Widely Conserved Signaling Pathways in the Establishment...

Widely Conserved Signaling Pathways in theEstablishment of Cell Polarity

Luke Martin McCaffrey and Ian G. Macara

Department of Microbiology, Center for Cell Signaling, University of Virginia School of Medicine,Charlottesville, Virginia 22908-5077

Correspondence: [email protected]

How are the asymmetric distributions of proteins, lipids, and RNAs established and main-tained in various cell types? Studies from diverse organisms show that Par proteins,GTPases, kinases, and phosphoinositides participate in conserved signaling pathways toestablish and maintain cell polarity.

The asymmetric distribution of proteins,lipids, and RNAs is necessary for cell fate

determination, differentiation, and specializedcell functions that underlie morphogenesis (StJohnston 2005; Gonczy 2008; Knoblich 2008;Macara and Mili 2008; Martin-Belmonte andMostov 2008). A fundamental question ishow this asymmetric distribution is establishedand maintained in different types of cells andtissues. The formation of a specialized apicalsurface on an epithelial cell seems quite differ-ent from the specification of axons versusdendrites in a neuron, or the asymmetricdivision of a nematode zygote. Yet, remarkably,a conserved molecular toolbox is used through-out the metazoa to establish and maintaincell polarity in these and many other contexts.This toolbox consists of proteins that arecomponents of signal transduction pathways(Goldstein and Macara 2007; Assemat et al.2008; Yamanaka and Ohno 2008). However,our understanding of these pathways, and theirintersection with other signaling networks,

remains incomplete. Moreover, the regulationand cross talk between the polarity proteinsand other signaling components varies fromone context to another, which complicates thetask of dissecting polarity protein function.Nonetheless, rapid progress is being made inour understanding of polarity signaling, whichis outlined in this article, with an emphasis onthe Par proteins, because these proteins playmajor roles integrating diverse signals thatregulate cell polarity (Fig. 1) (see Munro andBowerman 2009; Prehoda 2009; Nelson 2009).

PAR PROTEINS: INTERPRETERS OFCELL POLARITY

The establishment of cell polarity can bedissected into four primary components:(1) breaking symmetry; (2) establishing corticallandmarks; (3) polarizing the cytoskeleton; and(4) amplifying and maintaining the polarizedstate. In many different systems, the Par pro-teins, small GTPases, and the phosphoinositides

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and their regulators are among the conservedfactors that play fundamental roles in each ofthese stages. The Par (partition defective)genes were initially identified in an elegantscreen by Jim Priess and Ken Kemphues formaternal-effect genes that are embryonic lethalin Caenorhabditis elegans (Kemphues et al.1988). Eventually, seven genes required forasymmetric cell division of the zygote wereidentified. The products of the Par genes arequite diverse in function. Par-1 and Par-4encode serine/threonine-directed proteinkinases (Guo and Kemphues 1995; Watts et al.2000); Par-2 is a RING finger domain proteinthat may function as an E3 ubiquitin ligase(Levitan et al. 1994); Par-3 and Par-6 are PDZ(PSD95/Dlg/ZO1) domain-containing pro-teins with scaffolding or adaptor functions(Etemad-Moghadam et al. 1995; Hung andKemphues 1999); Par-5 is a 14-3-3 protein thatbinds to phosphorylated serine and threonineresidues (Morton et al. 2002); and PKC-3 is anatypical protein kinase C (aPKC). With theexception of Par-2, all of the Par proteins andaPKC are conserved throughout the Metazoa.Strikingly, most of these polarity proteins showa polarized distribution within the zygote, andare dependent on one another for their localiz-ation (Tabuse et al. 1998). Par-1 and Par-2 are

restricted to the posterior of the zygote cortex,whereas Par-3, Par-6, and aPKC are restrictedto the anterior cortex (although they are alsopresent in the cytoplasm) (Schneider andBowerman 2003; Munro 2006).

Par-3, Par-6, and aPKC can form a physicalcomplex, sometimes called the Par complex(Joberty et al. 2000; Lin et al. 2000; Wodarzet al. 2000), which has been identified inall animal cells that have been examined(Goldstein and Macara 2007). Par-6 and aPKCassociate through their amino-terminal PB1domains (Hirano et al. 2005), and Par-6 inhibitsthe basal activity of aPKC, but also can act as atargeting subunit for the kinase, recruitingsubstrates for phosphorylation (Yamanaka et al.2001). One of these substrates is Par-3. Par-6binds to Par-3, through a PDZ–PDZ inter-action, but aPKC can also bind through itskinase domain directly to the carboxy-terminalhalf Par-3, and phosphorylate a Ser residue(Nagai-Tamai et al. 2002). Importantly, Par-3,Par-6, and aPKC do not form a constitutivecomplex. Their interactions are regulated bymultiple protein kinases, by small GTPases,and by competition for other binding partners(Fig. 1). These regulators can alter the subcellu-lar distribution of the polarity proteins, andtheir function.

Microtubules/Spindle orientation

Phosphoinositidesignaling

Par3/Par6/aPKC

Numb/Notch

Tyrosine kinases

Insc/Pins

Rac/Rho

GEFs/GAPs

Actin cytoskeleton

Par1/Par5

Cdc42

TGFb-R

LglAurora kinase

UbiquitinE3 ligases

ROCK

LIMKPTEN

Wnt/DishevelledEphrinB1

p75NTR

Endocytosis

Figure 1. An overview of Par complex signaling, showing inputs (bottom) and outputs (top) with cellularfunctions that are targeted by these pathways (italics).

L.M. McCaffrey and I.G. Macara

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FACTORS THAT CONTROLPAR-3 LOCALIZATION

It is likely that the Par proteins provide criticalspatial information during cell polarization.A key question, therefore, concerns how thePar proteins themselves are localized. It is prob-ably fair to say that we do not yet know thecomplete answer to this question in any celltype, or for any polarity protein. Moreover,the mechanisms may be partly conserved andpartly dependent on context. For example,in the C. elegans zygote, Par-3 association withthe anterior cortex requires the expression ofPar-6, aPKC, and Par-2 (Etemad-Moghadamet al. 1995; Hung and Kemphues 1999); butin Drosophila neuroblasts and embryonic epi-thelial cells, Par-3 apical localization is inde-pendent of aPKC (Harris and Peifer 2005).And in mammalian epithelial cells, Par-3 isnot apical but is associated with tight junctions(Izumi et al. 1998; Joberty et al. 2000).

Protein localization often involves distinctsteps that can include transport, delivery,anchoring at the destination, and active exclu-sion from other areas of the cell. Transport cansimply involve passive diffusion, or directedmovement along the cytoskeleton. Alter-natively, the mRNA for the protein might betransported to the destination site, where itis locally translated. Anchors can includephospholipids, cytoskeletal elements, or morespecific protein complexes.

Par-3 transport: In the case of Par-3, trans-port and anchoring both appear to involvethe cytoskeleton in at least certain cell types(Fig. 2). For instance, in the Drosophila embryo,Par-3 is transported in a basal-to-apical direc-tion by dynein, presumably along microtubules,and is anchored in an actin-dependent mannerto apical adherens junctions (Harris and Peifer2005). Par-3 in turn recruits the Par-6/aPKCcomplex to the cortex at the apical surface,where another polarity protein, Crumbs, sepa-rates Par-3—which remains at the adherensjunctions—from Par-6 and aPKC, which assumea more apical localization (Harris and Peifer2005). In mammalian neurons, microtubulesare also important for the movement of Par-3

to the growing tips of axons. Par-3 interactsboth with the kinesin Kif3a and themicrotubule-associated protein APC (adeno-matous polyposis coli) (Nishimura et al. 2004;Shi et al. 2004). Neuronal microtubules areoriented with their plus ends toward thetips of nascent neurites. In undifferentiatedneurites, APC is phosphorylated by GSK3-b,which decreases the affinity of APC for micro-tubules. However, PI3-kinase is localized at thetips of axons, where it can phosphorylate andinactivate GSK3b, allowing APC to positivelyregulate microtubule growth and bundlingand provide a route for Kif3a-mediated trans-port of Par-3 along the microtubule tracksto the tips of axons (Shi et al. 2004). In theC. elegans zygote, in contrast, centrosomes andmicrotubules may play early roles in triggeringPar asymmetry (Tsai and Ahringer 2007), but aflow of cortical actin is more directly involvedin segregating the initial, uniformly corticalanterior Par protein complex to one pole(Munro et al. 2004; Munro and Bowerman2009).

Membrane attachment via phospholipids:How is Par-3 maintained at the plasma mem-brane once delivered? Although this questionhas not yet been definitively answered, multipledistinct mechanisms are probably involved,some that are general and some that are morespecific. So far, they all appear to involve thePDZ domains of Par-3. First, Par-3 has beenreported to bind directly to phosphoinositidesthrough its PDZ-2 domain, which is essentialfor membrane localization (Wu et al. 2007).This phospholipid association is unusualbecause most PDZ domains recognize specificcarboxy-terminal peptides, or loops, withinpolypeptides. Also, the interaction is not spe-cific for PIP2 or PIP3, so it is unable to makeuse of spatial information in the asymmet-ric distribution of these phospholipids. Theassociation might, however, provide a general,low-affinity cortical targeting function, whichcan be refined by further interactions withspecific proteins (Fig. 2). It is interestingthat Par-3 also interacts with the phosphoi-nositide phosphatase PTEN, which generatesPIP2 from PIP3, but whether this somehow

Widely Conserved Signaling Pathways

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regulates the lipid/Par-3 association is notknown (Wu et al. 2007; Feng et al. 2008).

A role for oligomerization: Oligomerizationof Par3 is also important for cortical targeting.The amino-terminal conserved region 1 (CR1)is necessary for self-association of Par-3 intohigher-order complexes (Benton and Johnston2003a; Mizuno et al. 2003; Feng et al. 2007).The CR1 domain is not sufficient for membraneattachment, because the isolated fragment isdiffuse throughout the cytoplasm. However, itis essential for membrane localization andfunction of Par-3 in both mammalian epithelialcells and Drosophila. It is also not clear howoligomerization of Par-3 maintains it at theplasma membrane, but oligomerization mightact to complement a weak phosphoinositidebinding (Fig. 2).

Anchoring to membrane proteins: Enrich-ment in a specific region of the cell cortex ismediated by direct interactions with proteinsvia the PDZ domains of Par-3. We refer to thisas anchoring, although of course the individualprotein molecules can be associating and disas-sociating from their attachment site in a highlydynamic fashion. In mammalian epithelia,cell–cell junctions can act as a primary land-mark that precedes the establishment ofpolarity. Initial contacts between adjacent cellsform through the Ca2þ-dependent dimeriza-tion of E-cadherin, as well as via nectin, toform primordial junctions. Through ZO-1,nectin recruits another Ca2þ-independent adhe-sion molecule, junction adhesion molecule(JAM), to junctions (Fukuhara et al. 2002).Par-3 is recruited to junctions through a direct

JAM

JAM

PP1a

PIP2/3

Par1

Par1

PIP2/3

Kif3

Microtubules

1. Delivery to cortex

P

2. Oligomerization and weak association with membrane via phospholipids

3. Specific association with tight junctions via transmembrane anchors

4. Release from cortex by Par-1mediated phosphorylation and 14-3-3 (Par-5) binding

Par5

P

Par5

Figure 2. Mechanisms for the transport, cortical association, and anchoring of Par3 in mammalian cells. It is notyet known if all of these mechanisms operate in any one cell type, and additional processes, such as RNAlocalization, might conceivably play roles in certain circumstances. Junctional adhesion molecule (JAM) isshown as an example of a transmembrane protein to which Par-3 can be anchored, but others undoubtedlyexist, such as the neurotrophin receptor, p75NTR, in mammalian Schwann cells (Chan et al. 2006).





interaction between the first PDZ domain ofPar-3 with the carboxy-terminal PDZ-bindingmotif of JAM (Fig. 2) (Ebnet et al. 2000;Itoh et al. 2001). As the primordial junctionsmature, they separate into distinct junctions,lateral adherens junctions, and more apicaltight junctions. Because Par-6 also binds to thefirst PDZ domain of Par-3, its association ispresumably mutually exclusive with that ofJAM, but no competition has been reported.A similar mechanism occurs in the mouseneuroepithelium, where Par-3 is recruited toearly junctions; however, in this case, Par-3 isapparently recruited through a direct inter-action with nectin-1 or nectin-3, and Par-3does not bind to JAM (Takekuni et al. 2003).In the Drosophila wing disc epithelium, Par-3is also recruited to junctions through its PDZdomain, by interacting with the adherensjunction proteins b-catenin and Echinoid,an immunoglobulin-domain transmembraneprotein (Wei et al. 2005).

Localized mRNA translation: To date, thereare no reports of localized Par-3 mRNA, butthe transcripts for two other polarity genes,Crumbs and Stardust, are enriched in theapical region of Drosophila epithelial cells(Horne-Badovinac and Bilder 2008; Li et al.2008). Both proteins are exclusively apical, arerequired for epithelial apical/basal polarity,and form a complex with one another. It is ofparticular interest that the Crumbs transcriptis localized because Crumbs is a transmembraneprotein that would normally have to transitthe Golgi to be delivered in vesicles to theapical surface. An interesting speculation isthat Crumbs is delivered by a different mechan-ism, to initiate apicalization, and that it sub-sequently directs the delivery of other apicalproteins to maintain apical identity.

Stardust is a scaffold protein that canassociate with Par-6 and a poly-PDZ domainprotein called Patj. The mammalian homologis called Pals1 (Roh et al. 2002). An alternati-vely spliced coding exon of Stardust containsthe mRNA localization signal, which enablesthe transcript to be transported in a dynein-dependent manner to the apical region(Horne-Badovinac and Bilder 2008). Dynein

is also important for Crumbs mRNA localiza-tion, and is essential for apico–basal polariza-tion in the follicle epithelium. Strikingly, theStardust splice variant is only produced earlyin development, whereas in mature epithelia,the mRNA is not localized. The physiologicalsignificance of this change is not yet under-stood, and it is not known if similar mecha-nisms pertain to the mammalian homologsfor these polarity proteins.

Active exclusion: None of the mechanismsdescribed above stably anchors Par-3 at theplasma membrane. FRAP experiments showthat the protein is highly dynamic in Drosophilaembryonic cells (Mayer et al. 2005), and it isquickly released from tight junctions on digi-tonin permeabilization of MDCK cells (un-published observation). Therefore, the targetingand retention mechanisms for Par-3 describedabove are insufficient to maintain its polar-ized distribution within the cell. Active mecha-nisms have been discovered, however, thatprevent Par-3 from accumulating in inappropri-ate areas of the cortex. The Par complex, consist-ing of Par-3, Par-6, and aPKC is often localizedin a complementary pattern to that of Par-1,and through mutual phosphorylations, the twokinases, aPKC and Par-1, exclude each otherfrom the complementary region of the cortex.Par-1 directly phosphorylates Par-3 (Bentonand Johnston 2003b), and these phosphorylatedresidues act as docking sites for 14-3-3 (alsoknown as Par-5), which binds and destabilizesthe membrane association of Par-3 (Fig. 2).This mechanism can therefore exclude Par-3(and associated aPKC and Par-6) from corticalregions that contain Par-1. Mammalian Par-3is phosphorylated by Par-1 on serine 144(S144, which is the same as S151 in Drosophila)(Hurd et al. 2003a). S144/S151 are located nearCR1, which is necessary for oligomerization ofPar-3. Binding of 14-3-3 to S151 inhibits self-oligomerization (Benton and Johnston 2003b),which may reduce phosphoinositide associ-ation, causing the protein to dissociate fromthe plasma membrane (Fig. 2).

Conversely, aPKC can phosphorylate Par-1,which both inhibits Par-1 kinase activity anddisassociates it from the plasma membrane.





Two distinct mechanisms have been identified,one in which aPKC directly phosphorylatesPar-1 on Thr 595 (T595) (Hurov et al. 2004),and an indirect mechanism by which aPKC acti-vates protein kinase D (PKD), which phosphor-ylates Par-1 on Ser 400 (S400) (Watkins et al.2008). Phosphorylation of T595 reduces thekinase activity and displaces Par-1 from themembranes. Furthermore, phosphorylation ofS400 recruits 14-3-3, which displaces Par-1from the membrane. The phosphorylation andbinding of 14-3-3 to Par-3 can be reversed byprotein phosphatase 1a (PP1a) (Traweger et al.2008), allowing recycling of Par-3 to appropriatecortical sites. This spatial antagonism probablyoperates in the C. elegans zygote to maintainthe distinct anterior–posterior distributions ofPar-3/aPKC/Par-6 and Par-1, as well as in epi-thelial cells of Drosophila and mammals, andmay be a conserved and widely applied methodfor excluding proteins from specific areas ofthe cell cortex. For example, the cell fate deter-minants Numb and Miranda, and the polarityprotein Lgl, are also removed from the plasmamembrane by aPKC-dependent phosphory-lations (Betschinger et al. 2003; Smith et al.2007; Atwood and Prehoda 2009; Prehoda2009). An Arf-GAP that is involved in conver-gent extension during Xenopus gastrulation isphosphorylated by aPKC/Par-6, which mayalter its subcellular distribution (Hyodo-Miuraet al. 2006). We predict that manyotherexamplesof the same mechanism occur, involving notonly aPKC and Par-1, but other protein kinasesthat target 14-3-3 consensus sites.

POLARITY SIGNALING THROUGHPAR-3/PAR-6/APKC

Signaling through small GTPases: There aretwo intrinsically asymmetric components ofcells: microtubules and actin filaments (seeMullins 2009). Both are vectorial polymers,and their organization is, therefore, of funda-mental importance to cell polarization. Thisorganization is dynamic and is highly regulatedby signaling networks that respond to bothexternal and internal cues. Central to these sig-naling networks are the Rho family GTPases,

which cycle between GTP-bound (on) andGDP-bound (off ) states and function as timedmolecular switches. It is not too surprising,therefore, that polarity proteins both regulateand are regulated by these GTPases. Given theenormity of the cytoskeleton and Rho researchfields, we will focus on those aspects that arerelated to Par proteins.

The Cdc42 GTPase was first identified inSaccharomyces cerevisiae, where it controls budsite establishment (Johnson 1999). Landmarkproteins that localize to the future bud siterecruit both Cdc42 and a guanine nucleotideexchange factor (GEF) that catalyzes the for-mation of the GTP-bound state of Cdc42(Chang and Peter 2003; see Slaughter et al.2009). In a classic feedback loop, Cdc42-GTPcan in turn recruit an adapter protein that stabil-izes the GEF and further enriches Cdc42 at thebud site (Butty et al. 2002). An unlocalizedGTPase activating protein (GAP) switches offany Cdc42-GTP that diffuses away from thebud site, helping to maintain its polarized local-ization. Additionally, the Cdc42-GTP stimulatesactin polymerization at the bud site, which canalso recruit more Cdc42 (Wedlich-Soldneret al. 2003). The anchored actin filaments thentransport vesicles that deliver membrane pro-teins for growth of the new bud.

Cdc42 as an upstream regulator of Par-6/aPKC: Cdc42 is a pivotal component of thepolarity machinery in yeast, and its conserva-tion throughout the evolution of the metazoaled to the expectation that it would playsimilar roles in animal development. Indeed,the injection of dominant–negative Cdc42mutants disrupted polarized migration inmammalian fibroblasts, and the discoverythat Cdc42-GTP binds to Par-6 provideda likely mechanism by which the GTPasemight control cell polarization. Par-6 containsa partial CRIB domain, which is conservedamong most of the downstream targets ofCdc42, and interacts with a region of theGTPase that undergoes a GTP-dependentswitch in conformation (Garrard et al. 2003).It is still unclear, however, exactly how Par-6behaves as an effector for Cdc42. One functionis to relieve the inhibition on aPKC activity





exerted by Par-6. Cdc42 can in this wayactivate aPKC. Recently, EphrinB1 was foundto compete with Cdc42 for binding to Par-6,blocking tight junction formation (Lee et al.2008). Tyrosine phosphorylation of the Ephrinreleases it from Par-6. In this way, signalingthrough tyrosine kinase receptors could impactaPKC activity and consequent cell polaritydecisions.

In addition to counteracting the inhibitionof aPKC by Par-6, Cdc42 can also recruit thePar-6/aPKC complex to specific regions of thecell cortex where the GTPase is activated (seePrehoda 2009). For example, in Drosophilaneuroblasts, Cdc42 mutants mislocalize Par-6/aPKC to the cytoplasm (Atwood et al. 2007),and in mammalian epithelial cells, knockdownof Cdc42 can partially mislocalize aPKC fromthe apical cortex (Martin-Belmonte et al.2007). However, robust Cdc42 localization inneuroblasts also requires Par-6, suggesting theexistence of a positive feedback loop thatreinforces their positioning. Moreover, thereare other mechanisms for aPKC and Par-6 local-ization: A dynamin-associated protein, Dap160,can bind to aPKC and stimulate its kinaseactivity, and in neuroblasts dap160 mutants,the aPKC is delocalized from the apical cortex(Chabu and Doe 2008). In the C. eleganszygote, Cdc42 is not essential for the initialanterior enrichment of Par-6, though the asym-metry is lost later during the first cell division,suggesting that other factors set up the polarity(Aceto et al. 2006); whereas in mammalianepithelial cells, Par-6 localization to the apicalsurface probably involves its association withPals1 and/or Crumbs, rather than Cdc42(Gao et al. 2002a; Hurd et al. 2003b).

Another member of the GTPase family,Rho-1, has been implicated in this process.Following fertilization of the C. elegans oocyte,a GAP called CYK-4 has been reported toassociate with the male pronucleus at the pre-sumptive posterior cortex and locally inacti-vates Rho-1 (Jenkins et al. 2006). However,CYK-4 is also a component of a complexinvolved in cytokinesis, and in this situation,at least, it is specific for Rac inactivation ratherthan Rho-1 (Canman et al. 2008). Two other

RhoGAPs are present in C. elegans but seem tohave a distinct function from CYK-4 becausethey control the ratio of the anterior/posteriordomain rather than its formation (Schmutzet al. 2007; Schonegg et al. 2007). Rho-1 GTPnormally activates a downstream kinase thatphosphorylates myosin light chain, increasingcontractility, but the accumulation of the GAPdepletes Rho-GTP and relaxes actomyosinat the posterior cortex. In addition, a GEF forthe Rho-1 GTPase is excluded from the pos-terior cortex, restricting Rho-GTP produc-tion to the anterior end of the zygote (Motegiand Sugimoto 2006). The Rho-GTP stimulatesanterior myosin contractility, which generatesa cortical actin flow, translocating Par-6,aPKC, Par-3, and Cdc42 to the anterior end ofthe zygote. Cdc42 then maintains this distri-bution of the Par proteins.

The Rho GTPase may play a distinct rolein mammalian cells by controlling the associ-ation of Par-3 with Par-6/aPKC. Nakayamaet al. found that a protein kinase downstreamof RhoA (ROCK) can phosphorylate Par-3on a threonine residue (T833) adjacent to theaPKC-binding site in the carboxyl terminusof Par-3, and that this phosphorylationblocks the association with aPKC and Par-6(Nakayama et al. 2008). However, this site isnot conserved in C. elegans.

A key remaining question concerns howCdc42-GTP is localized. Presumably, a GEFactivates the GTPase in a temporally or spatiallyrestricted manner to ensure that Par-6 recruit-ment is localized correctly. Spatially restrictedGAPs could also function to exclude Cdc42-GTP from certain regions of the cell, by con-verting it to the GDP-bound state. Indeed,during radial polarization in the early C.elegans embryo, a GAP called PAC-1 localizesto the basolateral membranes, whereas Par-6is restricted to the contact-free cell surfaces,most likely because Cdc42-GTP is destroyedby the PAC-1 at other surfaces (Andersonet al. 2008; see Munro and Bowerman 2009).In MDCK cells, annexin2, a Ca-dependent reg-ulator of actin dynamics, has been proposedto recruit Cdc42-GTP to the apical surface(Martin-Belmonte et al. 2007), but it remains





unclear how the annexin-bound Cdc42 couldalso bind Par-6. Nonetheless, this model couldpotentially dispense with a localized GEF orGAP. So far, no Cdc42-specific GEF has beenimplicated in Par-6 recruitment for any organ-ism, but RNAi screens are likely to resolve thisissue in the near future.

Rho GTPases as downstream effectors ofPar-3/Par-6/aPKC: Because actin dynamicsplays a key role in cell polarization and is regu-lated by Rho GTPases, one might expect theseGTPases to be modulated in some way by Parproteins. At least in mammalian cells, this ideahas been validated by several laboratories,which have identified a number of differentmechanisms (Fig. 1). First, the carboxy-terminalregion of Par-3 has been shown to bind to a RacGEF, called Tiam1 (Chen and Macara 2005;Mertens et al. 2005; Nishimura et al. 2005;Zhang and Macara 2006). Under some circum-stances, including the formation of tight junc-tions by MDCK epithelial cells, and theformation of excitatory synapses by hippocam-pal neurons, Par-3 sequesters Tiam1 to preventits inappropriate activation of Rac (Chen andMacara 2005; Zhang and Macara 2006). Thesilencing of Par-3 in these contexts causes a con-stitutive increase in Rac-GTP levels, which inturn disrupts junction and synapse formation.Both of these processes are actin-mediated, andpresumably the inappropriate activation of Raccauses misorganization of actin filaments atthe cell cortex. In other cell types, however,Par-3 might recruit Tiam1 to sites within thecell where Rac needs to be activated. In suchcases, loss of Par-3 might reduce Rac activationat these sites. For example, silencing of Par-3or Tiam1 expression in keratinocytes reducestheir ability to sustain polarized migration(Pegtel et al. 2007). Cdc42 might regulateRac activation through association with thePar-6/aPKC/Par-3 complex, perhaps byactivat-ing aPKC to phosphorylate Par-3 or Tiam1.Whatever the case, Par-3 can be defined as ascaffold protein that recruits signaling com-ponents to specific locations in the cell, enablingspatial control of the signal output.

Through quite distinct mechanisms, Par-6can regulate RhoA activity. First, via its

association with aPKC, Par-6 can activate aRhoGAP called p190, thereby reducingRho-GTP levels (Zhang and Macara 2008).This pathway is important in controllingsynapse density in hippocampal neurons.Elevated expression of Par-6 increases dendriticspine density, whereas silencing of Par-6 reducesspine density. However, the molecular linkagebetween Par-6/aPKC and the p190 RhoGAPis still unknown.

A second, different mechanism by whichPar-6 can affect Rho is through associationwith Smad-ubiquitin regulatory factor 1(Smurf1), an E3 ligase that down-regulates theTGFb signaling pathway (Fig. 1). In additionto targeting Smads for degradation, Smurf1can also ubiquinate RhoA (Wang et al. 2003).TGFb type II receptor can—at least in somecell types—bind to and phosphorylate Par-6on a conserved carboxy-terminal serine residue(S345) (Ozdamar et al. 2005). This phosphoryl-ation appears to stimulate the association ofPar-6 (or aPKC) with Smurf1, which mediatesthe localized ubiquitination and destructionof RhoA. In NMuMG cells, loss of RhoAcan cause the dissolution of tight junctionsand an epithelial–mesenchymal transition.However, these effects might be cell-typespecific because, for example in MDCK cells,dominant–negative RhoA expression had noeffect on tight junctions (Bruewer et al. 2004).

Cross talk between Wnt signaling and Parproteins: Planar cell polarity signaling is sti-mulated by Wnt ligands that activate seventransmembrane receptors in the Frizzledfamily (Simons and Mlodzik 2008; see Vladaret al. 2009). Downstream of these receptors,an adapter protein called Dishevelled (Dvl)can activate RhoA and the downstream kinaseROCK, which, as described above, phosphory-lates Par-3. In addition, however, Dvl is atarget for phosphorylation by Par-1, and candirectly associate with aPKC (Sun et al. 2001;Ossipova et al. 2005). The association withaPKC stabilizes and activates the kinase, andhas been implicated in axonal differentiationand polarized migration of mammalian cells(Schlessinger et al. 2007; Zhang et al. 2007).Moreover, the Drosophila Frizzled receptor is





phosphorylated and inhibited by aPKC, whichis recruited to Frizzled through another polarityprotein, Patj (Djiane et al. 2005). During planarcell polarity signaling, aPKC is down-regulated,whereas Par-3 expression is elevated. Clearly,there are multiple mechanisms for cross talkbetween planar cell polarity and Par polaritysignaling pathways, and a deeper understand-ing of these interactions will be an importantfocus for future work.

Signaling through protein kinases: Most sig-naling pathways use protein kinases either toamplify, attenuate, or direct information. It isnot surprising, therefore, to find that phos-phorylation plays an important role in regulat-ing the association of Par-3 and Par-6 with theireffectors and with the cell cortex (Figs. 1–3).

Several serine/threonine kinases target Par-3,and many of these phosphorylations are con-served throughout the animal kingdom. As dis-cussed above, Par-1 phosphorylates a conservedSerine in the carboxyl terminus of Par-3, pre-venting oligomerization and inducing disasso-ciation from the membrane (Fig. 2) (Bentonand Johnston 2003b); and aPKC phosphory-lates a conserved Serine in the carboxyl termi-nus of Par-3, releasing aPKC from its adjacentbinding site (Nagai-Tamai et al. 2002). Untilrecently, the function of this latter phos-phorylation was obscure, but recent studiesin mammalian cells, using knockdown andreplacement of Par-3 with nonphosphory-latable mutants, has shown that the Par-3/aPKC interaction is essential for Par-3 function(Horikoshi et al. 2009; McCaffrey and Macara2009). In mammary morphogenesis, a Par-3mutant that is unable to bind aPKC is mis-localized and cannot rescue normal ductalgrowth in the absence of the endogenousPar-3 (McCaffrey and Macara 2009). In addi-tion, the Rhoactivated kinase, ROCK, can phos-phorylate a nearby Threonine, also disruptingaPKC binding (Nakayama et al. 2008). How-ever, an interaction between Par-6/aPKC isstill possible with Par-3 through its PDZdomains (Fig. 3). We speculate that after Par-3has recruited Par-6/aPKC—to the tight junc-tion in mammalian epithelial cells—the phos-phorylation and release somehow enables

Par-6/aPKC to be handed off to an apicalpolarity complex consisting of Crumbs, Pals1,and Patj (Fig. 3). This complex is essential forapical membrane establishment, and for tightjunction formation (Roh et al. 2003; Straightet al. 2004; Shin et al. 2005). Par-6 can interactdirectly with all three members of the complexin a mutually exclusive manner. The interactionbetween Par-6 with Pals1 inhibits the inter-action of Pals1 with Patj; however, the biologicalsignificance of this effect has not yet beenestablished.

Importantly, the mitotic regulatory kinaseAurora-A can phosphorylate Par-6 (Fig. 3)(Wirtz-Peitz et al. 2008). This is a key step inensuring asymmetric stem cell divisions in theDrosophila embryo. At the onset of mitosis,Aurora-A phosphorylates Par-6 within theamino-terminal PB1 domain that binds toaPKC, releasing Par-6 and relieving its inhi-bition of aPKC (see Prehoda 2009). The acti-vated aPKC phosphorylates Lgl, which causesit to dissociate from the Par-6/aPKC complexand allows the interaction of the complex withPar-3. Par-3 then acts to recruit Numb as a sub-strate for aPKC. Phosphorylation of Numb byaPKC causes it to be released from the cellcortex (Smith et al. 2007; Wirtz-Peitz et al.2008). Because the Par complex is initially asym-metric, the loss of Numb occurs at only one sideof the mitotic cell, so one daughter will inheritNumb whereas the other does not. Numb nega-tively regulates the cell fate-determining tran-scription factor Notch, ensuring that only onedaughter cell responds to Notch signaling.Interestingly, the Lgl polarity protein seemshere to function as a buffer, to suppress thephosphorylation of Numb until the appropriatetime in the cell cycle. The recruitment of Numbby Par-3 seems to be a conserved function,because in migrating mammalian fibroblaststhe same mechanism is used to release Numbfrom the cell cortex, thereby regulating theinternalization of integrins (Nishimura andKaibuchi 2007). It will be interesting to knowwhether buffering of signaling components isthe principal function of Lgl and if it is impor-tant for controlling Numb phosphorylationduring cell migration.





The Par complex can also regulate down-stream protein kinases. As discussed previously,Par-6 inhibits aPKC, whereas aPKC phosphor-ylates and inhibits the Par-1 kinase, and triggersits disassociation from the cell cortex, therebyexcluding it from the apical domain (Hurovet al. 2004; Kusakabe and Nishida 2004). Aregion in the carboxyl terminus of Par-3 bindsto the LIMK2 protein kinase, and attenuatesLIMK2 kinase activity (Chen and Macara2006). The principal function of LIMK2 is tophosphorylate and inhibit cofilin, an actin-severing protein that is essential for actin remo-deling during junction formation (Ivanov et al.2004, MBC), so Par-3 can locally alter actindynamics through this mechanism.

Tyrosine kinases in particular have diversi-fied in higher organisms and undertake manyroles throughout development. Par-3 is atarget of Src family tyrosine kinases, in responseto epidermal growth factor (EGF) receptor acti-vation (Wang et al. 2006). Phosphorylationof Par-3 on a tyrosine residue in the carboxy-terminal region (Y1127) delays tight junctionassembly in mammalian epithelial cells. Thiseffect has been ascribed to a reduction in theassociation of Par-3 with LIMK2 (Wang et al.2006). It is not yet clear how universal thismechanism is, however, because constitutiveSrc activation, or EGF treatment, causes anepithelial–mesenchymal transition and theloss of tight junctions, which is the opposite

aPKC Par6

aPKC

P

Par6

aPKC

P

Crb/Pals1

Par6

aPKC

PP

P

Par6 Lgl

Par6

AurA

Lgl

P

Par6

Cdc42

Par6

aPKC

LglCdc42

Lgl

P

P

Par6

Numb

Par6

NumbaPKC

aPKCaPKC

aPKC

aPKC

Lgl

Figure 3. A model for the interactions of the Par complex with other polarity proteins and protein kinases.This model synthesizes data from a number of laboratories, and attempts to reconcile observations thataPKC can bind directly to Par-3, but also can bind indirectly through Par-6. The direct association isinhibited by phosphorylation; whereas the indirect association is inhibited by competition for Par-6 by theCrumbs/Pals1 complex and by the Lgl polarity protein. In addition, the schematic shows Par-3 functioningto recruit other targets for aPKC, such as Numb, and the regulation of Par-6 by the Cdc42 GTPase and theAurora A kinase.





of what would be predicted from these data.Nonetheless, high-throughput phosphoproteo-mics screens have identified multiple tyrosinephosphorylation sites in the carboxyl terminusof Par-3, and Par-6 can associate (indirectly)with ErbB2 (Aranda et al. 2006), suggestingthat there may be other inputs into the polaritycomplex that have yet to be understood.

In this regard, another study has also iden-tified a link between the Par proteins andSrc kinase, although with the opposite effectof destabilizing cell adhesions. In a stableblood-testis-barrier, Par-6 is in a complex withPals1 and JAM-C at cell–cell adhesions (Wonget al. 2008). During adhesion remodeling,however, Pals1, presumably with Par-6, associ-ates with Src and is sequestered away fromJAM-C, which weakens the cell–cell junctions.In this system, Par-3 and Par-6 appear tocooperate in regulating some aspects of cell–cell adhesion. Depletion of Par-3, but notPar-6, disrupts tight junction formation,whereas depletion of Par-6 reduces N-cadherinlevels. Depletion of either Par protein causes dis-ruption of JAM-A at adhesions. It would beinteresting to determine whether Par-3 is partof the Par-6/Pals1/Src complex and whetherPar-3 is tyrosine phosphorylated in this system,which might weaken cell–cell adhesionsduring blood-testis-barrier remodeling.

Phosphoinositides as polarity signals: Thefirst clear demonstrations that phosphoino-sitides function in cell polarity arose fromstudies on the chemotaxis of Dictyosteliumand mammalian neutrophils (see Orlando andGuo 2009). These cells can translate extraordi-narily shallow chemoattractant gradients intosteep front–rear polarity, and phosphoinosi-tides play a crucial role in establishing thispolarity, with high levels of the lipid kinasePI-3K and its product, PIP3, accumulating atthe leading edge where the highest chemoattrac-tant concentration is sensed (Devreotes andJanetopoulos 2003; Charest and Firtel 2006).The lipid phosphatase PTEN is excluded fromthe leading edge, but is associated with thelateral and rear plasma membranes, leading torestricted PIP3 at the leading edge of migratingcells (Charest and Firtel 2006). Somewhat

surprisingly, however, there is no evidence thatthe Par polarity proteins play any role in chemo-taxis by these actively migrating cells. Yet, inother contexts, Par proteins do seem to interactwith the phosphoinositide signaling pathway.For example, the PDZ domains of Par-3bind directly to PTEN (Feng et al. 2008).In Drosophila photoreceptors, Par-3 recruitsPTEN to the nascent zonula adherens and pro-motes conversion of PIP3 to PIP2 in that mem-brane domain (Pinal et al. 2006). It has beenreported that mammalian Par-3 recruits PTENto cell–cell contacts in MDCK cells (Fenget al. 2008), but Par-3 is almost exclusively loca-lized to tight junctions in these cells, and PTENis mostly apical. In C. elegans, the Par proteinsmediate enrichment of a phosphoinositidekinase (PI-4P kinase) at the posterior of theone-cell embryo, which is required for normalspindle movements (Panbianco et al. 2008).Interestingly, this asymmetric localization alsodepends on casein kinase, which is enrichedat the opposite, anterior end of the zygote.Presumably, the PI-4PK generates PIP2 at theposterior, which somehow interacts withthe machinery involved in aster microtubuleattachment to the cortex. However, the under-lying mechanisms by which Par proteins regu-late the PI-4K and casein kinase distributionsremain to be discovered.

We should also consider the possibility thatPar proteins operate downstream of phospho-inositide signaling. For example, Par-3 PDZdomain 2 can bind directly to phosphoino-sitides (Wu et al. 2007), and, in the Drosophilaoocyte, loss of phosphatidylinositol-5 kinaseactivity, which produces PIP2 at the plasmamembrane, causes mislocalization of Par-3,Par-1, and Lgl (Gervais et al. 2008). However,this effect might be quite indirect, because themicrotubule and actin cytoskeletons are alsodisorganized (Gervais et al. 2008).

There is substantial evidence that thephosphoinositides play a central role in defin-ing polarized membrane domains. Spatiallyrestricted activation of PI3-kinase can locallygenerate its product, PIP3, which may recruitand regulate downstream effectors that canbind to this lipid through a plekstrin homology





(PH) domain. PTEN can counteract the effectof PI3-K by dephosphorylating PIP3 to PIP2.In neurons, local activation of PI3-kinaseat the growing tips of neurites induces thegrowth of axons (Shi et al. 2003; Menageret al. 2004), whereas overexpression of PTENdisrupts the polarization of the axon. Elegantexperiments in MDCK epithelial cells haverecently highlighted the key role played byphosphoinositides in specifying membraneidentity (Gassama-Diagne et al. 2006; Martin-Belmonte et al. 2007; Martin-Belmonte andMostov 2008). These cells display a highly asym-metric distribution of these lipids, in whichPIP3 is completely excluded from the apicalsurface but is abundant on the basolateralsurface, whereas PIP2 is present throughoutthe plasma membrane but is enriched at theapical surface. This spatial asymmetry resultsfrom the localization of PI3-kinase to adherensjunctions, via binding to E-cadherin (Pece et al.1999), whereas the phosphatase PTEN isrestricted to the apical surface. Remarkably,the addition of exogenous PIP3 to the apicalsurface of such cells transiently induces apatch of membrane with basolateral character-istics, whereas apical markers are excluded(Gassama-Diagne et al. 2006). Conversely,addition of exogenous PIP2 directly to the baso-lateral surface of MDCK cysts relocalizes apicalmarkers to this region. Mostov and colleagueshave proposed that Annexin-2 binds to PIP2 atthe apical surface and recruits or activatesCdc42, which in turn recruits Par-6/aPKC(Gassama-Diagne et al. 2006). However, as dis-cussed previously, there are other mechanismsthat recruit Par-6/aPKC to the apical surfaceindependently of Cdc42, including Pals1binding. Moreover, the proposed mechanismis apparently independent of Par-3, yet Par-3 isknown to be required for apical localizationof aPKC (Hirose et al. 2006; McCaffrey andMacara 2009), so there are key aspects to thissystem that have yet to be understood. Anadditional puzzle is that in Drosophila retinalepithelial cells, the phosphoinositide distri-bution is the inverse of that seen in MDCKcells, such that PIP3 is concentrated on theapical surface, where it activates the protein

kinase Akt. Par-3 recruits PTEN to the cell–cell junctions, leading to destruction of PIP3

along the lateral membrane (Pinal et al. 2006).An important goal for future studies is to under-stand the cross talk between the Par proteinsand localized phosphoinositide metabolism.

CONCLUDING REMARKS

Understanding cell polarization is one of themajor goals of cell biology and will inevitablyhave a broad impact not only at the level ofbasic science but also in understanding diseasessuch as cancer and neurological degeneration.Work from many laboratories has uncovered acomplicated web of signaling systems that sur-round and intersect with the Par proteins, yetwe still understand very little about what thePar proteins do, how they are localized, howtheir various interactions are regulated, andwhich signaling components operate in whichcontexts. After all, the organization of a polar-ized cell is a formidably complicated processthat involves cytoskeletal remodeling, mem-brane traffic, RNA localization, and proteincomplex assembly and disassembly, with feed-back to gene expression and protein turnover.It is conceivable that the Par proteins participatein all of these processes, either directly orindirectly. It is worth remembering, however,that although the polarity machinery canwork in a cell autonomous fashion, the Pargenes do not exist, as far as is known, in any uni-cellular organism, which suggests that a key rolefor the Par proteins is to facilitate, mediate, orinterpret cell–cell and cell-matrix interactions.The tissue context might, therefore, be expec-ted to modulate Par protein functions. Evenin vitro, the context can exert importantinfluences. For example, Par-6 and aPKC areconcentrated at tight junctions of epithelialcells grown in two-dimensional cultures, butare spread over the apical surface in three-dimensional cultures. Within the much morecomplicated environment of an organism,such influences are likely to be widespread andmight alter signaling inputs to and outputsfrom the Par proteins. It will be of interest in





the future to determine whether these effectscontrol morphogenesis, and to what extentdifferential Par function contributes to pheno-typic variation both between different celltypes in one organism, and between species.

It will also be important to understandwhy Par genes have multiplied in the vertebratelineage. For example, vertebrates possess threeisoforms of Par-6 (Joberty et al. 2000). Theyall possess the same core properties, and—at least in cultured mammalian neurons—areinterchangeable (Zhang and Macara 2008).Yet, they are differentially expressed, possesssubtle biochemical distinctions (Gao andMacara 2004), and in zebrafish, are biologicallyunique (Munson et al. 2008). There are alsomultiple splice variants of the Par proteinsthat presumably operate differentially incertain contexts, but we are a long way fromunderstanding their physiological significance(Gao et al. 2002b). As an example, the chimpan-zee (and probably other mammals) expresses atleast 24 splice variants of Par-3. Some of theselack only a few amino acids, whereas otherslack entire domains. Various splice variants inthe mouse are differentially expressed, but wedo not know why. We lack sufficient data tofully understand Par proteins even in thesimpler metazoa, let alone in vertebrates. Forthis reason, the use of diverse model organismswill continue to be essential into the foreseeablefuture.

ACKNOWLEDGMENTS

Work in our laboratory was supported by aresearch grant from the National Institutes ofHealth (GM090702) to I.M. L.M.M is therecipient of a Postdoctoral fellowship from theCanadian Institutes of Health Research.

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2009; doi: 10.1101/cshperspect.a001370Cold Spring Harb Perspect Biol Luke Martin McCaffrey and Ian G. Macara PolarityWidely Conserved Signaling Pathways in the Establishment of Cell

Subject Collection Symmetry Breaking in Biology

SymmetryCytoskeletal Mechanisms for Breaking Cellular

R. Dyche MullinsCell Division in Tissue HomeostasisPolarity in Stem Cell Division: Asymmetric Stem

al.Yukiko M. Yamashita, Hebao Yuan, Jun Cheng, et

Symmetry Breaking in BiologyRong Li and Bruce Bowerman Budding Yeast

Symmetry Breaking in the Life Cycle of the

Brian D. Slaughter, Sarah E. Smith and Rong Li

Cell's CompassPlanar Cell Polarity Signaling: The Developing

AxelrodEszter K. Vladar, Dragana Antic and Jeffrey D.

Neuronal PolaritySabina Tahirovic and Frank Bradke

Cellular Polarity in Prokaryotic OrganismsJonathan Dworkin Polarity

Membrane Organization and Dynamics in Cell

Kelly Orlando and Wei Guo

ArabidopsisDivisions in Mechanisms Regulating Asymmetric Cell Symmetry Breaking in Plants: Molecular

Philip N. BenfeyJalean J. Petricka, Jaimie M. Van Norman and

DevelopmentCaenorhabditis elegansCellular Symmetry Breaking during

Edwin Munro and Bruce Bowerman

Polarity and ChemotaxisThe Signaling Mechanisms Underlying Cell

Fei Wang

OogenesisDrosophilaSymmetry Breaking During Siegfried Roth and Jeremy A. Lynch

Asymmetric Division Neuroblasts DuringDrosophilaPolarization of

Kenneth E. PrehodaEstablishment of Cell PolarityWidely Conserved Signaling Pathways in the

Luke Martin McCaffrey and Ian G. MacaraPhysical Model of Cellular Symmetry Breaking

Jasper van der Gucht and Cécile SykesShaping Fission Yeast with Microtubules

Fred Chang and Sophie G. Martin

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