Par6B and atypical PKC regulate mitotic spindle orientation during epithelial morphogenesis

Cdc42 plays an evolutionarily conserved role in promoting cell polarity and is indispensable during epithelial morphogenesis. To further investigate the role of Cdc42, we have used a three-dimensional matrigel model, in which single Caco-2 cells develop to form polarized cysts. Using this system, we previously reported that Cdc42 controls mitotic spindle orientation during cell division to correctly position the apical surface in a growing epithelial structure. In the present study, we have investigated the specific downstream effectors through which Cdc42 controls this process. Here, we report that Par6B and its binding partner, atypical protein kinase C (aPKC), are required to regulate Caco-2 morphogenesis. Depletion or inhibition of Par6B or aPKC phenocopies the loss of Cdc42, inducing misorientation of the mitotic spindle, mispositioning of the nascent apical surface, and ultimately, the formation of aberrant cysts with multiple lumens. Mechanistically, Par6B and aPKC function interdependently in this context. Par6B localizes to the apical surface of Caco-2 cysts and is required to recruit aPKC to this compartment. Conversely, aPKC protects Par6B from proteasomal degradation, in a kinase-independent manner. In addition, we report that depletion or inhibition of aPKC induces robust apoptotic cell death in Caco-2 cells, significantly reducing both cyst size and number. Cell survival and apical positioning depend upon different thresholds of aPKC expression, suggesting that they are controlled by distinct downstream pathways. We conclude that Par6B and aPKC control mitotic spindle orientation in polarized epithelia and, furthermore, that aPKC coordinately regulates multiple processes to promote morphogenesis.     

Numb

Epithelial to mesenchymal transition (EMT) is a critical event in embryogenesis and plays a fundamental role in cancer progression and metastasis1. Numb has been shown to play an important role in the proper functions of Par protein complex and in cell-cell junctions, both of which are associated with EMT. However, the role of Numb in EMT has not been fully elucidated. Recently, we showed that Numb is capable of binding to both Par3 and E-cadherin. Intriguingly, the interaction of Numb with E-cadherin or the Par protein complex is dynamically regulated by tyrosine phosphorylation induced by HGF or Src. Knockdown of Numb by shRNA in MDCK cells led to an lateral to apical translocation of E-cadherin and β-catenin, active F-actin polymerization, mis-localization of Par3 and aPKC, a decrease in cell-cell adhesion, and an increase in cell migration and proliferation. These data suggest a diverse role for Numb in regulating cell-cell adhesion, polarity and migration during EMT.     

Numb regulates cell–cell adhesion and polarity in response to tyrosine kinase signalling

Epithelial‐mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E‐cadherin (E‐cad) through its phosphotyrosine‐binding domain (PTB) and thereby regulates the localization of E‐cad to the lateral domain of epithelial cell–cell junction. Moreover, Numb engages the polarity complex Par3–aPKC–Par6 by binding to Par3 in polarized Madin‐Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E‐cad and Par3 and associates preferably with aPKC–Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF‐induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral‐to‐apicolateral translocation of E‐cad and β‐catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF‐induced cell scattering, a decrease in cell–cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell–cell adhesion and a sensor of HGF signalling or Src activity during EMT.     

Mammalian Lgl forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity

BACKGROUND: Epithelial cells have apicobasal polarity and an asymmetric junctional complex that provides the bases for development and tissue maintenance. In both vertebrates and invertebrates, the evolutionarily conserved protein complex, PAR-6/aPKC/PAR-3, localizes to the subapical region and plays critical roles in the establishment of a junctional complex and cell polarity. In Drosophila, another set of proteins called tumor suppressors, such as Lgl, which localize separately to the basolateral membrane domain but genetically interact with the subapical proteins, also contribute to the establishment of cell polarity. However, how physically separated proteins interact remains to be clarified. RESULTS: We show that mammalian Lgl competes for PAR-3 in forming an independent complex with PAR-6/aPKC. During cell polarization, mLgl initially colocalizes with PAR-6/aPKC at the cell-cell contact region and is phosphorylated by aPKC, followed by segregation from apical PAR-6/aPKC to the basolateral membrane after cells are polarized. Overexpression studies establish that increased amounts of the mLgl/PAR-6/aPKC complex suppress the formation of epithelial junctions; this contrasts with the previous observation that the complex containing PAR-3 promotes it. CONCLUSIONS: These results indicate that PAR-6/aPKC selectively interacts with either mLgl or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity.     

aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb

In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. Here, we identify in vivo phosphorylation sites of mammalian Numb and show that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions.     

Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes.     

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell–cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.     

EphrinB reverse signaling in cell-cell adhesion

Cell-cell adhesion is a critical process for the formation and maintenance of tissue patterns during development, as well as invasion and metastasis of cancer cells. Although great strides have been made regarding our understanding of the processes that play a role in cell-cell adhesion, the precise mechanisms by which diverse signaling events regulate cell and tissue architecture is poorly understood. In this commentary we will focus on the Eph/ephrin signaling system, and specifically how the ephrinB1 transmembrane ligand for Eph receptor tyrosine kinases sends signals affecting cell-cell junctions. In a recent study using the epithelial cells of early stage Xenopus embryos, we have shown that loss- or gain-of function of ephrinB1 can disrupt cell-cell contacts and tight junctions. This study reveals a mechanism where ephrinB1 competes with active Cdc42 for binding to Par-6, a scaffold protein central to the Par polarity complex (Par-3/Par-6/Cdc42/aPKC) and disrupts the localization of tight junction-associated proteins (ZO-1, Cingulin) at tight junctions. This competition reduces aPKC activity critical to maintaining and/or forming tight junctions. Finally, phosphorylation of ephrinB1 on specific tyrosine residues can block the interaction between ephrinB1 and Par-6 at tight junctions, and restore tight junction formation. Recent evidence indicates that de-regulation of forward signaling through EphB receptors may play a role in metastatic progression in colon cancer. In light of the new data showing an effect of ephrinB reverse signaling on tight junctions, an additional mechanism can be hypothesized where de-regulation of ephrinB1 expression or phosphorylation may also impact metastatic progression.     

The WD40 protein Morg1 facilitates Par6–aPKC binding to Crb3 for apical identity in epithelial cells

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6–aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6–aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6–aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6–aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.     

Numb: A new player in EMT

Epithelial to mesenchymal transition (EMT) is a critical event in embryogenesis and plays a fundamental role in cancer progression and metastasis.¹ Numb has been shown to play an important role in the proper functions of Par protein complex and in cell-cell junctions,^2,3 both of which are associated with EMT.^4,5 However, the role of Numb in EMT has not been fully elucidated. Recently, we showed that Numb is capable of binding to both Par3 and E-cadherin. Intriguingly, the interaction of Numb with E-cadherin or the Par protein complex is dynamically regulated by tyrosine phosphorylation induced by HGF or Src. Knockdown of Numb by shRNA in MDCK cells led to a lateral to apical translocation of E-cadherin and β-catenin, active F-actin polymerization, mis-localization of Par3 and aPKC, a decrease in cell-cell adhesion and an increase in cell migration and proliferation. These data suggest a diverse role for Numb in regulating cell-cell adhesion, polarity and migration during EMT.⁶Key words: Numb, E-cadherin, tyrosine kinase, cell polarity, adhesion, EMTNumb was originally identified as a gene required for cell fate determination during neuroblast division and sensory organogenesis.⁷ Recently, a number of proteins involved in cell polarity, cell-cell adhesion and tumorigenesis have been identified as binding partners for Numb. These include the Par3-Par6-aPKC polarity complex, E-cadherin, integrin, Notch, WNT and p53.^8–10 Although these new data implicate Numb in multiple signaling pathways, questions remain as to how the various interactions are regulated and in which biological context they occur. Interestingly, most binding partners of Numb are involved in one way or another in the onset and/or progression of cancer. For instance, the Par complex is involved in regulating the formation and stability of tight junction whereas E-cadherin is a key component of the adherens junction in epithelial cells. Understandably, deregulation of the Par protein complex and/or E-cadherin is implicated in EMT. A variety of stimuli have been identified to induce EMT,¹ including transforming growth factor-β (TGFβ), hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and activation of tyrosine kinase Src. During the progression of EMT, non-motile epithelial cells gradually lose their apical-basal polarity and cell-cell junctions and become mesenchymal cells with an ability to migrate away from the primary site to surrounding tissues.¹ Therefore, the study of Numb interactions with the Par protein complex and E-cadherin in the context of EMT provides a good point of entry to decode the complex signaling network mediated by Numb.Based on data obtained from Madin-Darby canine kidney (MDCK) cells,⁶ we propose a model in which Numb regulates epithelial polarity and cell-cell adhesion in EMT (Fig. 1). In epithelial cells, Numb binds to E-cadherin or the Par protein complex via Par3 under a normal physiological condition to stabilize adherens and tight junctions. Under the influence of an extracellular cue, such as HGF, the Met receptor recruits and activates one of its downstream components, c-Src. C-Src subsequently phosphorylates the DNVYYY motif on E-cadherin, resulting in Numb dissociation from phosphorylated E-cadherin. Numb then binds and sequesters phosphorylated aPKC and Par6, while phosphorylated Par3 is released from the Par complex. The Numb-aPKC-Par6 complex remains on the plasma membrane or in the cytoplasm, whereas Par3 is transported into the nucleus (by an unknown mechanism). Phosphorylated E-cadherin is relocated to an apico-lateral domain accompanied with active F-actin polymerization. Enhanced F-actin polymerization, together with reduced cell-cell adhesion and increased cell proliferation, promotes cell migration (Fig. 1).Open in a separate windowFigure 1A model depicting the role of Numb in epithelial-mesenchymal transition (EMT). In epithelial cells, Numb stabilizes both E-cadherin based adherens junctions through a PTB domain binding to NVYY motif on E-cadherin, and Par protein complex on tight junctions by interacting with Par3. During the early stage of EMT, c-Met recruits the tyrosine kinase c-Src upon its activation by HGF. C-Src phosphorylates E-cadherin on the NVYY motif, which leads to dissociation of Numb from E-cadherin and the translocation of E-cadherin to an apical domain. Numb forms a complex with phosphorylated aPKC and Par6, whereas phosphorylated Par3 is transported into the nucleus. Enhanced F-actin polymerization, together with reduced cell-cell adhesions, promotes the transition to mesenchymal cells.While our work in MDCK epithelial cells identifies a key role for Numb in regulating the sub-cellular localizationsand functions of E-cadherin and the Par protein complex, it raises a number of unaddressed questions. First, biochemical data obtained from temperature sensitive src mutant (ts-src) MDCK cell line suggest that tryosine phosphorylation plays a critical role in regulating the dynamic interactions of Numb with E-cadherin or the Par protein complex. Using a peptide array screen, we found that a conserved DNVYYY motif in E-cadherin is the binding site for phosphotyrosine binding (PTB) domain of Numb. Interestingly, peptide analogs in which a Tyr residue in the YYY triad is replaced by a pTyr were deficient in binding. This suggests that the Numb PTB domain may act in a phosphotyrosine-independent manner in EMT signal transduction events, and that the interaction of Numb with VIEW E-cadherin is negatively regulated by tyrosine kinase signaling. An intriguing question is why E-caderin harbors a conserved YYY triad⁶ when phosphorylation of one Tyr is sufficient to eliminate Numb binding. Furthermore, when and where are the YYY triad phosphorylated during EMT? It is likely that one or more tyrosine residues are phosphorylated by Src, depending on the duration of stimulus that a cell receives during EMT. Phosphorylated E-cadherin may be targeted to an alternative location in the cell by the E3 ligase Hakai which mediates the endocytosis and degradation of E-cadherin¹¹ after its phosphorylation and dissociation from Numb. To complicate the issue, Par3 and aPKC themselves are also shown to be tyrosine phosphorylated by HGF treatment or Src activation in our studies, although the precise phosphorylation sites on either protein remain to be determined. In a previous report, a high-throughput phosphoproteomic screen has identified multiple tyrosine phosphorylation sites in the carboxyl terminus of Par-3.¹² Phosphorylation of Y1127 in Par-3 reduced its interaction with LIMK2 and delayed tight junction assembly in mammalian epithelial cells with a constitutive Src activation or under EGF treatment.¹² A recent study, which shows that the SH2 domain of C-terminal Src kinase (Csk) binds to the Y1127 site on Par3, adds another layer of complexity to the scenario.¹³ In the same vein, Src has been shown to associate with aPKC and phosphorylate the latter in PC12 cells.¹⁴ Par6 appears also to be subjected to Src regulation as recruitment of Src by Pals leads to Pals1 and Par6 to be sequestered away from JAM-C of desmosome adhesions in the blood-testis-barrier, causing a disruption in cell adhesion.¹⁵ It will be of interest to determine whether Par6 is directly phosphorylated by Src in this case.¹⁵ Together, these studies suggest a role for tyrosine phosphorylation, besides serine/threonine phosphorylation, in regulating the functions of various polarity proteins.Second, our biochemical and immunfluorescence data suggest that the interactions of Numb with E-cad/Par complex are spatially and temporally regulated in response to HGF treatment. However, the molecular mechanisms for some remarkable phenotypes remain poorly defined. For instance, shRNA-mediated Numb knockdown caused a dramatic apicolateral mis-localization of E-cadherin and β-catenin. Dynamic cellular localization may be regulated by endocytosis and post-translational modifications such as phosphorylation. In keeping with this, Numb can directly bind to several endocytic proteins, including AP2 and Eps15, through its conserved DPF and NPF motifs.¹⁶ Our data suggest that Numb is responsible for targeting E-cadherin to correct localization on the basolateral membrane, but we cannot rule out the possibility that other proteins may be involved in this process. One candidate that may facilitate E-cadherin localization is Rab11. Previous studies revealed that in newly polarized MDCKII cells, Rab11 mutation causes an apical mis-localization of E-cadherin and aberrant actin localization, while leaving ZO-1 localization unchanged. This mirrors the phenotype we observed in the Numb shRNA cells.¹⁷ In support of the notion that Rab11 and Numb may be functionally related, Rab11 and Numb are segregated selectively in the pIIb daughter cell during the division of a sensory organ precursor (SOP).¹⁸ It is likely that the mis-localization of E-cadherin is caused by an abnormal enodocytosis of E-cadherin in the absence of Numb.In contrast, the Par protein complex provides critical spatial information in the formation of tight junctions. Treatment of MDCK cells by HGF caused Par3 to dissociate from Par6-aPKC, suggesting that the Par3-Par6-aPKC complex¹⁹ is dynamically regulated. Additionally, both Par3 and aPKC are found to translocate to the nucleus following HGF treatment. A key question, therefore, is how the localizations of these polarity proteins themselves are regulated during EMT. Through a PB1-PB1 domain heterodimarization,²⁰ Par-6 binds to aPKC and thereby inhibits its catalytic. Moreover, Par6 also helps recruit substrates to aPKC,²⁰ one of which is Par3. Par6 binds to Par3, through a PDZ-PDZ interaction, but aPKC can also bind directly to Par3 through its kinase domain, and phosphorylates a Ser residue on Par3.²¹ Importantly, depending on the cellular context, Par3, Par6 and aPKC may not form a constitutive complex.²⁰ For example, in Drosophila neuroblasts and embryonic epithelial cells, Par3 apical localization is independent of aPKC,²² while in mammalian epithelial cells, Par3 is not apical but is associated with tight junctions.²³ Interactions of these polarity proteins are dynamically regulated by multiple protein kinases, small GTPases, or competition from other binding partners.¹⁹ The dynamic change of components of the Par complex leads to the different sub-cellular distribution of these individual polarity proteins, and thereby alters their functions.¹⁹ We showed that activation of Src kinase or HGF treatment reduces the association of Par3 with aPKC, but does not change the binding between aPKC and Par6. In support of this, a similar observation is made in MDCK cells with activation of tyrosine kinase ErbB2, although neither Par3 nor Par6 is a substrate of ErbB2.²⁴ The dynamic assembly and dissolution of the Par3-Par6-aPKC complex in response to intracellular cues or extracellular stimuli may be a general mechanism used by metazoan to control cell polarity and movement.Lastly, active F-actin polymerization is one of most striking morphologies we observed in the Numb-shRNA cells. Given that the Numb-shRNA cells exhibit a faster rate of migration and wound healing than control cells, it is likely that the active F-actin polymerization is caused by aberrant activation of the Rho family GTPase activity. Small GTPases of the Rho family control organization of cytoskeleton, cell motility, cell growth, morphogenesis, cytokinesis and trafficking.²⁵ The most common members of small Rho GTPases are RhoA, Rac1 and Cdc42. RhoA is responsible for the activation of stress fibers and cell contractility.²⁵ Rac1 activation leads to polymerization of filamentous actin, which results in lamellipodium formation and membrane ruffling at the leading edge of migrating cells.²⁵ Cdc42 activation causes the formation of filopodia, long finger-like protrusions at the edges of lamellipodia.²⁵ A Rho family of GTPase exists in two states: a GDP-bound inactive state and a GTP-bound active state. The switch between the two states is regulated by a large group of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs).²⁵ GEFs and GAPs typically contain interaction domains which direct the enzymes to specific sub-cellular locations and help recruit upstream/downstream partners to affect processes such as actin cytoskeleton, cell polarity, microtubule dynamics and membrane transport.²⁵ It is often difficult to pinpoint exactly which member of the Rho family GTPases leads to a specific phenotype due to the overlapping functions of the Rho GTPase members. However, several observations have suggested that certain small Rho GTPases are essential for the establishment of the apico-basal polarity and likely interplay with Numb. Deletion of Cdc42 abolished normal localization of aPKC, Par6 and Numb in neuroepithelium.²⁶ Par6 binds to GTP-bound Cdc42 through a Rho GTPase-bindingCdc42/Rac-interactive binding (CRIB) domain.^23,27 Another line of evidence is that Numb binds to intersectin, a Cdc42 GEF, and enhances the GEF activity of intersectin, leading to activation of Cdc42 in vivo.²⁸ It appears that Numb both regulates and is regulated by Cdc42 temporally and spatially. Par3 has also been reported to sequester Tiam1, a Rac GEF, to inhibit Rac activation in hippocampal neurons.²⁹ Nevertheless, Par3 recruits Tiam1 to activate Rac in keratinocytes.³⁰ Thus, the dynamic interplay between Rho GTPases and the Par protein complex may be cell type-dependent. It will also be of interest in the future to determine whether other GEFs/GAPs interact with the Par protein complex, and whether and how these interactions are modulated by Numb.A more detailed understanding of the role of Numb in EMT will undoubtedly provide valuable insights into the molecular basis of cancer metastasis and suggest novel therapeutic strategies for cancer.     

Neph-Nephrin proteins bind the Par3-Par6-atypical protein kinase C (aPKC) complex to regulate podocyte cell polarity

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their foot processes and the interposed slit diaphragm. So far, very little is known about the guidance cues and polarity signals required to regulate proper development and maintenance of the glomerular filtration barrier. We now identify Par3, Par6, and atypical protein kinase C (aPKC) polarity proteins as novel Neph1-Nephrin-associated proteins. The interaction was mediated through the PDZ domain of Par3 and conserved carboxyl terminal residues in Neph1 and Nephrin. Par3, Par6, and aPKC localized to the slit diaphragm as shown in immunofluorescence and immunoelectron microscopy. Consistent with a critical role for aPKC activity in podocytes, inhibition of glomerular aPKC activity with a pseudosubstrate inhibitor resulted in a loss of regular podocyte foot process architecture. These data provide an important link between cell recognition mediated through the Neph1-Nephrin complex and Par-dependent polarity signaling and suggest that this molecular interaction is essential for establishing the three-dimensional architecture of podocytes at the kidney filtration barrier.     

非典型蛋白激酶C复合物在细胞极性建立中的作用

极性是多数细胞的共同特征,是细胞分化和细胞行使正常功能的基础,细胞极性的建立对于生物体的生长发育至关重要。过去十年的研究显示,进化上保守的非典型蛋白激酶C(aPKC)复合物在许多生物的多种细胞中都参与了细胞极性的建立,并且在其中扮演着相当重要的角色,这为揭示极性建立的机制提供了重要的线索。以线虫合子前-后极(anterior-posterior)的形成、哺乳动物和果蝇上皮细胞顶-底极(apical-basal)的建立以及果蝇神经母细胞不对称分裂中细胞命运决定子的分配这3个典型的极性过程为主线,综述了aPKC复合物在细胞极性建立中的作用,并探讨其中的分子机制。     

Par‐aPKC‐dependent and ‐independent mechanisms cooperatively control cell polarity,Hippo signaling,and cell positioning in 16‐cell stage mouse embryos

In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par‐aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE‐specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16‐cell stage embryos. Similar to 32‐cell stage embryos, disruption of the Par‐aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32‐cell stage embryos, 16‐cell stage embryos with a disrupted Par‐aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p‐ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16‐cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8‐cell stage embryos form polar–apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16‐cell stage is regulated by both Par‐aPKC‐dependent and ‐independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling.     

A polarity complex of mPar-6 and atypical PKC binds,phosphorylates and regulates mammalian Lgl

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.     

Transforming growth factor-β1 induces epithelial-to-mesenchymal transition in human lung cancer cells via PI3K/Akt and MEK/Erk1/2 signaling pathways

Metastasis of tumor cells is associated with epithelial-to-mesenchymal transition (EMT), which is a process whereby epithelial cells lose their polarity and acquire new features of mesenchyme. EMT has been reported to be induced by transforming growth factor-??1 (TGF-??1), but its mechanism remains elusive. In this study, we performed a study to investigate whether PI3K/Akt and MAPK/Erk1/2 signaling pathways involved in EMT in the human lung cancer A549 cells. The results showed that after treated with TGF-??1 for 48?h, A549 cells displayed more fibroblast-like shape, lost epithelial marker E-cadherin and increased mesenchymal markers Vimentin and Fibronectin. Moreover, TGF-??1-induced EMT after 48?h was accompanied by increased of cell migration and change of Akt and Erk1/2 phosphorylation. In addition, EMT was reversed by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126, which suggested that A549 cells under stimulation of TGF-??1 undergo a switch into mesenchymal cells and PI3K/Akt and MAPK/Erk1/2 signaling pathways serve to regulate TGF-??1-induced EMT of A549 cells.     

Willin and Par3 cooperatively regulate epithelial apical constriction through aPKC-mediated ROCK phosphorylation

Apical-domain constriction is important for regulating epithelial morphogenesis. Epithelial cells are connected by apical junctional complexes (AJCs) that are lined with circumferential actomyosin cables. The contractility of these cables is regulated by Rho-associated kinases (ROCKs). Here, we report that Willin (a FERM-domain protein) and Par3 (a polarity-regulating protein) cooperatively regulate ROCK-dependent apical constriction. We found that Willin recruits aPKC and Par6 to the AJCs, independently of Par3. Simultaneous depletion of Willin and Par3 completely removed aPKC and Par6 from the AJCs and induced apical constriction. Induced constriction was through upregulation of the level of AJC-associated ROCKs, which was due to loss of aPKC. Our results indicate that aPKC phosphorylates ROCK and suppresses its junctional localization, thereby allowing cells to retain normally shaped apical domains. Thus, we have uncovered a Willin/Par3-aPKC-ROCK pathway that controls epithelial apical morphology.     

Phosphorylation-dependent binding of 14-3-3 to Par3beta, a human Par3-related cell polarity protein

Mammalian Par3alpha and Par3beta/Par3L participate in cell polarity establishment and localize to tight junctions of epithelial cells; Par3alpha acts via binding to atypical PKC (aPKC). Here we show that Par3beta as well as Par3alpha interacts with 14-3-3 proteins in a phosphorylation-dependent manner. In the interaction, Ser-746 of Par3beta and the corresponding residue of Par3alpha (Ser-814) likely play a crucial role, since replacement of these residues by unphosphorylatable alanine results in a loss of interacting activity. The mutant Par3 proteins with the replacement are correctly recruited to tight junctions of MDCK cells and to membrane ruffles induced by an active form of the small GTPase Rac in HeLa cells. Thus, the interaction with 14-3-3 appears to be dispensable to Par3 localization. Consistent with this, the Par3alpha-14-3-3 interaction does not inhibit the Par3alpha-aPKC association required for the Par3alpha localization, although the aPKC-binding site lies close to the Ser-814-containing, 14-3-3-interacting region.     

aPKC phosphorylates JAM-A at Ser285 to promote cell contact maturation and tight junction formation

The PAR-3-atypical protein kinase C (aPKC)-PAR-6 complex has been implicated in the development of apicobasal polarity and the formation of tight junctions (TJs) in vertebrate epithelial cells. It is recruited by junctional adhesion molecule A (JAM-A) to primordial junctions where aPKC is activated by Rho family small guanosine triphosphatases. In this paper, we show that aPKC can interact directly with JAM-A in a PAR-3-independent manner. Upon recruitment to primordial junctions, aPKC phosphorylates JAM-A at S285 to promote the maturation of immature cell-cell contacts. In fully polarized cells, S285-phosphorylated JAM-A is localized exclusively at the TJs, and S285 phosphorylation of JAM-A is required for the development of a functional epithelial barrier. Protein phosphatase 2A dephosphorylates JAM-A at S285, suggesting that it antagonizes the activity of aPKC. Expression of nonphosphorylatable JAM-A/S285A interferes with single lumen specification during cyst development in three-dimensional culture. Our data suggest that aPKC phosphorylates JAM-A at S285 to regulate cell-cell contact maturation, TJ formation, and single lumen specification.     

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.