting phenomenonhas obvious implications for signaling between cell junctionsand the nucleus.

p120 family structureThe p120 relatives share several structural features. Each hasten Arm repeats, which are organized identically and containinterruptions within repeats 4, 6 and 9 (see Figs 2 and 3). Fig.3 shows the sequence and predicted boundaries of each p120Arm repeat according to the recently elucidated β-catenincrystal structure (Huber et al., 1997). An alignment of allknown p120 family members is shown in Fig. 4 and highlightsthe sequence conservation shared by these proteins. Theinterruptions noted above are thought to represent looped-outstructures positioned between helices H2 and H3 – as occursin β-catenin (Huber et al., 1997). In the loop within Arm repeat6, all of the p120 relatives contain motifs postulated to act asnuclear localization sequences. In ARVCF, there appears to bea bipartite nuclear-localization signal (NLS; Imamura et al.,1998), whereas p120, p0071 and δ-catenin/NPRAP have ahighly conserved basic motif (CW/FGxKKxKxKK).Interestingly, the presence of p120 exon C alters this basicmotif (Keirsebilck et al., 1998). Although it is suggested thatthese basic motifs play a role in nuclear localization (Lu et al.,1999), indirect data suggest otherwise (Mariner et al., 2000).The plakophilins also contain basic motifs within this loop, andseveral lines of evidence suggest that they also are not required


Fig. 4. CLUSTAL W alignment of human p120ctn1ABC, ARVCF, δ-catenin/NPRAP, p0071 and plakophilins 1a, 2a and 3. Dark-boxedareas designate amino acid identity between at least 4 out of theseven sequences. Light gray regions show similarity rather thanidentity. The conservation between family members in the centralArm domain is clear, whereas divergence is evident in the N- and C-terminal ends. A conserved coiled-coil domain in the extreme N-terminal domain of p120, ARVCF, δ-catenin/NPRAP and p0071 isdepicted. A putative PDZ-binding domain at the extreme C-terminalof ARVCF, δ-catenin/NPRAP and p0071 (but not p120) is alsoshown. Closer inspection, coupled with functional data, justifiesfurther division into two subgroups consisting of the top foursequences (the p120 family) and the bottom three sequences (theplakophilin family). Database accession numbers: p120ctn1ABC:AF062341; ARVCF: U51269; δ-catenin/NPRAP: U96136; p0071:X81889, plakophilin 1a: Z34974; plakophilin 2a: X97675;plakophilin 3: AF053719.

Fig. 5. p120 binds to theconserved cadherinjuxtamembrane domain(JMD), a region highlyconserved among type I(classical) and type IIcadherins. β-catenin orplakoglobin binds to the so-called catenin-binding domain(CBD). Triple-alanine (AAA)substitution mutations(indicated) spanning the E-cadherin JMD define a coreregion (the p120-binding core)within the JMD that isrequired for interaction withp120 in yeast two-hybridassays (Thoreson et al., 2000;+ denotes normal p120 binding; – denotes loss of p120 binding). The triple substitution mutant 764 (EED/AAA) has been tested in mammaliancells and shown to uncouple p120 binding in vivo.

E-cadN-cadP-cadR-cad4VE-cad5cad6cad8cad10OB-cad11cad12cad14cad15cad18

R A V V K E P L L P P E - D D T R D N V Y Y Y D E E G G G E E D Q D - F D L S Q L H R GE R Q A K Q L L I D P E - D D V R D N I L K Y D E E G G G E E D Q D - Y D L S Q L Q Q PK R K I K E P L L L P E - D D T R D N V F Y Y G E E G G G E E D Q D - Y D I T Q L H R GE R H T K Q L L I D P E - D D V R E K I L K Y D E E G G G E E D Q D - Y D L S Q L Q Q PR L R K Q A R A H G K S V P E I H E Q L V T Y D E E G G G E M D T T S Y D V S V L N S VR Q R K K E P L I I S K - E D I R D N I V S Y N D E G G G E E D T Q A F D I G T L R N PR H Q K N E P L I I K D D E D V R E N I I R Y D D E G G G E E D T E A F D I A T L Q N PR Q R K K E P L I L S K - E D I R D N I V S Y N D E G G G E E D T Q A F D I G T L R N PR R Q K K E P L I V F E E E D V R E N I I T Y D D E G G G E E D T E A F D I A T L Q N PR R Q K K H T L M T S K - E D I R D N V I H Y D D E G G G E E D T Q A F D I G A L R N PR R S K K E P L I I S E - E D V R E N V V T Y D D E G G G E E D T E A F D I T A L R N PQ S R G K G L L H G P Q - D D L R D N V L N Y D E Q G G G E E D Q D A Y D I S Q L R H PR R S K K E P L I I S E - E D V R E N V V T Y D D E G G G E E D T E A F D I T A L R N P

AAA AAA AAAAAA AAAAAAAAAAAAAAA

JMD CBDEC E-cadherin

(p120 binding)734 775

Core Binding Region


for nuclear translocation (Klymkowsky, 1999; Schmidt et al.,1997).

A highly conserved N-terminal motif in p120, ARVCF,p0071 and δ-catenin/NPRAP encodes a putative coiled-coildomain. Coiled-coil motifs are typically involved in protein-protein interactions. This region is spliced out in p120 isoforms2, 3 and 4, whose translation begins at downstream ATGs.Given that p120 type 1 isoforms are prevalent in motile celltypes (e.g. fibroblasts), they might be important for promotingmore dynamic cadherin interactions. Roles in homo- or hetero-dimerization can also be envisioned, although such amechanism would pertain selectively to type 1 isoforms.Alternatively, this domain could be responsible for therecruitment of novel effectors to the cadherin complexes. Adifferent conserved region is present in the N terminus of allplakophilins (Bonne et al., 1999) and might be involved indesmosomal localization.

In the C-terminal end, ARVCF, δ-catenin/NPRAP andp0071, but not p120, share a third region of similarity. Theconserved sequence DSWV is a PDZ-binding motif (type I;Fanning and Anderson, 1999), which in the case of δ-catenin/NPRAP binds to S-SCAM, a PDZ-domain-containing scaffold protein (Ide et al., 1999). Interactionsbetween p120 family members and scaffold proteins couldplay a role in the recruitment of signaling molecules tocadherin junctions, and/or in promoting cadherin clustering –an event important in cadherin-mediated adhesion, which wediscuss in detail later.

REGULATION OF THE p120-CADHERININTERACTION

Stoichiometry of the cadherin-p120 interactionThe relatively weak interaction between cadherins and p120 indetergent cell lysates led investigators to estimate that only 5-20% of total cellular p120 is in complex with cadherins (Ozawaand Kemler, 1998; Shibamoto et al., 1995; Papkoff, 1997;Staddon et al., 1995). In contrast, immunofluorescence analysisgenerally indicates precise colocalization of p120 withendogenous cadherins (Reynolds et al., 1994; Thoreson et al.,2000), which suggests a significantly higher stoichiometry.Recent evidence strongly favors the latter view. In cadherin-deficient cell lines (e.g. A431D, MDA-231 and L-cells), p120is stranded in the cytosol, which is consistent with thehypothesis that cadherins are the only p120 receptor in themembrane (Thoreson et al., 2000; Ohkubo and Ozawa, 1999).Expression of intact E-cadherin, but not of an E-cadherinmutant containing minimal substitutions that uncouple p120association, results in efficient recruitment of p120 to cell-celljunctions. Moreover, biochemical fractionation of thesetransfected cell lines in the absence of detergents shows that>90% of the p120 fractionates with membranes in the presenceof wild-type E-cadherin (Thoreson et al., 2000). In contrast,p120 fractionates with the cytosol when the same experimentis carried out with p120-uncoupling E-cadherin mutants.Therefore, cadherins are both necessary and sufficient forrecruitment of p120 to membranes, and the in vivostoichiometry of p120 in cadherin complexes is likely to behigh and similar to that of β-catenin (Nathke et al., 1994;Papkoff, 1997).

p120 binds to the cadherin juxtamembrane domain Early work showed that immunoprecipitates of p120 containedβ-catenin and vice versa, suggesting that the two proteins bindsimultaneously to different regions of the cadherin cytoplasmictail (Reynolds et al., 1994; Daniel and Reynolds, 1995).Subsequent studies demonstrate that p120 associates with thecadherin juxtamembrane domain (JMD; Finnemann et al.,1997; Lampugnani et al., 1997; Ozawa and Kemler, 1998; Yapet al., 1998; Aono et al., 1999; Ohkubo and Ozawa, 1999;Thoreson et al., 2000), whereas β-catenin and plakoglobin bindto the so-called catenin-binding domain (CBD; Nagafuchi andTakeichi, 1989; Ozawa et al., 1990; Stappert and Kemler, 1994;see Fig. 5). Yeast two-hybrid data and mutational analysespoint to a stretch of ~10 residues that makes up the core of theJMD and constitutes the critical p120-binding sequence(Thoreson et al., 2000; Ohkubo and Ozawa, 1999). Anengineered triple-alanine substitution, termed 764 EED/AAA,in this region of E-cadherin effectively uncouples the cadherin-p120 interaction in mammalian cells (Thoreson et al., 2000).The core p120-binding domain is the most highly conservedregion among classical cadherins, which is consistent with theconcept that p120 plays a fundamental role in regulation ofthese proteins.

Potential roles of the cadherin juxtamembranedomainIncreasing evidence indicates that the JMD and the CBD ofcadherins contribute different functions. Early deletion studiesindicate that the CBD and its association with β-catenin, α-catenin and the actin cytoskeleton, are required for cadherin-mediated adhesion (Nagafuchi and Takeichi, 1989; Ozawa etal., 1990; Fujimori and Takeichi, 1993; Barth et al., 1997;Kawanishi et al., 1995; Watabe et al., 1994). Subsequently,Kintner (1992) demonstrated that overexpression of amembrane-tethered N-cadherin JMD construct inhibitsectodermal cell-cell adhesion in Xenopus embryos. Dominantnegative constructs lacking either the JMD or the CBDindependently blocked adhesion, which suggests that thesedomains have separate but indispensable roles. Interestingly, inthe JMD construct used by Kintner in these studies, a‘proximal’ JMD sequence was deleted; the recently identified‘core’ p120-binding region was left intact (Thoreson et al.,2000). Thus, the observed effects may not be due to the failureof the JMD deletion to selectively eliminate binding to p120.Interestingly, a peptide mimicking the ‘proximal’ JMD region(and lacking the core JMD p120-binding site) reportedly bindsto endogenous N-cadherin and apparently displaces thetyrosine kinase Fer from the N-cadherin complex (Balsamo etal., 1999; Lilien et al., 1999). These data suggest that the JMDcontains two binding motifs, a ‘distal’ one that binds p120 anda ‘proximal’ one that may be involved in cadherin dimerizationand/or binding to Fer. Although the full spectrum of molecularinteractions supported by these domains and their function inadhesion remain to be elucidated, the reported interactionbetween Fer and p120 (Kim and Wong, 1995; Rosato et al.,1998) suggests that these proteins may act in concert tomodulate adhesion. For clarity subsequent references to theJMD in this review will refer to the p120-binding region.

In other work, the JMD has been implicated in the ability ofectopically expressed cadherin to suppress invasion andmotility. Chen et al. (1997) have shown that introduction of a

1326

JMD-deleted E-cadherin in WC5 cells (which lack cadherinexpression) induces aggregation, but is unable to suppressmotility. The authors suggest that the adhesion and motilityfunctions of the cadherin cytoplasmic domain can befunctionally uncoupled. As these studies were conducted incells expressing a temperature-sensitive Src mutant, it is notyet clear whether the data are broadly relevant.

The JMD also appears to be required for neuronal outgrowthmediated by N-cadherin attachment, since overexpression ofdominant negative constructs containing the JMD, but not theCMD, were inhibitory (Riehl et al., 1996). N-cadherinhomophilic binding induces neurite extension in a mannerdependent on intracellular signaling by the FGF receptor(Saffell et al., 1997), which is thought to bind to the N-cadherinextracellular domain. One possible interpretation is that N-cadherin binding between adjacent cells promotes theactivation of FGF receptor tyrosine kinase signaling byinducing clustering of the receptors at areas of cell-cell contact.Further work in these systems to clarify the role of p120 iswarranted.

Two recent reports appear to contradict the idea that the JMDis involved in cell-cell adhesion: using dominant negative JMDconstructs, Zhu and Watt (1996), and Nieman et al. (1999) didnot detect an inhibition of adhesion measured by aggregationassays. A potentially unifying hypothesis is that the JMDmodulates the transition from weak to strong adhesion – thesetwo processes relate to the ability of cadherin-dependent cell-cell aggregates to withstand distracting forces (e.g. shearforces, such as pipetting through a narrow aperture). Indeed, ifoverexpression of membrane-tethered JMD constructs blockstrong, but not weak adhesion, the aggregation assays used inthe later studies would not be predicted to show an inhibitoryeffect, because they do not discriminate between these adhesivestates.

Early studies suggested that dominant negative cadherinsthat contain both the JMD and the CBD but lack theextracellular domain block adhesion by sequestering catenins.However, several reports indicate that the dominant negativeeffect of the CBD is not due to sequestration of classicalcatenins (i.e. α-catenin, β-catenin and plakoglobin; Fujimoriand Takeichi, 1993; Zhu and Watt, 1996; Nieman et al., 1999;Troxell et al., 1999) but rather to downregulation ofendogenous cadherin expression (Zhu and Watt, 1996; Niemanet al., 1999; Troxell et al., 1999). Nonetheless, dominantnegative effects of cadherin cytoplasmic domains containingboth the JMD and the CBD cannot always be ascribed todownregulation of endogenous cadherin expression (Kintner,1992; Fujimori and Takeichi, 1993; Troxell et al., 1999). Insome cases, the dominant negative effect of these constructsmight be mediated by the JMD and its ability to competitivelyuncouple p120 from endogenous cadherins. β-Catenin and α-catenin are stabilized by cadherin binding; thus, their levels inmost normal cells are strongly dependent on binding tocadherins. Dominant negative cytoplasmic cadherin constructsmay simply stabilize increased levels of these proteins. Bycontrast, the amount of p120 is not significantly changed bythe presence or absence of ectopic E-cadherin (Thoreson et al.,2000; Papkoff, 1997). Therefore, dominant negative constructsmay inhibit adhesion largely through sequestration of thelimited amount of available p120.

Interestingly, membrane-tethered cadherin cytoplasmic

domains expressed at levels that do not affect endogenouscadherin expression localize at cell-cell junctions and inhibitadhesion in a manner consistent with inhibition of endogenouscadherin clustering (Fujimori and Takeichi, 1993; Troxell et al.,1999). It is not yet clear whether the adhesive defect is due todirect inhibition of cadherin dimerization or clustering or,alternatively, to the inhibition of cadherin function throughdominant negative interactions with p120. Nonetheless, thedata suggest that the cadherin cytosolic domain (Fujimori andTakeichi, 1993; Amagai et al., 1995; Katz et al., 1998), andspecifically the JMD (Navarro et al., 1998; see below), isresponsible for the selective recruitment of membrane-associated cadherins into areas of cell-cell contact.

CONTROVERSIES REGARDING THE ROLE OF p120IN ADHESION

Experiments focused specifically on the role of p120 inmediating JMD function have led to two results that appearinitially to be diametrically opposed. The first stronglyimplicates the JMD and p120 in cadherin clustering and theinduction of strong adhesion independently of the CBD,whereas the second implicates p120 in negative regulation ofcell-cell adhesion.

‘Activation’ of p120 and positive effects on cell-celladhesionStable expression in cadherin-deficient CHO cells of a C-cadherin construct lacking the CBD induced strong adhesion,despite the lack of any detectable association of the proteinwith the actin cytoskeleton (Yap et al., 1998). This strongadhesion was dependent on the clustering of cadherinsmediated by contact with immobilized extracellular cadherinfragments. A construct lacking the JMD did not induceclustering, despite evidence that the mutated cadherin couldassociate with the cytoskeleton. Interestingly, the clusteringpotential of the JMD appeared to be activated by thehomophilic interaction between cadherin extracellular domains(formation of adhesive or trans dimers) and was not observedin the absence of cadherin trans association (Yap et al., 1998).Thus, cadherin clustering in vivo might be secondary to theformation of adhesive cadherin dimers, and requires the JMDbut not the CBD (or the interaction with the actin cytoskeletonthat the CBD mediates). Coupled with the demonstration thatp120 associates with the JMD construct that was used in theseexperiments, these data strongly implicate p120 in ligand-dependent activation of cadherin clustering. An active p120-mediated clustering process is also consistent with laser-trapping experiments measuring the kinetics of cadherin lateraldiffusion. The kinetic data argue that cadherin clustering is anactive process occurring too rapidly to be dependent on passivediffusion alone (Kusumi et al., 1999).

Overexpression in CHO cells of a VE-cadherin constructlacking the CBD also induces aggregation independently ofcytoskeletal association (Navarro et al., 1995). This CBD-negative protein localizes appropriately to cell-cell junctionsand recruits p120 (Navarro et al., 1995; Lampugnani et al.,1997). In related experiments, Navarro et al. (1998) show thatVE-cadherin selectively excludes N-cadherin from cell-celljunctions when co-expressed in cadherin-deficient CHO cells,



and provide evidence that VE-cadherin binds to p120 withgreater affinity than does N-cadherin. Interestingly, VE-cadherin constructs lacking the JMD (and therefore unable tobind to p120) cannot exclude N-cadherin (Navarro et al., 1998),which suggests that p120 plays a role in this phenomenon. Animplication is that high-affinity binding of p120 to VE-cadherin, and/or low-affinity binding of p120 to N-cadherin,results in differential recruitment of these cadherins to theadherens junction. High-affinity interactions with p120 mayfavor selective clustering of VE-cadherin at cell-cell contacts,and affinity modulation might be further regulated by post-translational modification. The data generated in this modelsystem reflect the natural scenario in endothelial cells, in whichboth cadherins are present, and VE- but not N-cadherin isfound at the adherens junction.

In two separate cadherin-deficient cell model systems, p120-uncoupled E-cadherin mutants failed to induce the strongadhesion observed in cells transfected with wild-type E-cadherin (Thoreson et al., 2000), which is consistent with theobservations reviewed above. Minimal triple-alanine mutants(e.g. E-cad 764 EED/AAA, see Fig. 5) within the p120-bindingregion were used to reduce the chance that the observed effectsare due to conformational artifacts or disruption of othercadherin-protein interactions, such as the above-noted potentialassociation between N-cadherin and Fer. In aggregation assays,the p120-uncoupled cadherins failed to induce compactioneven after overnight incubation, and aggregates of cellsexpressing p120-uncoupled cadherins readily dissociated tosingle cells upon pipetting, unlike their wild-type-cadherin-expressing counterparts. Correlating with the loss ofcompaction, colonies of cells expressing the mutant cadherinsexhibited a disruption of the cortical actin cytoskeleton. Thissuggests that p120-induced events (presumably clustering) areprerequisite for the organization of the actin cytoskeleton inepithelial cells.

The above evidence suggests that the clustering ofcadherins at cell-cell junctions requires the JMD and, byimplication, p120. On the basis of these studies, we havedefined the apparent activity of p120 that is associated withclustering (and the strengthening of adhesion) as ‘activation’of p120.

Inactivation of p120 and negative effects on cell-celladhesionA second group of studies appears to contradict the aboveresults and suggests that p120 plays a crucial role in thedisassembly of adherens junctions. In Colo205 cells, theknown components of the E-cadherin complex are essentiallyintact, but the cells remain rounded and largely non-adherentin cell cultures (Aono et al., 1999). Interestingly, p120 isconstitutively hyperphosphorylated in these cells. Brieftrypsinization under conditions that protect cadherins, ortreatment with the serine/threonine kinase inhibitorstaurosporine, correlates with decreased p120 phosphorylationand restored adhesiveness (Aono et al., 1999). Moreover,expression of an N-terminally deleted p120 construct that lacksmost of the phosphorylation sites also restores adhesion. Oneinterpretation is that an aberrant constitutive signaling event inColo205 cells leads to inactivation of p120’s ability to promoteclustering and adhesion. Although Colo205 cells are clearlyexceptional, experiments in K-562 leukemia cells and murine

L-fibroblasts also suggest that p120 can block adhesion(Ozawa and Kemler, 1998; Ohkubo and Ozawa, 1999). In K-562 cells, expression of membrane-tethered E-cadherinextracellular domains promotes aggregation, whereas similarconstructs containing the JMD, but lacking the CBD, blockaggregation. Moreover, minimal mutation of the JMD in thelatter construct restores adhesiveness (Ozawa and Kemler,1998). Inhibition of adhesion again correlated withhyperphosphorylation of p120, and N-terminally deleted p120restored adhesiveness (Ohkubo and Ozawa, 1999). Theseresults implicate p120 in inhibition of cell-cell adhesion.

Mechanisms and models The above data indicate that the JMD and p120 can mediateboth positive and negative regulation of adhesion. One obviousproblem is how to integrate the seemingly inconsistent resultsin one all-encompassing model. The most likely explanation isthat trans binding between cadherins is somehow linked to thepositive effect of the JMD (p120 activation; Yap et al., 1998;Navarro et al., 1998; Thoreson et al., 2000), whereasintracellular signaling can induce its negative function (Aonoet al., 1999; Ohkubo and Ozawa, 1999). Rapid activation ofadhesion in Colo205 cells by brief trypsinization might, forexample, result from clipping of a receptor and subsequent lossof an aberrant constitutive signaling event acting ultimately onp120. Thus, p120 could act as a switch that induces cadherinclustering and strong adhesion when activated, and alsomediate junction disassembly following signaling eventsleading to its inactivation. Although there is evidence thatserine or tyrosine phosphorylation constitutes the switchbetween these forms (Aono et al., 1999; Ohkubo and Ozawa,1999), this has not yet been proven, and the terms activationand inactivation cannot be assumed to reflect changes inphosphorylation status.

Fig. 6 illustrates a p120-centric view of cadherin-mediatedadhesion that summarizes our interpretation of these data andpossible mechanisms of p120 action. Cadherins are thought toexist both as monomers and as lateral (or cis) dimers in theplasma membrane. On the basis of the N-cadherin-repeatcrystal structure and other data (Shapiro et al., 1995; Nagar etal., 1996; Pertz et al., 1999; Tamura et al., 1998), it has beenproposed that the Ca2+-dependent homophillic association ofadhesive trans dimers (connecting adjacent cells) occurs afterthe association of lateral dimers. Strengthening of adhesion iscorrelated with both cadherin clustering and association withthe actin cytoskeleton – experimentally separable eventsrelated to JMD and CBD functions, respectively. As mentionedearlier, the formation of adhesive dimers might activate theclustering potential of p120 and the JMD. The mechanism ofthis activation is not known, but it could involve simpleconformational changes affecting p120 binding or an adhesion-sensitive signaling cascade (phosphorylation?) ultimatelyimpacting on p120 function. Under normal conditions, p120activation induces clustering and results in significantenhancement of adhesive strength. When p120 is constitutivelyinactivated, adhesive dimers cannot cluster and revert back toform lateral dimers, which are not adhesive. In this view, theCBD mediates subsequent or possibly concomitantimmobilization of cadherin complexes by actin crosslinking.The latter might not be necessary for strong adhesion, per se,but is probably a key factor in compaction – a term used to

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reflect an even-tighter consolidation of cellular aggregates(Adams et al., 1996, 1998).

Fig. 6 also shows three potential mechanisms by which p120could affect adhesion. In the simplest scenario, activated p120mediates the physical interaction between cadherins by directhomodimerization, or heterodimerization involving other p120family members (Fig. 6A). Although this scenario is intuitively

attractive, p120 does not interact with itself in yeast two-hybridassays (Daniel and Reynolds, 1995) or in direct GST-pulldownassays (Yap et al., 1998). In the second scenario, p120 mediatescadherin clustering by interacting with unidentifiedcrosslinking proteins (Fig. 6B). For example, in neuronalsynapses the p120 relative δ-catenin/NPRAP interacts directlywith S-SCAM (Ide et al., 1999), a scaffold protein implicated


Fig. 6. A p120-centric model forcadherin-mediated adhesion. Bothmonomers and lateral (cis) dimers arethought to exist at the plasmamembrane. Formation of lateral dimers(stage I) is believed to temporallyprecede the formation of adhesive(trans) dimers (stage II) betweencadherins on adjacent cells. Adhesivedimers cluster (stage III) at areas of cell-cell contact, which results in theestablishment of strong adhesion.Although diffusion alone might bringtrans-dimers on adjacent cells intocontact, several lines of evidencesuggest that the cytoplasmic domain isrequired for proper targeting of thesecomplexes to cell-cell junctions.Evidence suggests that cadherinclustering requires ligand binding (trans-adhesive dimerization), an intact JMD(presumably to coordinate p120binding) and RhoA, but not the CBDand/or α-catenin-mediated interactionwith the actin cytoskeleton (see text). Inall of the models, formation of anadhesive dimer leads somehow to‘activation’ of p120, which facilitatessubsequent cadherin clustering. Theswitch that regulates the activated (blue)and inactivated (orange) states is not yetclear. Possible mechanisms includechanges in p120 phosphorylation,conformation or binding-partnerinteractions. The interaction betweencadherins and other catenins through theCBD (depicted in dark green) is omittedhere for simplicity. Three possiblemodels are proposed for p120-inducedclustering. In the first (A; direct p120dimerization), direct homodimerizationof 120 is responsible for the clusteringof adhesive dimers. Heterodimerizationof p120 with other p120 familymembers is a related possibility. In thesecond model (B; indirect p120dimerization), clustering is alsomediated by a dimerization mechanism,but unknown crosslinking proteins arepostulated. Finally, the third model(C; cadherin-mediated dimerization)predicts that binding of activated p120 to cadherin adhesive dimers induces conformational changes in the cadherin itself, which subsequentlypromotes clustering. This model is supported by evidence that the extracellular and/or transmembrane domains can in fact dimerize on their own butare regulated somehow by intracellular interactions. Finally, the last stage of cadherin-mediated adhesion, actin crosslinking (stage IV), is probablycoordinated with clustering but further requires the CBD, Rac1 and Cdc42, and direct association with the actin cytoskeleton. The distinctionbetween clustering (stage III) and actin crosslinking (stage IV), both of which result in so-called strong adhesion in aggregation assays, may berelevant for distinguishing the specific role of p120 in the complex.

p120"Activation"

PM

[A]Direct p120dimerization

[B]Indirect p120dimerization

[C] Cadherin-mediated dimerization

CadherinMonomer

Lateraldimerization

Adhesivedimerization

Cadherin clustering

Stage I Stage II

Stage III

Ca2+

Stage IV Actin crosslinking


in the clustering of membrane receptors. Crosslinking toscaffold proteins might therefore facilitate the clusteringprocess. This scenario is necessarily vague because it invokesadditional p120-binding partners that have not yet beenidentified. In the third scenario, association of adhesive dimerswith activated p120 leads to conformational changes in thecadherins themselves, which in turn facilitate cadherin-clustering mechanisms involving the extracellular,transmembrane, or proximal juxtamembrane domains (Fig.6C). Inside-out signaling analogous to that which occurs withintegrins (for review see Kolanus and Zeitlmann, 1998; Faulland Ginsberg, 1996; Tozer et al., 1996) may be involved. Theability of the extracellular domains to support clustering hasbeen proposed on the basis of structural data (Shapiro et al.,1995; Nagar et al., 1996) but is not supported by other evidence(Fujimori and Takeichi, 1993; Katz et al., 1998; Troyanovsky,1999). However, Huber et al. (1999) recently showed that thecadherin transmembrane domain can homodimerize, andBalsamo et al. (1999) reported that peptides mimicking theproximal JMD bind cadherins. All of these models predictincreased adhesion due to p120 activation and are notnecessarily mutually exclusive.

Rho family GTPases and cadherin-mediatedadhesionAn important consideration in all of these models is theincompletely understood role of the Rho family of GTPases incadherin function (Braga et al., 1997; Takaishi et al., 1997;Braga et al., 1999; Jou and Nelson, 1998). RhoA transientlylocalizes to cell-cell junctions upon induction of calcium-dependent adhesion (Takaishi et al., 1995; Kotani et al., 1997),which raises the possibility that it is recruited to junctionsand activated following the formation of adhesive dimers. Inaddition, several studies indicate that Rac1 and Cdc42activities mediate the crosslinking of cadherins with theactin cytoskeleton (Braga et al., 1997; Jou and Nelson, 1998;Takaishi et al., 1997; Kodama et al., 1999). RhoA is thoughtto act at an earlier step, because inhibition of RhoA by C3exotransferase expression blocks both cadherin localization atareas of cell-cell contact (Braga et al., 1997; Zhong et al., 1997;Takaishi et al., 1997) and Rac1-mediated actin recruitment(Braga et al., 1997). Furthermore, forced clustering ofcadherins on antibody-coated beads abolishes the ability of C3to inhibit Rac1-mediated actin recruitment (Braga et al., 1997),which suggests that RhoA is required for cadherin clustering.Together, these data suggest potential links between RhoA,p120 and the JMD in cadherin clustering.

p120 PHOSPHORYLATION

p120 is a superb Src substrate both in vivo and in vitro(reviewed by Daniel and Reynolds, 1997). Althoughconstitutive tyrosine phosphorylation of p120 in cellsexpressing activated Src correlates with cell transformation(Reynolds et al., 1989), we cannot yet distinguish the directeffects of p120 phosphorylation from the many other eventsassociated with Src activation. Multiple lines of evidencesuggest that tyrosine phosphorylated p120 binds with increasedaffinity to various cadherins (Kinch et al., 1995; Skoudy et al.,1996b; Papkoff, 1997; Calautti et al., 1998). There is also

evidence that transient tyrosine phosphorylation of p120 occursin nascent cell-cell junctions (Calautti et al., 1998; Lampugnaniet al., 1997; Kinch et al., 1997), although, under steady-stateconditions, tyrosine phosphorylation of p120 is generally notdetected. p120 is also tyrosine phosphorylated in response toEGF, PDGF, CSF-1, VEGF and NGF (Downing and Reynolds,1991; Shibamoto et al., 1995; Daniel and Reynolds, 1997;Esser et al., 1998), but it is not clear whether the implicatedreceptors phosphorylate p120 directly or through therecruitment of associated tyrosine kinases, such as Src.Nonetheless, the numerous reports linking cadherin complexeswith Src, receptor tyrosine kinases, and a variety of tyrosinephosphatases (reviewed by Daniel and Reynolds, 1997), arguestrongly for an important role for tyrosine phosphorylation inthe regulation of cadherin function. Discriminating the directeffects of tyrosine phosphorylation of p120 and β-catenin oncadherin function will probably require the mapping andsubsequent mutation of the relevant sites.

In most cells, p120 is extensively phosphorylated on serineresidues (and to a lesser extent on threonine residues; Downingand Reynolds, 1991; Ratcliffe et al., 1997, 1999; Aono et al.,1999; Ohkubo and Ozawa, 1999). In fact, even the receptortyrosine kinase ligand EGF induces extensive serinephosphorylation of p120 (Downing and Reynolds, 1991). p120isoforms generally migrate diffusely on polyacrylamide gels,mostly because of serine phosphorylation, and phosphatasetreatment collapses the bands into sharply migrating species(Staddon et al., 1995; Aono et al., 1999; Ohkubo and Ozawa,1999; Thoreson et al., 2000). Early work demonstrated thatonly membrane-associated Src can phosphorylate p120(Reynolds et al., 1989). The observation that tyrosinephosphorylation and serine/threonine phosphorylation of p120require recruitment of p120 to cell membranes throughassociation with cadherins is consistent with this (Reynolds etal., 1994; Ohkubo and Ozawa, 1999; Thoreson et al., 2000).Conversely, when p120 is stranded in the cytoplasm (e.g. inmetastatic cell lines that have lost cadherin expression), it isvirtually unphosphorylated (Ohkubo and Ozawa, 1999;Thoreson et al., 2000) and probably unavailable to receivemembrane-associated signals. The level of serinephosphorylation of p120 can be reduced by the kinase inhibitorstaurosporine (Ratcliffe et al., 1997, 1999; Aono et al., 1999),and by either inhibition or activation of protein kinase C, whichimplicates a PKC-modulated protein kinase (Ratcliffe et al.,1997, 1999). As mentioned earlier, two important studies nowimplicate serine dephosphorylation as a mechanism for theactivation of p120’s adhesive function (Aono et al., 1999;Ohkubo and Ozawa, 1999). However, given that concreteevidence of the relevant phosphorylation events is lacking,other events mediated by the N-terminal domain of p120 mightalso account for these effects. The mapping of specific serinephosphorylation sites and identification of respective kinaseswill be crucial for an understanding of how these eventsmodulate p120 function.

NUCLEAR SIGNALING

p120 was recently added to the list of Arm-repeat proteins thathave dual localization at cell-cell junctions and in the nucleus(Van Hengel et al., 1999; Mariner et al., 2000). Perhaps the

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best-known example is β-catenin, which plays a key role inboth adhesion and signaling (reviewed by Ben-Ze’ev andGeiger, 1998, and references therein). A novel transcriptionfactor, Kaiso, binds directly to the p120 Arm domain,providing the first candidate for a nuclear target of a p120family member (Daniel and Reynolds, 1999). Kaiso belongs toa growing POZ/ZF (Pox virus and zinc finger) superfamily oftranscription factors (reviewed by Bardwell and Treisman,1994; Albagli et al., 1995), members of which arecharacterized by a conserved N-terminal hydrophobic domainof ~120 residues (the POZ domain) and C-terminal C2H2-typezinc-finger motifs (the ZF domain). Many POZ/ZF proteins aresequence-specific transcriptional repressors that apparentlyrecruit histone-deacetylase complexes via their POZ domains(Grignani et al., 1998; Lin et al., 1998; David et al., 1998).Although the functional significance of the Kaiso-p120interaction is currently speculative, the known activities of theother POZ/ZF proteins in development and cancer make Kaisoan attractive signaling candidate for a conveyor of informationrelated to cell-cell adhesion. Van Hengel et al. (1999) havedemonstrated that p120 enters the nucleus. Moreover, anuclear-exclusion signal that appears to mediate p120 exportis present in alternatively spliced forms of p120 containingexon B. The simplest hypothesis, therefore, is that p120interacts with Kaiso in the nucleus, thereby modifying itspredicted transcriptional activity. Further analysis of thisinteraction awaits the characterization of Kaiso transcriptionaltargets, which will also allow elucidation of the roles of p120and cadherin in Kaiso-mediated effects. It is likely that othermembers of the p120 subfamily also have nuclear partners. Aninteresting observation is that the nuclear localization of p120is enhanced in cadherin-deficient cells (Van Hengel et al.,1999). Thus, aberrant regulation of Kaiso by p120 mightcontribute in previously unsuspected ways to the pleiotropiceffects of cadherin loss in metastatic cells.

ECTOPIC p120 OVEREXPRESSION

Overexpression studies offer an obvious avenue for testing thebiological functions of novel proteins. p120-overexpressionstudies have been hindered because of unexplained problemsin generating stable cell lines. Interestingly, transientoverexpression of p120 in fibroblasts induces severe branchingof cellular processes, resulting in a so-called dendriticphenotype (Reynolds et al., 1996). Morphological changes,albeit less dramatic, are also induced in other cell types.Overexpression of other Arm-domain proteins, including theclosest relative of p120, ARVCF (Mariner et al., 2000), haslittle effect in fibroblasts. Moreover, deletions in the Arm-repeat domain completely block the effect (Reynolds et al.,1996). A similar phenotype is observed under a variety ofconditions that disrupt RhoA-mediated contractility (Jalink etal., 1994; Kranenburg et al., 1999), further suggesting afunctional link between p120 and Rho signaling.

Ventral overexpression of p120-1A or 1N in Xenopusembryos does not induce Wnt signaling or the duplicate axisformation associated with ventral overexpression of β-catenin.In contrast, overexpression of p120 in dorsal blastomeresperturbs gastrulation (Geis et al., 1998; Paulson et al., 1999),apparently because of reduced cell motility, reduced adhesion

between blastomeres, and ectodermal defects. These datasuggest that p120 affects morphogenetic events, which isconsistent with a role for p120 in cell-cell adhesion.

p120 EXPRESSION IN TUMORS

Although knockout data are not yet available, analysis of p120expression in human tumors suggests that p120 plays animportant role in malignancy. Loss of p120 expression has beendemonstrated in a variety of human tumors and in some casesis statistically linked to an aggressive tumor phenotype (Dillonet al., 1998; Gold et al., 1998; Shimazui et al., 1996; Skoudy etal., 1996a; Syrigos et al., 1998; Valizadeh et al., 1997). Bycontrast, Jewhari et al. (1999) report striking upregulation ofp120 expression in ~66% of gastric carcinomas. In addition,expression of p120 isoforms is remarkably heterogeneous inhuman tumor cell lines (Wu et al., 1998; Skoudy et al., 1996a;Keirsebilck et al., 1998), but the significance of this observationis unknown. One possibility is that misexpressed p120 isoformspromote tumor progression by dysregulating cadherin-mediatedadhesion, or promoting cell motility and invasion. Finally, thecorrelation of constitutive p120 phosphorylation with defects inColo205 cell adhesion (Aono et al., 1999) suggests thatsignaling pathways contribute to malignancy through directmodification of p120 function.

FUNCTIONAL ROLES OF p120 FAMILY MEMBERS

Competition among p120 relatives for cadherinbindingA largely unexplored issue involves the potential functionalcrosstalk between p120 and other closely related familymembers. The existence of p120 relatives suggests that theyhave functional similarities and differences that play out indifferent cell types. For example, p120 family members maycompete for cadherin binding. δ-catenin/NPRAP, like p120,interacts directly with the JMD (Lu et al., 1999), and thebinding of ARVCF and p120 to E-cadherin is apparentlymutually exclusive (Mariner et al., 2000). One clue comes fromthe selective expression of δ-Catenin/NPRAP/neurojungin inneuronal tissues, which suggests that it has cell-type specificfunctions. In addition, although ubiquitously expressed,ARVCF appears to be present in most adult tissues at extremelylow levels (Mariner et al., 2000). Thus, it is unlikely to act asa direct p120 competitor in vivo. Thus far, p120 appears to bethe most abundant member of the family in most cell types,which suggests that it is the dominant player in cadherinfunction. However, p0071 is also believed to be abundant andis a potential, although largely unstudied, rival for this role(Hatzfeld and Nachtsheim, 1996). An important area of futureresearch is to determine whether these family members bind tothe same or different cadherins and whether (and if so how)competition for the JMD imparts functional diversity oncadherin-mediated adhesion.

Specific functional roles of individual p120 relativesTable 1 summarizes the expression patterns, cellularlocalizations, binding partners and chromosomal locations ofall known p120 family members. Specific functions have been



suggested for particular p120 relatives and plakophilins. Forexample, it is possible that p0071 has dual roles in bothadherens junctions and desmosomes (Hatzfeld andNachtsheim, 1996). Furthermore, p0071 (Stahl et al., 1999;Tanahashi and Tabira, 1999) and δ-catenin/NPRAP (Levesqueet al., 1999; Zhou et al., 1997; Tanahashi and Tabira, 1999)interact with presenilin 1, a protein that when mutated leads tofamilial Alzheimer’s disease (Nishimura et al., 1999); thisraises the possibility that p120 family members are involved inneurodegeneration. In addition, Sirotkin et al. (1997) havelinked the gene that encodes ARVCF to Velo-Cardio-Facialsyndrome, whereas δ-catenin/NPRAP maps to a regioninvolved in Cri-du-chat syndrome (cat cry; Bonne et al., 1998).Although the involvement of ARVCF or δ-catenin/NPRAP inthese syndromes has not been confirmed, it is interesting thatboth syndromes exhibit developmental facial and heart defects.

As mentioned earlier, p120 induces a striking branchingphenotype when overexpressed in fibroblasts. δ-Catenin/NPRAP induces morphological changes in MDCK cells(increased spreading, and formation of lamelipodia andfilopodia), negatively regulates cadherin adhesiveness andincreases motility in response to HGF (Lu et al., 1999).Although these phenotypic changes are similar to those inducedby p120 in MDCK cells (unpublished observation), δ-catenin/NPRAP does not induce branching in fibroblasts (Q. Lu and K.Kosik, personal communication). Interestingly, overexpressionof the plakophilin 1 Arm domain but not overexpression offull-length plakophilin 1 induces a similar phenotype inepithelial cells (M. Hatzfeld, personal communication),whereas overexpression of ARVCF in fibroblasts or epithelialcells does not alter cell morphology (Mariner et al., 2000). Thesignificance of the observation that p120 is particularly difficultto express stably in cells is unclear: stable expression of bothARVCF (Mariner et al., 2000) and δ-catenin/NPRAP (Lu et al.,1999) has been accomplished in a variety of cell lines. Clearly,there are important differences between these proteins, and eachis likely to contribute unique functions at cell junctions and inthe nucleus.

CONCLUSIONS

It is now clear that p120 is a major cadherin-binding partnerand the likely mediator of JMD function. Further elucidationof the mechanism of p120 action in cadherin-mediatedadhesion will undoubtedly clarify our understanding ofprocesses involved in cadherin-dependent tissuemorphogenesis and related issues in metastatic cells. It is likelythat p120 has both positive and negative roles in cadherinadhesion, functions that are probably regulated by post-translational modifications. Genetic studies in mice andother organisms are likely to provide further insight on thespecific functions of p120 and family members in cell-celladhesion and morphogenesis. Detailed mapping of p120phosphorylation sites and identification of new p120-bindingpartners and effectors are also important goals for futureprogress. Another interesting and largely unexplored issue isthe significance of the apparently universal dual localization ofp120 family members in junctions and nuclei. The relativestability of p120 and its cytoplasmic/nuclear localization incadherin-deficient cells raises the possibility that aberrantp120/Kaiso signaling contributes to the pleiotropic effects ofcadherin loss in metastatic cells. An important possibility isthat the interplay between oncogenes, catenins and nuclearsignaling is responsible for tumor-cell defects associated withthe contact inhibition of cell growth, a hallmark of malignancythat remains unexplained at the molecular level.

REFERENCES

Adams, C. L., Nelson, W. J. and Smith, S. J. (1996). Quantitative analysisof cadherin-catenin-actin reorganization during development of cell-celladhesion. J. Cell Biol. 135, 1899-1911.

Adams, C. L., Chen, Y. T., Smith, S. J. and Nelson, W. J. (1998).Mechanisms of epithelial cell-cell adhesion and cell compaction revealed byhigh-resolution tracking of E-cadherin-green fluorescent protein. J. CellBiol. 142, 1105-1119.

Aho, S., Rothenberger, K. and Uitto, J. (1999). Human p120ctn catenin:

Table 1. p120 family expression and localizationSubcellular Chromosomal

Expression localization Binding partners localization

p120 ctn Ubiquitous, high level expression. Adherens junctions, nucleus Classical cadherins, Kaiso, 11q11||Not in B/T cells BP180*, FER kinase‡

ARVCF Ubiquitous, low level expression Adherens junctions, nucleus Classical cadherins 22q11 (region deleted in Velo-cardio-facial syndrome)

δ-Catenin/NPRAP Neuroepithelium, neuroendocrine Adherens junctions, nucleus Classical cadherins, presinilin 1, 5p15 (region involved in tissues S-SCAM Cri-du-chat Syndrome

P0071 Ubiquitous expression Desmosomes and adherens Presinilin 1¶ 2q23-q31junctions§

PKP 1 Epithelial tissues, especially Desmosomes, nucleus Desmoplakin, cytokeratins, 1q32 (linked to ectodermal stratified epithelia Dsg 1 dysplasia/skin fragility

syndrome)PKP 2 Ubiquitous expression Nucleus, desmosomes Desmoplakin, cytokeratins 12p11, 12p13 (pseudogene)PKP 3 Stratified and single layered epithelia. Desmosomes, nucleus Not identified 11p15

Not hepatocytes, foreskin fibroblasts, sarcoma-derived cells

*p120ctn reportedly interacts with the hemidesmosomal protein BP180 (Aho et al., 1999) but the significance of this interaction is unknown.‡p120ctn interacts with FER kinase either directly or indirectly (Kim and Wong, 1995). §It is not known whether p0071 is also found in the nucleus.¶Direct association of p0071 with components of the desmosomes or adherens junctions has not been reported to date. ||For chromosomal localization of p120 family members see Bonne et al. (1998) and references therein.

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p120 catenin roles in adhesion and signaling · 2001. 5. 3. · p120 catenin roles in adhesion and...

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