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Regulation of Par6 by extracellular signals
Regulation of Par6 by extracellular signals Rohit Bose1,2 and Jeffrey L Wrana1,2 The critical regulator of polarity, Par6, is a key member of a multi-component polarity complex that controls a variety of cellular processes such as asymmetric cell division, establishment of epithelial apico-basal polarity, and polarized cell migration. Recently, we have come to understand how regulation of the Par6 interactome by extracellular cues such as integrin and transforming growth factor b signalling regulates cell motility and tight junction dissolution. These studies have begun to elucidate how signalling to the polarity complex might regulate pathological processes such as tumour cell invasion and metastasis. Addresses 1 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada 2 Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada Corresponding author: Wrana, Jeffrey L ([email protected])
Current Opinion in Cell Biology 2006, 18:206–212 This review comes from a themed issue on Cell regulation Edited by Claude Prigent and Bruno Goud Available online 20th February 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.02.005
Introduction In recent years, major advances have been made in the identification of proteins that regulate cellular polarity. Numerous multi-protein polarity complexes have been shown to control processes such as epithelial apico-basal polarity, cell migration and asymmetric cell division [1–5]. However, to understand the dynamics of polarity, we must determine not only the composition of these complexes, but also how they are regulated to effect morphological changes. One critical regulator of polarity is partitioning-defective 6 (Par6). Par6 is an adapter protein that engages in many protein–protein interactions that are spatiotemporally regulated to control polarity. Here we review how Par6 functions as an effector of signal transduction pathways to control polarized cell migration and apico-basal polarity, particularly as a target of transforming growth factor b (TGFb).
The Par6 polarity complex Par6 was originally identified as a gene that regulates the asymmetric division of the C. elegans zygote [6], and over Current Opinion in Cell Biology 2006, 18:206–212
the past decade it has emerged as a key player in a plethora of cellular polarity processes in organisms ranging from worms to humans [1–4]. These include asymmetric cell division, neuronal polarity, apico-basal polarity of epithelial cells, and polarized cell movement. There are three mammalian Par6 isoforms, A, B and C, that all share the same protein–interaction domains. Little is known about their relative expression but mRNAs of all three are simultaneously expressed in polarized mammalian epithelial cells (R Bose, JL Wrana, unpublished). These isoforms display different subcellular localizations in polarized epithelia and thus may have distinct functions. Par6A predominantly co-localizes with tight junction markers, whereas Par6B is localized to the cytosol [7]. Par6C localizes to both tight junctions and the cytosol [7]. Par6 functions as part of a protein complex and contains three domains that enable it to interact with the other members of the complex (Figure 1). At the N terminus, there is the Phox/Bem1 (PB1) domain, which binds to other PB1-domain-containing proteins such as atypical protein kinase C (aPKC) [8,9]; next, there is the Cdc42– Rac interaction binding (CRIB) motif, which binds the Cdc42 or Rac GTPases strictly in their activated GTPbound state [8,9]; and finally, there is the PSD-95/Dlg/ ZO-1 (PDZ) domain, which can bind PDZ domains in other proteins such as Par3 [8,9]. Tandem affinity purification (TAP)-tagging of Par6 and subsequent mass spectrometry analysis of endogenous interactors has confirmed these interactions and revealed that Par6 is imbedded in an extensively interconnected network of protein–protein interactions (Figure 2) [10]. Par6 is thus a hub [11–13] in a large polarity network that is evolutionarily conserved from worms to mammals [1–4].
Par6 complex activation and polarized cell migration When cells engage in directed cell migration, the microtubule network is organized such that the microtubuleorganizing centre (MTOC) and Golgi apparatus are oriented between the leading edge and the nucleus [14]. This can facilitate the delivery of membrane precursors and cytoskeletal regulators towards the leading edge. During cell migration, MTOC polarization is regulated by the Cdc42–Par6–aPKC complex [14,15–17], but how exactly does the cell use spatial cues to control this complex and the orientation of the MTOC? One pathway involves the integrins, cell-surface receptors that regulate cellular motility. Integrin binding to extracellular matrix (ECM) components at the leading edge of the cell leads to their clustering in structures called focal complexes; this clustering activates their intracellular signalling pathways [18]. In migrating astrocytes, integrin signalling in www.sciencedirect.com
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Figure 1
Schematic of the domain structure of Par6. Par6 contains three protein interaction domains. At the N terminus is the Phox/Bem1 (PB1) domain, which binds to other PB1-domain-containing proteins such as atypical protein kinase C (aPKC); next, there is the Cdc42–Rac interaction binding (CRIB) motif, which binds the Cdc42 or Rac GTPases strictly in their activated GTP-bound state; and finally, there is the PSD-95/Dlg/ZO-1 (PDZ) domain, which can bind PDZ domains in other proteins such as Par3. The TGFb-dependent phosphorylation site, serine 345, is shown.
protrusions activates Cdc42, which in turn binds and activates aPKC scaffolded on Par6 at the leading edge (Figure 3) [15]. This link between Cdc42 binding and aPKC activation is supported by genetic and biochemical studies in the fly and worm, as well as mammalian cells [19,20,21]. Assembly of the Cdc42–Par6–aPKC complex
leads to phosphorylation of glycogen synthase kinase 3b (GSK3b) on its inhibitory serine 9 site. This, in turn, blocks adenomatous polyposis coli (APC) phosphorylation, thereby enabling APC to associate with the growing plus ends of microtubules [22]. This occurs concomitantly with Cdc42- and aPKC-dependent recruitment of Discs Large (Dlg), which interacts with microtubule-bound APC to reorient the MTOC in migrating cells, possibly by linking the leading edge of the plasma membrane to the microtubule network [23]. In addition to the orientation of cell migration, the Par6 polarity complex may regulate the actual movement of cells themselves. Migrating cells form cellular processes such as filopodia and lamellipodia in the direction of movement and the formation of these structures is regulated by the Cdc42 and Rac1 GTPases, respectively [24,25]. In contrast, the closely related GTPase, RhoA, regulates the formation of focal adhesions and actin stress-fibres. Coordinated, localized regulation of these GTPases is critical for effective cell movement [26]. Par6 also plays a role in this process, since it is localized to the tips of cellular protrusions where it can recruit the E3 ubiquitin ligase Smad ubiquitination regulatory factor-1 (Smurf1, originally identified as a negative regulator of TGFb-mediated Smad signalling), which can locally degrade RhoA [27]. This promotes protrusive activity by restricting the inappropriate localized activation of RhoA in Cdc42- or Rac-induced membrane protrusions.
Figure 2
Par6 is a hub in a polarity network. The Par6 protein-interaction network reveals that Par6 (orange) is part of a highly interconnected network that is shown as known physical interactions (lines) between Par6 and numerous Par6 partners represented as nodes. The core, evolutionarily conserved polarity complex is shown in green, components of the TGFb pathway in light blue, the apical CRB–PALS–PATJ complex in pink and the basolateral Lgl–Dlg–Scrib complex in yellow. OCLN, occludin. www.sciencedirect.com
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Figure 3
Par6 regulates polarized cell migration in response to extracellular cues. In migrating cells, integrins at the leading edge bind to ECM components, leading to the formation of focal complexes. This integrin clustering activates Cdc42, which in turn activates aPKC scaffolded on Par6. The Cdc42–Par6–aPKC complex leads to phosphorylation of GSK3b on its inhibitory serine 9 site, which blocks APC phosphorylation to allow APC interaction with membrane-localized Dlg. This latter interaction orients the MTOC towards the direction of movement. The Par6 polarity complex also coordinates the cross-regulation between Rho GTPases at the leading edge. The Cdc42–Par6–aPKC complex can bind and recruit the E3 ubiquitin ligase Smurf1 to locally degrade RhoA in cellular protrusions, thereby promoting protrusive activity.
Par6 may thus function to mediate cross-talk between Rho GTPases during cell migration.
Par6 and the regulation of epithelial polarity In addition to cell motility, Par6 has been shown to be a critical regulator of epithelial polarity. One of the structural hallmarks of a fully polarized epithelial cell is the presence of tight junctions, which confer apico-basal polarity to the cell [28]. Par6, which localizes to tight junctions, has been shown to regulate junctional stability [1–4], but the mechanisms are poorly understood. For example, overexpression of Par6 or activated Cdc42 inhibits tight junction formation [29], whereas the Par6 polarity complex can also promote tight junction formation by recruiting Tiam1, a Rac-specific guanine nucleotide exchange factor (GEF) [30]. Thus, Par6 can regulate both the loss and formation of tight junctions. Par6-dependent control of epithelial apico-basal polarity is mediated via its interactions with two other tri-protein complexes [1,3]. One consists of the transmembrane protein Crumbs (CRB), protein associated with lin-seven (PALS) and PALS1-associated tight-junction protein (PATJ). This complex is located apically to Par6 and genetic and biochemical data suggest that the two complexes cooperate to maintain each other’s subcellular localization and establish apical polarization [1,31–33]. Current Opinion in Cell Biology 2006, 18:206–212
In contrast, the second Par6-interacting complex, consisting of Lethal Giant Larvae (Lgl), Discs Large (Dlg) and Scribble (Scrib) is located basolaterally to the Par6 polarity complex, and is proposed to negatively regulate the establishment of the apical membrane [1,34,35]. Thus, the balance between opposing gradients of CRB–PALS–PATJ and Lgl–Dlg–Scrib complexes may restrict the Par6 complex to tight junctions, thereby coordinating the establishment of epithelial apico-basal polarity.
TGFb and Par6 TGFb is a growth factor that can serve as a tumour suppressor or a tumour promoter, depending on the situation [36]. TGFb signals through two transmembrane serine-threonine kinases, the type II (TbRII) and type I (TbRI) receptors. The classically described TGFb pathway begins with the binding of the TGFb ligand to the constitutively active TbRII, which in turn binds and phosphorylates TbRI. This activates the Smad pathway to regulate gene transcription. Many of the tumour suppressor functions of TGFb are mediated by transcription in this manner [37]. However, advanced tumours become refractory to TGFb-mediated growth inhibition and, in these tumours, TGFb is proposed to promote invasion and metastasis [36]. Interestingly, addition of TGFb to several polarized epithelial cell types causes them to www.sciencedirect.com
Regulation of Par6 by extracellular signals Bose and Wrana 209
Figure 4
acquire a fibroblastoid phenotype in a process known as epithelial-to-mesenchymal transition, or EMT [36]. This process involves the dissolution of epithelial cell–cell junctions and the loss of apico-basal polarity, as well as a change from an epithelial to a fibroblastoid gene expression program. Importantly, EMT is correlated with the progression of carcinomas to an invasive and metastatic state. TGFb-dependent EMT is known to require both Smad-dependent and Smad-independent pathways [38]. In particular, recent evidence indicates that TGFbdependent loss of tight junctions is mediated via Par6 phosphorylation by the TGFb receptor and appears to be independent of Smad-mediated transcriptional responses [39]. The mammalian-based LUMIER protein–protein interaction screen [40] was used to discover novel interacting partners of the TGFb type I receptor. Three of the identified proteins were: Par6C; occludin, a four-pass transmembrane protein that is a structural component of tight junctions; and p21-activated kinase 1 (PAK1), a Cdc42-activated kinase that is a key regulator of the actin cytoskeleton and cell migration [39,40]. Thus, the TGFb pathway can link the polarity complex with tight junction components and regulators of the actin cytoskeleton. Mouse mammary epithelial NMuMG cells can be used to study epithelial apico-basal polarity and tight junction homeostasis and undergo EMT upon TGFb stimulation. In fully polarized NMuMG cells, there are distinct distributions of cell-surface TGFb receptors: TbRII is localized to puncta distributed over the surface, whereas TbRI is strictly co-localized with tight junction markers [39]. Occludin is important for this restricted localization of TbRI [40], which may also involve other tetraspanin proteins such as the claudins. However, upon 30 min treatment with TGFb ligand, TbRII redistributes to the tight junctions, and this correlates with the observed ligand-dependant interaction of TbRII with occludin [40]. In tight junctions, Par6, which binds TbRI, is recruited to TbRII via its interactions with the type I receptor (Figure 4a). Within this complex, TbRII can directly phosphorylate Par6 on serine 345 (S345) (Figure 1, Figure 4b), which then remains bound to the receptor complex; this contrasts with the Smads, which dissociate upon phosphorylation. Preventing Par6 phosphorylation on serine 345 profoundly blocks TGFbdependent loss of tight junctions [39]. This process appears to be independent of Smad transcriptional regulation, although there remains the possibility that the Par6 polarity complex directly regulates Smad functions, as Smad3 has been shown to interact with Par3 [41]. Thus
The TGFb pathway. (a) Occludin (OCLN) restricts TbRI localization to the tight junctions, where it co-localizes with Par6. Upon TGFb stimulation, TbRII is recruited to the tight junctions and forms active TGFb receptor complexes. (b) TbRII phosphorylates Par6 on serine 345, stimulating www.sciencedirect.com
binding and recruitment of the E3 ubiquitin ligase Smurf1 to tight junctions. TbRII also phosphorylates and activates TbRI. (c) Smurf1 ubiquitinates and degrades RhoA, promoting tight junction dissolution, while activated TbRI can phosphorylate and activate the Smad transcriptional pathway. Together, these activities lead to TGFbdependent EMT. Current Opinion in Cell Biology 2006, 18:206–212
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active receptor complexes containing phosphorylated Par6 are assembled in tight junctions during TGFb signalling and are critical for EMT. How, then, does TGFb-mediated Par6 phosphorylation on S345 specifically control tight junction loss? The clues lie in how the Par6 polarity complex regulates cellular protrusions [27]. As described earlier, the Par6 polarity complex localized at the tips of fibroblast cellular protrusions recruits Smurf1, an E3 ubiquitin ligase belonging to the C2-WW-HECT family, to regulate localized RhoA degradation [27]. Similarly, in epithelial cells, Par6 is capable of binding and recruiting Smurf1 to tight junctions and this is dependent on TGFb-dependent phosphorylation on S345 (Figure 4b) [39]. In polarized epithelial cells, RhoA can function to maintain apicobasal polarity and cell–cell junctions, possibly via its stabilization of cortical actin [42,43]. Thus, localized degradation of RhoA, as was observed in Smurf1-dependent regulation of protrusive activity, would be consistent with the loss of tight junctions. Indeed, preventing Smurf1 targeting of RhoA inhibited TGFb-dependent EMT (Figure 4c) [39]. Although these studies focused on Smurf1, multiple C2-WW-HECT family members may function in regulating tight junction loss. For example, Itch, another C2-WW-HECT ligase, has been found to ubiquitinate and degrade occludin [44]. Thus, it is the TGFb-dependent phosphorylation of Par6 and the subsequent recruitment of Smurf1 that facilitates tight junction dissolution and EMT [39]. Moreover, these studies hint that the phosphorylation state of Par6 may well function as a switch. In the absence of TGFb stimulation, Par6 is predominantly phosphorylated on another site [39]. However, Par6 phosphorylated on S345 is missing this second phosphorylation site and Par6 phosphorylated on S345 interacts with Smurf1, whereas Par6 phosphorylated on the second site does not. These studies thus demonstrate the presence of distinct and possibly mutually exclusive phospho-isoforms of Par6. How phosphorylation switching of Par6 affects its polarity functions and regulates its interactions with proteins such as the CRB–PALS–PATJ or Lgl–Dlg– Scrib complexes is an important area of investigation. Is phosphorylation the sole mechanism by which TGFb activates Par6 and regulates EMT, or are other signals required, such as the activation of Cdc42 and aPKC? TGFb has been shown to activate Cdc42 [45] and a potential GEF involved is Ect2, which also binds to Par6 and is localized to tight junctions [46]. GTP– Cdc42 could also activate TGFb-receptor-bound Pak1, thereby regulating changes in the actin cytoskeleton and modulating cell motility [40,47]. Indeed, Pak5 has also been identified as yet another component of polarity complexes [48]. Thus, TGFb may coordinate the loss of tight junctions and EMT with cell movement. Current Opinion in Cell Biology 2006, 18:206–212
Given the connection between EMT and carcinoma progression, it will be interesting to define if and how the Par6–Smurf1 pathway regulates tumour progression to a metastatic state. This could be the mechanism by which TGFb is a pro-tumorigenic factor for late-stage cancers. Interestingly, Par6 has been shown to be upregulated downstream of the SRC3 oncogene in MCF7 breast cancer cells [49]. Furthermore, the aPKC iota isoform is an oncogene in human non-small-cell lung cancer [50]. Par6 also interacts with the Ras-like proteins Rin and Rit to potentiate cellular transformation [51]; this is particularly intriguing as oncogenic Ras co-operates with TGFb to cause EMT. Thus, Par6 may serve as a node to integrate the Ras and TGFb pathways during EMT. In addition, Par6 may contribute to TGFbinduced tumour invasion and metastasis beyond EMT as it is important for regulating polarized cell movement. EMT is also required for various embryological stages such as gastrulation, neurulation and neural crest development. The embryonic epithelia must lose their apical cell junctions in order for correct morphogenesis and developmental patterning to occur. The fact that Par6 and aPKC are required for vertebrate gastrulation [52] could reflect their role in mediating developmental EMT. In addition, the TGFb phosphorylation site on Par6 is maintained as a serine or threonine among every vertebrate isoform of Par6, but not among lower organisms such as the fly or worm [39]. Since tight junctions are vertebrate-specific structures, the phosphorylation site may have co-evolved with tight junctions, thereby enabling Par6 to function as a TGFb target during developmental EMT.
Conclusions The Par6 polarity complex is recruited by various signalling pathways, including the integrin and TGFb pathways, to regulate polarized cell motility and apico-basal epithelial polarity. To better understand Par6 biology, we must learn how these signalling pathways function to regulate the Par6 interactome, as well as its subcellular localization and activity. Clearly, post-translational mechanisms are critical for modulating the Par6 polarity complex. What are the identities of the kinases that phosphorylate Par6 and thus are responsible for the distinct phospho-isoforms? Do the TGFb receptors phosphorylate Par6A and B as well, given their altered cellular distributions? And which phosphatases de-phosphorylate Par6? Another gap in our understanding concerns how Par6 causes its downstream effects. For example, how does the Par6–Smurf1–RhoA pathway explicitly mediate tight junction dissolution? The answers will further our understanding of Par6 in developmental and homeostatic processes, as well as its potential role in tumour invasion and metastasis.
Acknowledgements This work was supported by grants from the Canadian Institutes of Health Research (CIHR), and National Cancer Institute of Canada. R.B. is www.sciencedirect.com
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supported by a CIHR M.D.-Ph.D. Studentship award. J.L.W. is a CIHR Investigator and an International Scholar of the Howard Hughes Medical Institute.
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Current Opinion in Cell Biology 2006, 18:206–212 This review comes from a themed issue on Cell regulation Edited by Claude Prigent and Bruno Goud Available online 20th February 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.02.005
Introduction In recent years, major advances have been made in the identification of proteins that regulate cellular polarity. Numerous multi-protein polarity complexes have been shown to control processes such as epithelial apico-basal polarity, cell migration and asymmetric cell division [1–5]. However, to understand the dynamics of polarity, we must determine not only the composition of these complexes, but also how they are regulated to effect morphological changes. One critical regulator of polarity is partitioning-defective 6 (Par6). Par6 is an adapter protein that engages in many protein–protein interactions that are spatiotemporally regulated to control polarity. Here we review how Par6 functions as an effector of signal transduction pathways to control polarized cell migration and apico-basal polarity, particularly as a target of transforming growth factor b (TGFb).
The Par6 polarity complex Par6 was originally identified as a gene that regulates the asymmetric division of the C. elegans zygote [6], and over Current Opinion in Cell Biology 2006, 18:206–212
the past decade it has emerged as a key player in a plethora of cellular polarity processes in organisms ranging from worms to humans [1–4]. These include asymmetric cell division, neuronal polarity, apico-basal polarity of epithelial cells, and polarized cell movement. There are three mammalian Par6 isoforms, A, B and C, that all share the same protein–interaction domains. Little is known about their relative expression but mRNAs of all three are simultaneously expressed in polarized mammalian epithelial cells (R Bose, JL Wrana, unpublished). These isoforms display different subcellular localizations in polarized epithelia and thus may have distinct functions. Par6A predominantly co-localizes with tight junction markers, whereas Par6B is localized to the cytosol [7]. Par6C localizes to both tight junctions and the cytosol [7]. Par6 functions as part of a protein complex and contains three domains that enable it to interact with the other members of the complex (Figure 1). At the N terminus, there is the Phox/Bem1 (PB1) domain, which binds to other PB1-domain-containing proteins such as atypical protein kinase C (aPKC) [8,9]; next, there is the Cdc42– Rac interaction binding (CRIB) motif, which binds the Cdc42 or Rac GTPases strictly in their activated GTPbound state [8,9]; and finally, there is the PSD-95/Dlg/ ZO-1 (PDZ) domain, which can bind PDZ domains in other proteins such as Par3 [8,9]. Tandem affinity purification (TAP)-tagging of Par6 and subsequent mass spectrometry analysis of endogenous interactors has confirmed these interactions and revealed that Par6 is imbedded in an extensively interconnected network of protein–protein interactions (Figure 2) [10]. Par6 is thus a hub [11–13] in a large polarity network that is evolutionarily conserved from worms to mammals [1–4].
Par6 complex activation and polarized cell migration When cells engage in directed cell migration, the microtubule network is organized such that the microtubuleorganizing centre (MTOC) and Golgi apparatus are oriented between the leading edge and the nucleus [14]. This can facilitate the delivery of membrane precursors and cytoskeletal regulators towards the leading edge. During cell migration, MTOC polarization is regulated by the Cdc42–Par6–aPKC complex [14,15–17], but how exactly does the cell use spatial cues to control this complex and the orientation of the MTOC? One pathway involves the integrins, cell-surface receptors that regulate cellular motility. Integrin binding to extracellular matrix (ECM) components at the leading edge of the cell leads to their clustering in structures called focal complexes; this clustering activates their intracellular signalling pathways [18]. In migrating astrocytes, integrin signalling in www.sciencedirect.com
Regulation of Par6 by extracellular signals Bose and Wrana 207
Figure 1
Schematic of the domain structure of Par6. Par6 contains three protein interaction domains. At the N terminus is the Phox/Bem1 (PB1) domain, which binds to other PB1-domain-containing proteins such as atypical protein kinase C (aPKC); next, there is the Cdc42–Rac interaction binding (CRIB) motif, which binds the Cdc42 or Rac GTPases strictly in their activated GTP-bound state; and finally, there is the PSD-95/Dlg/ZO-1 (PDZ) domain, which can bind PDZ domains in other proteins such as Par3. The TGFb-dependent phosphorylation site, serine 345, is shown.
protrusions activates Cdc42, which in turn binds and activates aPKC scaffolded on Par6 at the leading edge (Figure 3) [15]. This link between Cdc42 binding and aPKC activation is supported by genetic and biochemical studies in the fly and worm, as well as mammalian cells [19,20,21]. Assembly of the Cdc42–Par6–aPKC complex
leads to phosphorylation of glycogen synthase kinase 3b (GSK3b) on its inhibitory serine 9 site. This, in turn, blocks adenomatous polyposis coli (APC) phosphorylation, thereby enabling APC to associate with the growing plus ends of microtubules [22]. This occurs concomitantly with Cdc42- and aPKC-dependent recruitment of Discs Large (Dlg), which interacts with microtubule-bound APC to reorient the MTOC in migrating cells, possibly by linking the leading edge of the plasma membrane to the microtubule network [23]. In addition to the orientation of cell migration, the Par6 polarity complex may regulate the actual movement of cells themselves. Migrating cells form cellular processes such as filopodia and lamellipodia in the direction of movement and the formation of these structures is regulated by the Cdc42 and Rac1 GTPases, respectively [24,25]. In contrast, the closely related GTPase, RhoA, regulates the formation of focal adhesions and actin stress-fibres. Coordinated, localized regulation of these GTPases is critical for effective cell movement [26]. Par6 also plays a role in this process, since it is localized to the tips of cellular protrusions where it can recruit the E3 ubiquitin ligase Smad ubiquitination regulatory factor-1 (Smurf1, originally identified as a negative regulator of TGFb-mediated Smad signalling), which can locally degrade RhoA [27]. This promotes protrusive activity by restricting the inappropriate localized activation of RhoA in Cdc42- or Rac-induced membrane protrusions.
Figure 2
Par6 is a hub in a polarity network. The Par6 protein-interaction network reveals that Par6 (orange) is part of a highly interconnected network that is shown as known physical interactions (lines) between Par6 and numerous Par6 partners represented as nodes. The core, evolutionarily conserved polarity complex is shown in green, components of the TGFb pathway in light blue, the apical CRB–PALS–PATJ complex in pink and the basolateral Lgl–Dlg–Scrib complex in yellow. OCLN, occludin. www.sciencedirect.com
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Figure 3
Par6 regulates polarized cell migration in response to extracellular cues. In migrating cells, integrins at the leading edge bind to ECM components, leading to the formation of focal complexes. This integrin clustering activates Cdc42, which in turn activates aPKC scaffolded on Par6. The Cdc42–Par6–aPKC complex leads to phosphorylation of GSK3b on its inhibitory serine 9 site, which blocks APC phosphorylation to allow APC interaction with membrane-localized Dlg. This latter interaction orients the MTOC towards the direction of movement. The Par6 polarity complex also coordinates the cross-regulation between Rho GTPases at the leading edge. The Cdc42–Par6–aPKC complex can bind and recruit the E3 ubiquitin ligase Smurf1 to locally degrade RhoA in cellular protrusions, thereby promoting protrusive activity.
Par6 may thus function to mediate cross-talk between Rho GTPases during cell migration.
Par6 and the regulation of epithelial polarity In addition to cell motility, Par6 has been shown to be a critical regulator of epithelial polarity. One of the structural hallmarks of a fully polarized epithelial cell is the presence of tight junctions, which confer apico-basal polarity to the cell [28]. Par6, which localizes to tight junctions, has been shown to regulate junctional stability [1–4], but the mechanisms are poorly understood. For example, overexpression of Par6 or activated Cdc42 inhibits tight junction formation [29], whereas the Par6 polarity complex can also promote tight junction formation by recruiting Tiam1, a Rac-specific guanine nucleotide exchange factor (GEF) [30]. Thus, Par6 can regulate both the loss and formation of tight junctions. Par6-dependent control of epithelial apico-basal polarity is mediated via its interactions with two other tri-protein complexes [1,3]. One consists of the transmembrane protein Crumbs (CRB), protein associated with lin-seven (PALS) and PALS1-associated tight-junction protein (PATJ). This complex is located apically to Par6 and genetic and biochemical data suggest that the two complexes cooperate to maintain each other’s subcellular localization and establish apical polarization [1,31–33]. Current Opinion in Cell Biology 2006, 18:206–212
In contrast, the second Par6-interacting complex, consisting of Lethal Giant Larvae (Lgl), Discs Large (Dlg) and Scribble (Scrib) is located basolaterally to the Par6 polarity complex, and is proposed to negatively regulate the establishment of the apical membrane [1,34,35]. Thus, the balance between opposing gradients of CRB–PALS–PATJ and Lgl–Dlg–Scrib complexes may restrict the Par6 complex to tight junctions, thereby coordinating the establishment of epithelial apico-basal polarity.
TGFb and Par6 TGFb is a growth factor that can serve as a tumour suppressor or a tumour promoter, depending on the situation [36]. TGFb signals through two transmembrane serine-threonine kinases, the type II (TbRII) and type I (TbRI) receptors. The classically described TGFb pathway begins with the binding of the TGFb ligand to the constitutively active TbRII, which in turn binds and phosphorylates TbRI. This activates the Smad pathway to regulate gene transcription. Many of the tumour suppressor functions of TGFb are mediated by transcription in this manner [37]. However, advanced tumours become refractory to TGFb-mediated growth inhibition and, in these tumours, TGFb is proposed to promote invasion and metastasis [36]. Interestingly, addition of TGFb to several polarized epithelial cell types causes them to www.sciencedirect.com
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Figure 4
acquire a fibroblastoid phenotype in a process known as epithelial-to-mesenchymal transition, or EMT [36]. This process involves the dissolution of epithelial cell–cell junctions and the loss of apico-basal polarity, as well as a change from an epithelial to a fibroblastoid gene expression program. Importantly, EMT is correlated with the progression of carcinomas to an invasive and metastatic state. TGFb-dependent EMT is known to require both Smad-dependent and Smad-independent pathways [38]. In particular, recent evidence indicates that TGFbdependent loss of tight junctions is mediated via Par6 phosphorylation by the TGFb receptor and appears to be independent of Smad-mediated transcriptional responses [39]. The mammalian-based LUMIER protein–protein interaction screen [40] was used to discover novel interacting partners of the TGFb type I receptor. Three of the identified proteins were: Par6C; occludin, a four-pass transmembrane protein that is a structural component of tight junctions; and p21-activated kinase 1 (PAK1), a Cdc42-activated kinase that is a key regulator of the actin cytoskeleton and cell migration [39,40]. Thus, the TGFb pathway can link the polarity complex with tight junction components and regulators of the actin cytoskeleton. Mouse mammary epithelial NMuMG cells can be used to study epithelial apico-basal polarity and tight junction homeostasis and undergo EMT upon TGFb stimulation. In fully polarized NMuMG cells, there are distinct distributions of cell-surface TGFb receptors: TbRII is localized to puncta distributed over the surface, whereas TbRI is strictly co-localized with tight junction markers [39]. Occludin is important for this restricted localization of TbRI [40], which may also involve other tetraspanin proteins such as the claudins. However, upon 30 min treatment with TGFb ligand, TbRII redistributes to the tight junctions, and this correlates with the observed ligand-dependant interaction of TbRII with occludin [40]. In tight junctions, Par6, which binds TbRI, is recruited to TbRII via its interactions with the type I receptor (Figure 4a). Within this complex, TbRII can directly phosphorylate Par6 on serine 345 (S345) (Figure 1, Figure 4b), which then remains bound to the receptor complex; this contrasts with the Smads, which dissociate upon phosphorylation. Preventing Par6 phosphorylation on serine 345 profoundly blocks TGFbdependent loss of tight junctions [39]. This process appears to be independent of Smad transcriptional regulation, although there remains the possibility that the Par6 polarity complex directly regulates Smad functions, as Smad3 has been shown to interact with Par3 [41]. Thus
The TGFb pathway. (a) Occludin (OCLN) restricts TbRI localization to the tight junctions, where it co-localizes with Par6. Upon TGFb stimulation, TbRII is recruited to the tight junctions and forms active TGFb receptor complexes. (b) TbRII phosphorylates Par6 on serine 345, stimulating www.sciencedirect.com
binding and recruitment of the E3 ubiquitin ligase Smurf1 to tight junctions. TbRII also phosphorylates and activates TbRI. (c) Smurf1 ubiquitinates and degrades RhoA, promoting tight junction dissolution, while activated TbRI can phosphorylate and activate the Smad transcriptional pathway. Together, these activities lead to TGFbdependent EMT. Current Opinion in Cell Biology 2006, 18:206–212
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active receptor complexes containing phosphorylated Par6 are assembled in tight junctions during TGFb signalling and are critical for EMT. How, then, does TGFb-mediated Par6 phosphorylation on S345 specifically control tight junction loss? The clues lie in how the Par6 polarity complex regulates cellular protrusions [27]. As described earlier, the Par6 polarity complex localized at the tips of fibroblast cellular protrusions recruits Smurf1, an E3 ubiquitin ligase belonging to the C2-WW-HECT family, to regulate localized RhoA degradation [27]. Similarly, in epithelial cells, Par6 is capable of binding and recruiting Smurf1 to tight junctions and this is dependent on TGFb-dependent phosphorylation on S345 (Figure 4b) [39]. In polarized epithelial cells, RhoA can function to maintain apicobasal polarity and cell–cell junctions, possibly via its stabilization of cortical actin [42,43]. Thus, localized degradation of RhoA, as was observed in Smurf1-dependent regulation of protrusive activity, would be consistent with the loss of tight junctions. Indeed, preventing Smurf1 targeting of RhoA inhibited TGFb-dependent EMT (Figure 4c) [39]. Although these studies focused on Smurf1, multiple C2-WW-HECT family members may function in regulating tight junction loss. For example, Itch, another C2-WW-HECT ligase, has been found to ubiquitinate and degrade occludin [44]. Thus, it is the TGFb-dependent phosphorylation of Par6 and the subsequent recruitment of Smurf1 that facilitates tight junction dissolution and EMT [39]. Moreover, these studies hint that the phosphorylation state of Par6 may well function as a switch. In the absence of TGFb stimulation, Par6 is predominantly phosphorylated on another site [39]. However, Par6 phosphorylated on S345 is missing this second phosphorylation site and Par6 phosphorylated on S345 interacts with Smurf1, whereas Par6 phosphorylated on the second site does not. These studies thus demonstrate the presence of distinct and possibly mutually exclusive phospho-isoforms of Par6. How phosphorylation switching of Par6 affects its polarity functions and regulates its interactions with proteins such as the CRB–PALS–PATJ or Lgl–Dlg– Scrib complexes is an important area of investigation. Is phosphorylation the sole mechanism by which TGFb activates Par6 and regulates EMT, or are other signals required, such as the activation of Cdc42 and aPKC? TGFb has been shown to activate Cdc42 [45] and a potential GEF involved is Ect2, which also binds to Par6 and is localized to tight junctions [46]. GTP– Cdc42 could also activate TGFb-receptor-bound Pak1, thereby regulating changes in the actin cytoskeleton and modulating cell motility [40,47]. Indeed, Pak5 has also been identified as yet another component of polarity complexes [48]. Thus, TGFb may coordinate the loss of tight junctions and EMT with cell movement. Current Opinion in Cell Biology 2006, 18:206–212
Given the connection between EMT and carcinoma progression, it will be interesting to define if and how the Par6–Smurf1 pathway regulates tumour progression to a metastatic state. This could be the mechanism by which TGFb is a pro-tumorigenic factor for late-stage cancers. Interestingly, Par6 has been shown to be upregulated downstream of the SRC3 oncogene in MCF7 breast cancer cells [49]. Furthermore, the aPKC iota isoform is an oncogene in human non-small-cell lung cancer [50]. Par6 also interacts with the Ras-like proteins Rin and Rit to potentiate cellular transformation [51]; this is particularly intriguing as oncogenic Ras co-operates with TGFb to cause EMT. Thus, Par6 may serve as a node to integrate the Ras and TGFb pathways during EMT. In addition, Par6 may contribute to TGFbinduced tumour invasion and metastasis beyond EMT as it is important for regulating polarized cell movement. EMT is also required for various embryological stages such as gastrulation, neurulation and neural crest development. The embryonic epithelia must lose their apical cell junctions in order for correct morphogenesis and developmental patterning to occur. The fact that Par6 and aPKC are required for vertebrate gastrulation [52] could reflect their role in mediating developmental EMT. In addition, the TGFb phosphorylation site on Par6 is maintained as a serine or threonine among every vertebrate isoform of Par6, but not among lower organisms such as the fly or worm [39]. Since tight junctions are vertebrate-specific structures, the phosphorylation site may have co-evolved with tight junctions, thereby enabling Par6 to function as a TGFb target during developmental EMT.
Conclusions The Par6 polarity complex is recruited by various signalling pathways, including the integrin and TGFb pathways, to regulate polarized cell motility and apico-basal epithelial polarity. To better understand Par6 biology, we must learn how these signalling pathways function to regulate the Par6 interactome, as well as its subcellular localization and activity. Clearly, post-translational mechanisms are critical for modulating the Par6 polarity complex. What are the identities of the kinases that phosphorylate Par6 and thus are responsible for the distinct phospho-isoforms? Do the TGFb receptors phosphorylate Par6A and B as well, given their altered cellular distributions? And which phosphatases de-phosphorylate Par6? Another gap in our understanding concerns how Par6 causes its downstream effects. For example, how does the Par6–Smurf1–RhoA pathway explicitly mediate tight junction dissolution? The answers will further our understanding of Par6 in developmental and homeostatic processes, as well as its potential role in tumour invasion and metastasis.
Acknowledgements This work was supported by grants from the Canadian Institutes of Health Research (CIHR), and National Cancer Institute of Canada. R.B. is www.sciencedirect.com
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supported by a CIHR M.D.-Ph.D. Studentship award. J.L.W. is a CIHR Investigator and an International Scholar of the Howard Hughes Medical Institute.
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