Cell surface-associated mucins in signal transduction

Cell surface-associated mucins insignal transductionPankaj K. Singh and Michael A. Hollingsworth

Eppley Institute for Research in Cancer and Allied Diseases, Department of Biochemistry and Molecular Biology,

University of Nebraska Medical Center, Omaha, NE 68198-6805, USA

Review TRENDS in Cell Biology Vol.16 No.9

Mucins are heavily glycosylated high molecular weightglycoproteins, which are involved in the protection andlubrication of luminal epithelial surfaces. Transmem-brane mucins also engage in signal transduction,through extracellular domain-mediated ligand bindingor by interacting with receptors for growth and differ-entiation factors. The cytoplasmic tail of MUC1(MUC1CT), the best characterized of the transmembranemucins, is involved in several signaling pathways,including those involving Ras, b-catenin, p120 catenin,p53 and estrogen receptor a. MUC1CT also forms com-plexes with transcription factors, and then translocatesto the nucleus by an unknown mechanism, where it isbelieved to influence the transcription of their targetgenes. MUC1CT has also been proposed to localize tomitochondrial membranes under conditions of geno-toxic stress, where it attenuates the apoptotic pathwayin response and confers resistance to apoptosis-indu-cing drugs.

IntroductionMucins are high molecular weight glycoproteins expressedby secretory or polarized epithelia, lining the luminalsurfaces of respiratory, gastrointestinal and reproductivetracts [1]. The cellular expression pattern ofmucins has ledinvestigators to propose two classifications: membrane-boundmucins and secretorymucins. The secretorymucins,which lack a transmembrane domain and are secreteddirectly into the extracellular spaces, include MUC2,MUC5AC, MUC5B, MUC6, MUC7, MUC8 and MUC19.Themembrane-bound class ofmucins are type Imembraneproteins with single transmembrane domains and differ-ent lengths of cytoplasmic tail at the C-terminus. Themembrane-bound class includes MUC1, MUC3A, MUC3B,MUC4, MUC12, MUC13, MUC15, MUC16, MUC17 andMUC20. Membrane-bound mucins can be released fromcells through proteolytic cleavage, and many are producedin secreted forms that result from alternative mRNAsplice forms in which the transmembrane domains areeliminated.

Transmembrane mucins are postulated to serve assensors of the external environment, through extracellulardomain-mediated ligand binding or as a consequence ofaltered conformations that result from changes in externalbiochemical conditions (pH, ionic composition, physical

Corresponding author: Hollingsworth, M.A. ([email protected]).Available online 9 August 2006.

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interactions). Signals are transmitted to the interior ofthe cell through post-translational modifications of thecytoplasmic tail that include phosphorylation events, pro-teolytic events and perhaps other modifications. The cyto-plasmic tails of membrane-bound mucins are alsohypothesized to be involved in signaling events that con-tribute to the progression of cancer.

Of the cell-surface mucins, MUC1 is the best character-ized with respect to signal transduction, and is the focushere (Box 1). Signals are transmitted to the nucleus byassociation of the MUC1 cytoplasmic tail (MUC1CT) withagents of signal transduction that include b-catenin, p120catenin, p53 and estrogen receptor a (ER-a) (Figure 1).Several studies indicate that intercellular adhesion mole-cule 1 (ICAM-1) might serve as a ligand for MUC1, activat-ing outside-in signaling via the MUC1CT [2–4]. Thebinding of ICAM-1 toMUC1 can initiate calcium signaling,which is independent of the mitogen-activated protein(MAP) kinase pathway [2]. Another study showed thatbinding to Pseudomonas aeruginosa or its flagellin proteincan serve as an activator of MUC1 signaling by promotingMUC1CT phosphorylation-mediated activation of theMAP kinase pathway [5]. It is likely that there are otherunknown ligands for MUC1.

Signaling through MUC1CTMUC1CT contains 72 residues and harbors several sitesthat can be phosphorylated; these modulate the bindingaffinities of different classes of signal transduction ele-ments (Figure 2). Interactions of MUC1 with proteinkinases in several different signaling cascades are wellcharacterized and are discussed below.

Nuclear and nucleolar localization of MUC1CT and itsinteraction with ErbB family membersMUC1CT, as demonstrated by reciprocal coimmunopreci-pitations, associates with b-catenin, p120 catenin, p53 andER-a in the nucleus, where it has been hypothesized tomodulate transcriptional events. Chromatin immunopre-cipitation (ChIP) assays on MUC1-overexpressing breastcancer cells have shown that the MUC1CT resides onpromoter elements of genes regulated by p53. The associa-tion of MUC1 with transcriptional regulators at promoterssupports the hypothesis that the MUC1CT contributes totranscriptional regulation.

The MUC1CT lacks any known DNA-binding motif.Hence, it is speculated that the MUC1CT influences tran-scriptional regulation by helping to configure complexes

d. doi:10.1016/j.tcb.2006.07.006

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Box 1. Signaling through cytoplasmic tails of other

transmembrane mucins

Cytoplasmic tails of other mucins are currently poorly characterized.

The putative size of cytoplasmic tails on other cell surface-

associated mucins varies from 22 residues to 80 residues. A

comparison of the cytoplasmic tail sequences from all known

mucins indicates the presence of positively charged lysine–arginine-

rich motifs in the region juxtaposed to the plasma membrane. This

motif is present in MUC1 (sequence RRK), MUC3 (RRGR), MUC12

(RKRHR), MUC15 (KRK), MUC16 (RRRKK) and MUC17 (RSKR). The

known function of this positively charged cluster is to serve as a

spatial delimiting sequence for the hydrophobic domain, preventing

insertion of the remaining cytoplasmic region into the plasma

membrane. It can also serve as an imperfect or partial nuclear

localization signal or a potential motif for enzymatic cleavage of the

mucin cytoplasmic tail in response to concerted signaling events.

Nuclear localization has not been investigated for cytoplasmic tails

for mucins other than MUC1. Another conserved motif in the

cytoplasmic tails of many transmembrane mucins is the tyrosine-

based sorting signal for clathrin mediated endocytosis. This motif

[YXX(L/M/V/I/F)] has been shown to be crucial for clathrin-mediated

endocytosis of MUC1 (YHPM) [34]. Similar motifs are found in

MUC3 (YVAL), MUC12 (YNNF) and MUC17 (YSNF). Although the

phosphorylation status of the MUC1CT has been associated with

increased oncogenic potential of tumor cells, other mucin cytoplas-

mic tails remain uncharacterized in this regard. Identification of

phosphorylation status and binding partners of these mucins will

provide us with further insights into the signaling potential of cell

surface-associated mucins.

468 Review TRENDS in Cell Biology Vol.16 No.9

that include other factors, or it affects the affinity of thesecomplexes towards regulatory DNA elements. Moreover,there is not a well-defined nuclear transport signal in theMUC1CT but there is evidence for its translocation to thenucleus [6] by an unknown mechanism. Two hypothesesmight explain nuclear localization of the MUC1CT. Thefirst hypothesis posits a Notch-type proteolytic cleavage,followed by nuclear translocation [7] in association withother factors. Another possibility is endosomal uptake ofthe cell-surface form, followed by nuclear delivery of theendocytosed protein. The second mechanism has beendemonstrated for the nuclear translocation of ErbB2cell-surface protein [8] but would probably requireproteolytic processing of the full-length MUC1 protein intoa cytoplasmic tail fragment.

The ErbB family consists of four transmembrane recep-tors: ErbB1 [also known as epidermal growth factor recep-tor (EGFR)], ErbB2 (also known as Her2 or neu), ErbB3(also known as Her3) and ErbB4 (also known as Her4).These receptor tyrosine kinases (RTKs) differ in ligandspecificity. Ligand-dependent dimerization induces trans-phosphorylation of tyrosine residues in the activation loop,significantly enhancing kinase activity [9]. None of theknown ErbB family ligands bind Erb2, which is activatedby heterodimerization with other members of the ErbBfamily. Full-length MUC1, including the cytoplasmic tail,interacts with all fourmembers of the ErbB family of RTKs[10,11]. In tumor cells, MUC1 coimmunoprecipitates andcolocalizes with ErbB1, with and without the addition ofexogenous EGF, a well known ligand for the ErbB1 [12].Ligand-bound ErbB1, in turn, can phosphorylate theMUC1CT at tyrosine in the YEKV motif [12]. ErbB1-mediated phosphorylation of the MUC1CT enhances itsaffinity for the c-Src SH2 domain and b-catenin, and

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facilitates nuclear localization of the MUC1CT, presum-ably following proteolytic cleavage.

Heregulin (HRG; also known as neuregulin 1), a ligandfor ErbB3 and a stimulator of ErbB2 through ErbB2–ErbB3 heterodimerization, has been shown to enhancethe interaction of the MUC1CT with g-catenin and totarget the MUC1CT–g-catenin complex to the nucleolus[10]. The MUC1CT can be coimmunoprecipitated fromcomplexes that contain ErbB2, and this interaction isfurther strengthened by stimulation with HRG.

Mutation of an RRK motif to AAA in the MUC1CTabrogates nucleolar localization of theMUC1CT–g-catenincomplex, without altering complex formation between theMUC1CT and g-catenin. One plausible (albeit speculative)hypothesis is that HRG–ErbB3 binding mediates the acti-vation of ErbB2 and causes ErbB2 to phosphorylate theMUC1CT, which enhances the affinity of the MUC1CT forg-catenin and promotes cleavage of the MUC1CT at theRRK motif by an as yet undefined protease. The MUC1CTis translocated to the nucleolus in complex with g-catenin.Whether the RRK motif is a cleavage site, or disruption ofthe RRKmotif disrupts the accessibility of the cleavage siteto the protease responsible for the cleavage, is not known atthis time.

The role of the MUC1CT–g-catenin complex in thenucleolus is not clear. The nucleolus serves as a site forassembly of ribosomes, where synthesis and processing ofrRNA and the processing of some ribonucleoproteins iscarried out. The nucleolus might also serve as a site fortemporary sequestration of some components of the tran-scriptional regulatory machinery. It remains to be deter-mined if nucleolar localization represents temporarysequestration of factors or if the MUC1CT–g-catenin com-plex performs some other function.

Interaction of the MUC1CT with the Src family ofnon-RTKsThe Src family includes nine non-RTKs: Src, Yes, Fgr, Yrk,Fyn, Lyn, Hck, Lck and Blk, which share common sequenceand structural features. Of the nine family members, c-Src,Lyn and Lck can bind and phosphorylate the MUC1CT offull-length MUC1 at the tyrosine in the YEKV motif [13–17]. The c-Src SH2 domain interacts directly with theYEKV motif and inhibits the binding of glycogen synthasekinase 3b (GSK-3b), which can also phosphorylate theMUC1CT and reduce its interaction with b-catenin.c-Src–mediated phosphorylation at the tyrosine in theYEKVmotif can therefore facilitate binding with b-catenindirectly, and also prevent interaction with GSK-3b.

MUC1 influences c-Src-mediated signaling during poly-oma middle T-antigen-induced mammary tumorigenesis[13], where it facilitates the association of c-Src with thep85 subunit of phosphatidylinositol 3-kinase (PI 3-kina-se)and b-catenin. In this experimental system, theMUC1CT of full-length MUC1 associates with and coloca-lizes with focal adhesion kinase, a c-Src substrate and a keyplayer in integrin-mediated signaling. The interaction ofthe MUC1CT with focal adhesion kinase has been pro-posed to target c-Src to the cell periphery, which couldenable or enhance the spatial interaction of c-Src with itssubstrates.

Figure 1. MUC1 signaling. Different conditions at the cell surface, such as binding interactions with ICAM-1, Pseudomonas aeruginosa flagellin, changes in pH or other

biochemical conditions in extracellular space, might induce MUC1-mediated outside-in signaling. MUC1 signaling can be mediated through phosphorylation of the

MUC1CT by growth factor receptors such as the ErbB1. Phosphorylated MUC1CT can contribute to Grb2–Sos-mediated activation of the Ras–ERK pathway or activation of

phospholipase Cg (PLCg)-mediated signaling events. The MUC1CT is also postulated to be cleaved and to localize to the nucleus in complexes with b-catenin or g-catenin,

where it might facilitate the transcriptional activation of various proliferative genes, such as cyclin D1. Abbreviations: DAG, diacylglycerol; ER, endoplasmic reticulum;

NF-kB, nuclear factor kB; PKC, protein kinase C; PM, plasma membrane.

Review TRENDS in Cell Biology Vol.16 No.9 469

MUC1 and regulation of the LEF/TCF-dependent WntpathwayWnt signaling regulates the normal developmentalprogram and cell fate along the crypt–villus axis [18].Wnts, secreted glycoproteins that serve as ligands forthe frizzled seven-pass transmembrane receptors, prevent


the proteasomal degradation of b-catenin, which interactswith the lymphoid enhancer factor 1 (LEF)/T-cell factor(TCF) family of transcription factors to activate the tran-scription of Wnt target genes, including cyclin D1 and c-myc. GSK-3b and adenomatous polyposis coli (APC) aretwo other components of the Wnt cascade, which promote

Figure 2. Sequence of MUC1CT, showing the sites of binding by association partners and known phosphorylation sites. Proteins listed above the MUC1CT sequence

represent kinases capable of phosphorylating the MUC1CT. Proteins listed below the sequence represent MUC1CT interaction partners without any kinase activity; p53 and

ER-a bind to the region denoted by the orange line and b-catenin to that denoted by the pink line. The kinases and interaction partners represent several different signaling

cascades, suggesting a role for the MUC1CT as an integrator of various signaling pathways. Sequence variations of the MUC1CT have been reported in the literature: in

place of the C-terminal sequence . . .AAASANL, . . .AATSANL has been reported [47–49].


the proteasomal degradation of b-catenin. The MUC1CTcan directly interact with different components of the Wntsignaling pathway, including b-catenin, APC and GSK-3b.The MUC1CT contains a b-catenin binding motif(SXXXXXSSL), and affinity for this motif can be differen-tially altered by phosphorylation at specific sites. TheMUC1CT also associates with p120 catenin and facilitatesits nuclear localization [19].

The influence of interactions between the MUC1CT andAPC on interactions between the MUC1CT and b-catenin,or on the stability of b-catenin complexes, is not yet estab-lished; however, it is clear that interactions of theMUC1CT with different components of the Wnt signalingcascade are associated with the differing carcinogenic andmetastatic potential of several cancers. For example, theMUC1CT interacts with the APC protein in breast cancer[20]. This interaction is enhanced by stimulation withEGF. Knockout of MUC1 expression on the mouse mam-mary tumor virus (MMTV)–Wnt-1 transgenic backgrounddelayed onset of mammary tumorigenesis [21].

Treatment with the MUC1CT peptides (carrying wild-type GSK-3b- and b-catenin-binding motifs) enhancedinvasion of the MDA-MB-468 breast cancer cell line, irre-spective of the phosphorylation status of the peptide [21].MUC1CT peptides lacking either GSK-3b- or b-catenin-binding motifs did not enhance invasion by the cell line,suggesting a role for these interactions in promoting inva-sion. MUC1 expression was associated with increasedsteady-state levels of b-catenin in the cytoplasm andnucleus of breast carcinoma cells by blocking the GSK-3b-mediated phosphorylation of b-catenin [22]. It is pos-sible that the MUC1CT serves as a scaffold protein,enabling interaction between different regulators of theWnt cascade. Alternatively, the MUC1CT might competefor or sequester b-catenin, which is normally associatedwith cadherins at the adherens junctions. In some celltypes, the MUC1CT is involved in the transcriptionalactivation of b-catenin–TCF-binding sites and transcrip-tional activation of cyclin D1 [23].

The MUC1CT of full-length MUC1, along with b-cate-nin, colocalizes with fascin (an actin-binding protein)and vinculin at the sites of focal adhesion during cellinvasion into collagen matrices [21]. Fascin providesmechanical support to cell protrusions in migrating cells,


suggesting a role for the MUC1CT complex during cellmigration.

Regulation of the p53 pathway by the MUC1CTp53 is a major tumor suppressor that helps to regulateantiapoptotic functions, DNA repair and DNA recombina-tion. Most human tumors have either mutations in p53 ordefects in the p53 pathway. The MUC1CT (presumably acleaved form) associates with p53 and contributes tothe regulation of DNA damage-induced transcriptionalactivation of p21, a key player in the promotion ofcell-cycle progression and prevention of apoptosis, and toabrogation of the transcription of Bax, which serves proa-poptotic functions [24]. Hence, it is hypothesized that theMUC1CT serves as a switch, altering the transcriptionalprofiles of p53-dependent genes in response to genotoxicstress from apoptosis to growth arrest. Although no directDNA-binding activity has been shown for the MUC1CT, ithas been shown to occupy the promoter elements of p21 andBax genes in association with p53 [24]. This suggests thatthe MUC1CT can serve as a transcriptional coregulator,whichmight function bymodulating the affinity of differenttranscriptional regulators, such as p53 and LEF/TCF pro-teins, for their responsive promoter elements. Hence, it isproposed that the MUC1CT contributes to the regulation ofcell fate under both normal and malignant conditions.

Activation of the MAP kinase pathway by the MUC1CTThe MUC1CT is involved in activation of MAP kinasepathways, through interactions with ErbB receptors,where it potentiates their signaling by enhancing theactivation of extracellular-signal-regulated kinases(ERK) 1 and -2 in mouse mammary glands [11]. TheMUC1CT of full-length MUC1 also interacts with Grb2–Sos. One hypothesis is that the MUC1CT serves as anadaptor or a scaffold protein that recruits Grb2 to sites inclose proximity to ErbB1 in a MUC1CT phosphorylation-dependent manner. The Grb2 adaptor protein might inturn recruit the guanine nucleotide-releasing factor Sosthrough its SH3 domain. The MUC1CT has been shown tobe in complex with Grb2 and Sos [25], and the presence offull-length MUC1 enhances the association of ErbB1 withSos [11]. The Sos protein in these complexes might facil-itate activation of the Ras pathway via G-protein-coupled


receptors, eventually leading to activation of the MAPkinase pathway.

A role for MUC1CT phosphorylation in the activation ofthe MAP kinase pathway is further supported by studiesinvolving a chimera of theMUC1CTwith extracellular andtransmembrane domains of CD8 (TM-CD8–MUC1CT).Cells expressing the TM-CD8–MUC1CT chimera and sti-mulated with anti-CD8 antibody showed significant acti-vation of ERK1 and -2, which was abrogated by eitherdeletion of the MUC1CT (just the TM-CD8) or by a mutantconstruct carrying a phosphorylation-incompetentMUC1CT [26,27]. ERK activation in this system was abro-gated by a dominant negative Ras mutant or in the pre-sence of a MAPK kinase (MEK) inhibitor, demonstratingthe involvement of the MUC1CT in the Grb2–Sos–Ras–MEK–ERK signaling cascade.

The MUC1CT causes stabilization of ER-a.ERs, which exist in two main forms (ER-a and ER-b), areligand-based transcription factors which regulate genetranscription by binding to estrogen response elements(ERE) in DNA [28]. 17b-estradiol (E2) is the most commonER ligand, and exerts its effects on growth, developmentand homeostasis in different tissues and organs. In breastcancer, MUC1 enhances E2-dependent growth and survi-val by binding to and stabilizing ER-a, which blocks its

Figure 3. Hypothesized role for MUC1 and RTKs in signaling related to cellular polarity

RTKs localize to the basolateral surfaces. Under conditions of tissue injury, cell polarizati

trigger signaling cascades, informing the cell of the loss of polarity and culminating in a

Such interactions occur constitutively in tumor cells, which inherently lack any apica

metastasis. Once phosphorylated, the MUC1CT translocates to the nucleus by mech

transcriptional events.


proteasomal degradation [29]. The MUC1CT is present inER-a transcriptional complexes at the ERE in promoterregions of estrogen-responsive pS2 and cathepsin D genes,as shown by ChIP assays [29]. TheMUC1CT also enhancesER-a occupancy of ERE and facilitates the recruitment ofsteroid receptor coactivator 1 (SRC-1) and glucocorticoidreceptor-interacting protein 1 (GRIP1) transcriptionalcoactivators to ER-a transcriptional complexes. Hence,MUC1 facilitates E2-mediated transcriptional activation,growth and survival of breast cancer cells.

The MUC1CT: at the crossroads between differentsignaling pathwaysThe MUC1CT might facilitate unique interactions amongdifferent signaling pathways. Although a role for theMUC1CT as a scaffolding protein has not been clearlyestablished, its interaction with components of differentsignaling pathways raises the possibility that it mightserve as a platform for integrating cellular signalingnetworks. Simultaneous association of the MUC1CT,b-catenin and c-Src has been demonstrated and it is knownthat c-Src can phosphorylate b-catenin and activateb-catenin–TCF-mediated transcription [6,30,31]. Tyrosinekinase-mediated phosphorylation of the MUC1CTenhances b-catenin-mediated nuclear signaling andtranscriptional activation [6]. It is thus possible that the

. In normal polarized epithelial cells, MUC1 localizes to the apical surface, whereas

on is lost, which facilitates interaction of MUC1 with RTKs. These interactions might

lteration of transcription of genes that are intended to repair the injured epithelium.

l–basal polarity, and might use these signals to advantage during invasion and

anisms which are poorly understood, where it contributes to the regulation of


MUC1CT brings b-catenin and c-Src into close proximity toenable unique signaling by this interaction.

The MUC1CT binds to and is phosphorylated by GSK-3b, which might also phosphorylate b-catenin that wasbound to MUC1. GSK-3b-mediated phosphorylation of b-catenin can facilitate its proteasomal degradation, a pro-cess that is inhibited by Wnt signaling. However, furtherstudies are required precisely to define the role of theMUC1CT as a spatial regulator of interactions betweenb-catenin and regulatory kinases.

TheMUC1CTmight also serve as a bridge betweenEGF-mediated signaling and the Wnt signaling network. It hasbeen reported that EGF stimulation of cells enhances phos-phorylation of the MUC1CT at the tyrosine in the YEKVmotif, which enhances the affinity of the MUC1CT for b-catenin and APC [12,20]. The effect of interactions betweenthe MUC1CT and APC on b-catenin signaling is not fullyunderstood. Nonetheless, taken together, these findingssuggest a possible role of MUC1CT as an integrator ofsignals for EGF activation and the Wnt signaling cascade.

Regulation of MUC1 endocytosis by the signaling statusof the MUC1CTMUC1 is a heavily glycosylated transmembrane protein,and its membrane trafficking has been shown to be depen-dent on the glycosylation status of the extracellular subunit[32,33]. However, glycosylation-independent internaliza-tion of MUC1 is regulated by signaling motifs in theMUC1CT [34]. The tyrosine in the YHPM motif is crucialfor interaction with the clathrin adaptor protein AP-2,whereas tyrosine in the YTNP regulates interaction withthe SH2 domain of Grb2-signaling adaptor protein, whichparticipates in the early stages of internalization [34,35].The YHPM sequence is recognized as a YXXf tyrosine-based internalization motif by AP-2, which serves selec-tively to recruit cargo molecules into clathrin-coated pits[34]. However, the specific signals that induce and regulatethese interactions need further elucidation.

Palmitoylation of the two cysteines in theCQCRRKmotifof theMUC1CThasbeendetectedand shown to regulate therate of MUC1 recycling to the cell surface; however, it doesnot have a significant impact on the rate of endocytosis ofMUC1 [36]. Whether palmitoylation regulates the subcel-lular distribution of the MUC1CT has not been reported.

The MUC1CT: a regulator of oxidative stress-inducedapoptosisExpression of MUC1 has been shown to modulateapoptosis both in vivo and in vitro [24,37–42]. Uniparous(having produced a single litter) MMTV–MUC1 transgenicmice show decreased apoptosis postlactation in mammaryglands [38]. The MUC1CT activates the PI 3-kinase–Aktpathway and increases the expression of the antiapoptoticprotein Bcl-xL by a PI 3-kinase-independent mechanism invitro [42]. Overexpression of MUC1 attenuates theapoptotic response to araC and gemcitabine in 3Y1fibroblast cells. By contrast, MUC1-expressing cells aremore sensitive to FasL-induced apoptosis, as comparedwith cells lacking MUC1 [40].

MUC1 expression is transcriptionally activated by oxi-dative stress, and associated with attenuation of


endogenous and H2O2-induced intracellular levels of reac-tive oxygen species (ROS) [39] and reduction of the oxida-tive stress-mediated apoptotic response. MUC1 alsofacilitates dephosphorylation-mediated activation ofFOXO3a, a transcription factor that regulates the cellularresponse to oxidative stress in a PI 3-kinase–Akt-depen-dent fashion [41]. Moreover, expression of the MUC1CT issufficient for activation of FOXO3a, which helps todecrease intracellular ROS levels and attenuates ROS-induced apoptosis. Targeting of the MUC1CT to mitochon-dria (Box 2), which is enhanced byHRG–ErbB stimulation,might be responsible for the MUC1-mediated attenuationof stress-induced apoptosis [37]. MUC1 overexpression canalso attenuate cytochrome c release from mitochondria inresponse to cisplatin treatment. This response is not sup-ported by a MUC1CT mutant (Y46F), indicating a role forphosphorylation of the MUC1CT in the antiapoptoticresponse. Hence, it is clear that the MUC1CT contributessignificantly to the attenuation of stress-induced apoptosis.However, the precise role and regulation of the MUC1CTin the apoptotic response requires further study.

MUC1CT in triggering immune responsesMUC1 is expressed by some activated T cells, and has beenproposed as a downstream effector of T cell receptor liga-tion and T cell stimulation [43,44]. The MUC1CT of full-length MUC1 is bound and phosphorylated by Lck andZAP-70 tyrosine kinases, which are activated upon T cellactivation [15,16,45]. Hence, MUC1 can be envisioned tocontribute to signaling responses during immune activa-tion. The MUC1CT contains a potential immunoreceptortyrosine-based activation-like motif (YXXLX8YXXM),which is phosphorylated by ZAP70 at the tyrosine in theYHPMmotif and at the tyrosine in the YEKVmotif by Lck.These phosphorylation events enhance the affinity of theMUC1CT for b-catenin, thus connecting T cell activationwith signaling mediated by b-catenin. The MUC1CT alsoassociates with the kinase Lyn, which phosphorylates theMUC1CT at the tyrosine in the YEKV motif and therebystrengthens the affinity for b-catenin [17]. Interleukin 7(IL-7), a physiological activator of lymphocytes, furtherenhances the association of the MUC1CT with Lyn. IL-7stimulation of RPMI 8826myeloma cells enhanced nuclearlocalization of the MUC1CT and b-catenin, potentiallyenabling the activation of several b-catenin-regulatedgenes in this cell type. We speculate that one function ofMUC1-related signaling in activated immune cells isrelated to cell motility because activated immune cellsshow increased motility (so that they canmigrate to appro-priate areas of immune organs such as lymph nodes tofacilitate activation, maturation and expansion of theimmune response). Moreover, aberrant MUC1 expressionin malignant lymphocytes (e.g. myeloma or lymphoma)might contribute to altered expression of metastasis-asso-ciated genes, thus providingmalignant lymphocytes with asurvival or metastatic advantage.

Cellular architecture, mucins and signalingBoth MUC1 and MUC4 can interact with tyrosine kinases(Box 3), including the ErbB family of receptors and otherRTKs. However, these are expressed at the basolateral

Box 2. Mitochondrial localization of the MUC1CT

Mitochondria are primary sites for energy metabolism in cells, and

have a significant role in the apoptotic process. A controversial model

suggests that MUC1 localizes to mitochondria and affects the

apoptotic process [37] (Figure I). Mitochondrial localization of MUC1

is enhanced following stimulation with HRG, and is abrogated by

mutations in the tyrosine in the YEKV motif, indicating that

phosphorylation at this site might regulate translocation of the

MUC1CT. Furthermore, c-Src, which phosphorylates MUC1CT at the

same site, is activated by HRG treatment, and this, in turn, facilitates

the association of MUC1 with heat shock protein 70 (HSP70) [50].

Hence, HSP70 is hypothesized to facilitate the transport of the

MUC1CT to the mitochondria as an integral membrane protein.

Genotoxic stress also facilitates mitochondrial localization of the

MUC1CT. The mitochondrial localization of the MUC1CT is proposed

to attenuate the apoptotic pathway in response to genotoxic agents

and to provide resistance to apoptosis-inducing drugs.

Figure I.


surfaces of normal differentiated epithelial cells, whichprobably precludes physical interactions with MUC1 andMUC4 (expressed at the apical surface of normal epithelialcells) and consequent signaling. A loss of polarity in theepithelial cells, perhaps through a physical or molecularbreach in the lateral or basal aspects of the epithelial celllayer, or as a consequence of changes in cellular architec-ture through proliferative or inflammatory processes,would enable the RTKs and mucins to associate andengage in signaling, and as such would serve as a mechan-ism to inform the cell that polarity had been lost. In cancercells, which have lost polarity, MUC1 is phosphorylated onthe cytoplasmic tail by ErbB1 [11], and is reported toassociate with other ErbB members [10,11]. MUC4 isknown to interact with ErbB2 by binding to an extracel-lular portion of the molecule through interactions betweenan EGF-like domain on MUC4 and the cognate receptorportion of ErbB2 [1]. This leads us to propose that onefunction of interactions between receptor kinases


expressed at the basal surface of the cell, and cell surfacemucins expressed at the apical surface of polarized cells isto enable signaling pathways that inform the cell thatthere has been a loss of polarized architecture, and toactivate gene transcription that is designed to repair thisbreach and reestablish cell polarity (Figure 3). Theseinteractions might induce survival signals to counteractinduction of anoikis or related events. Presumably, tumorcells, which have lost many aspects of differentiated cel-lular architecture, are using the signals from this pathwayto enable their survival or to regulate processes of invasionand metastasis. Consistent with this hypothesis is thefinding that nonapical or cytoplasmic expression ofMUC1 is associated with poor prognosis in several cancers[46].

SummaryMucins are normally distributed on the apical cell surface ofepithelia, in contrast to adhesion molecules and growth

Box 3. MUC4 signaling

In contrast to MUC1, cell-surface mucins, such as MUC4, can engage

in signal transduction by cis-interactions with ErbB receptors on the

cell surface through specific domains that have homology to EGF,

and serve to either block, induce or modulate the activation of

signaling through these receptors [51]. These EGF-like domains

might be masked on MUC4 but become exposed upon proteolytic

cleavage or other molecular alterations that expose these domains

[52]. Interactions between MUC4 and ErbB2 have been widely

reported for different epithelial cell types [51,53,54] but the mechan-

ism by which this interaction modulates signaling is complex and

poorly understood. ErbB3 binds to its ligand neuregulin (NRG) and

forms a heteromeric complex with ErbB2. This leads to phosphor-

ylation-mediated activation of ErbB2. The ErbB2–MUC4 complex

forms soon after the synthesis of two proteins in the cell. The

presence of MUC4 in the ErbB2–ErbB3–NRG complex (now a

tetramer complex) stabilizes the complex at the cell surface by

preventing its internalization. MUC4 binding also facilitates hyper-

phosphorylation of ErbB2, leading to enhanced activation of the PI 3-

kinase–Akt pathway and Ras–ERK pathway in cancer cells lacking

polarity. This leads to transcriptional activation of cyclin D1 and

hence promotes cell proliferation. It is known that MUC4 can bind to

ErbB2 and block accessibility of Herceptin (Figure Ia), and that this

might explain Herceptin resistance in some patients with breast

cancer [51,54,55]. Another possibility is that MUC4 directs or

redirects the subcellular localization of ErbB2 either from basolateral

sites on epithelial cells [53] (Figure Ib), or releases it from internal

cytoplasmic sites to enable transport to the surface [56]. Once ErbB2

is at the cell surface in complex with MUC4, it is autophosphorylated

and activates p38 MAPK. The p38 MAPK phosphorylates Akt, which

in turn abrogates the p27KIP1-mediated inhibition of cyclin D1

expression. Another possibility is that that MUC4 modulates the

ability of ErbB2 to dimerize with other ErbB family members, such as

ErbB3, and thereby creates receptors with different functional

signaling capacities [57].

Figure I. MUC4 signaling. (a) Effect of MUC4 on ErbB localization, signaling and accessibility in a non-polarized cancer cell; (b) ErbB receptor localization in a polarized

epithelial cell (i) not expressing MUC4 and (ii) expressing MUC4.




factor receptors, which are localized on the basal and lateralsurfaces of polarized epithelia. Loss of polarized architec-ture enables association of mucins, RTKs and other signal-ingmolecules, which sends a signal to the nucleus regardingloss of polarity, status of motility and other morphogeneticconditions. These signals are predicted to incite changes ingene transcription that facilitate restructuring of thedamaged epithelia, regulatemotility and/or ensure survivalunder these stressful conditions. Tumor cells, which showsignificant loss of cell polarization, have usurped thesefunctions and utilize them to promote tumor cell invasion,metastasis, survival and proliferation.

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Cell surface-associated mucins in signal transduction