The first Scribbled-like protein to be characterized, Densin-180, was isolated from the rbat postsynaptic density (Apperson, 1996). This protein has a unique structure, in that it contains a set of leucine-rich repeats (LRRs) as well as a PSD-95/Dlg/ZO-1 (PDZ) domain; these domains are thought to mediate protein-protein interactions. Recently, further proteins containing both types of domains have been isolated from fly, worm, mouse and human. It seems an opportune time to select a collective name for this family of proteins. The name 'LAP (LRR And PDZ domain) proteins' is endorsed for proteins with this structure. The known LAP proteins contain 16 canonical LRRs located at the amino terminus of the protein, as well as a conserved, LRR-like region immediately carboxy-terminal to the LRRs. The LRRs from LAP proteins are more closely related to each other than to LRRs from other proteins. The known LAP proteins also contain either one or four PDZ domains. It may be useful to distinguish between these subfamilies by using the designations 'LAP1' and 'LAP4', respectively. Among eukaryotes for which complete genomic sequence is available, Saccharomyces cerevisiae (in which both LRR and PDZ domains can be found) contains no LAP proteins. C. elegans contains a single LAP1 protein, Let-413 (Legouis, 2000), whereas Drosophila contains both a LAP1 protein, dLAP (GenBank AAF58179), and a LAP4 protein, Scribbled (two alternative splice variants, GenBank AF190774 and AJ271647). Vertebrates are likely to contain at least three LAP proteins, because the LAP1 proteins Densin-180 (Bilder, 2000a) and Erbin (Borg, 2000) are both found in rat, whereas human hScribbled is a LAP4 protein (Bilder, 2000a and Nagase, 1995 -- two variants have been identified: GenBank AF271734 and AF240677). Further vertebrate LAP proteins may be identified as complete genomic sequence becomes available. Although analyses of these proteins are just beginning, one striking feature is their polarized localization along the cell membrane. This localization, in conjunction with the modular arrangement of the protein-protein interaction domains they contain, is consistent with genetic and molecular analyses, indicating that LAP proteins may play a key role in regulating the subcellular distribution of other proteins. Identification and further studies of LAP proteins will reveal what aspects of function, as well as of structure, are conserved within this family (Bilder, 2000c).
Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. The gene let-413 has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions; epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. It is suggested that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells (Legouis, 2000).
The correct assembly of junction components, such as E-cadherin and beta-catenin, into the zonula adherens is fundamental for the function of epithelia, both in flies and in vertebrates. In C. elegans, however, the cadherin-catenin system is not essential for general adhesion, raising the question as to the genetic basis controlling junction morphogenesis in nematodes. dlg-1, the C. elegans homolog of the Drosophila tumor-suppressor gene discs-large, plays a crucial role in epithelial development. DLG-1 is restricted to adherens junctions of all embryonic epithelia, which contrasts with the localization of the Drosophila and vertebrate homologs in septate and tight junctions, respectively. Proper localization of DLG-1 requires the basolateral LET-413 protein (identified as the Drosophila scrib ortholog), but is independent of the cadherin-catenin system. Embryos in which dlg-1 activity is eliminated by RNA-mediated interference fail to form a continuous belt of junction-associated antigens and arrest development. Loss of dlg-1 activity differentially affects localization of proteins normally enriched apically to the zonula adherens. While the distribution of an atypical protein kinase C (PKC-3) and other cytoplasmic proteins (PAR-3, PAR-6) is not affected in dlg-1 (RNAi) embryos, the transmembrane protein encoded by crb-1, the C. elegans homolog of Drosophila crumbs, is no longer concentrated in this domain. In contrast to Drosophila, however, crb-1 and a second crb-like gene are not essential for epithelial development in C. elegans. Together the data indicate that several aspects of the spatial organization of epithelial cells and its genetic control differ between flies, worms, and vertebrates, while others are conserved. The molecular nature of DLG-1 makes it a likely candidate to participate in the organization of a protein scaffold that controls the assembly of junction components into the zonula adherens (Bossinger, 2001).
An abundant protein of apparent molecular mass 180 kDa has been purified from the postsynaptic density fraction of rat forebrain. Amino acid sequences of three tryptic peptides generated from the protein were obtained. The sequences were used to design a strategy cloning the cDNA encoding the protein by polymerase chain reaction. The open reading frame of the cDNA encodes a novel protein of predicted molecular mass 167 kDa. The protein has been named densin-180. Antibodies raised against the predicted amino and carboxyl sequences of densin-180 recognize a 180 kDa band on immunoblots that is enriched in the postsynaptic density fraction. Immunocytochemical localization of densin-180 in dissociated hippocampal neuronal cultures shows that the protein is highly concentrated at synapses along dendrites. The message encoding densin-180 is brain specific and is more abundant in forebrain than in cerebellum. The sequence of densin-180 contains 17 leucine-rich repeats, a sialomucin domain, an apparent transmembrane domain, and a PDZ domain. This arrangement of domains is similar to that of several adhesion molecules, in particular GPIbalpha, which mediates binding of platelets to von Willebrand factor. It is proposed that densin-180 participates in specific adhesion between presynaptic and postsynaptic membranes at glutamatergic synapses (Apperson, 1996).
The ERBB receptors play a crucial role in morphogenesis and oncogenesis. A new PDZ protein, ERBIN (ERBB2 interacting protein), has been identified that acts as an adaptor for the receptor ERBB2/HER2 in epithelia. ERBIN contains 16 leucine-rich repeats (LRRs) in its amino terminus and a PDZ (PSD-95/DLG/ZO-1) domain at its carboxy terminus, and belongs to a new PDZ protein family. The PDZ domain directly and specifically interacts with ERBB2/HER2. ERBIN and ERBB2/HER2 colocalize to the lateral membrane of human intestinal epithelial cells. The ERBIN-binding site in ERBB2/HER2 has a critical role in restricting this receptor to the basolateral membrane of epithelial cells, as mutation of the ERBIN-binding site leads to the mislocalization of the receptor in these cells. It is suggested that ERBIN acts in the localization and signaling of ERBB2/HER2 in epithelia (Borg, 2000).
Densin-180, a brain-specific protein highly concentrated at the postsynaptic density (PSD), belongs to the LAP [leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains] family of proteins, some of which play fundamental roles in the establishment of cell polarity. To identify new Densin-180-interacting proteins, a yeast two-hybrid library was screened using the COOH-terminal fragment of Densin-180 containing the PDZ domain as bait, and MAGUIN-1 was isolated as a Densin-180-binding protein. MAGUIN-1, a mammalian homologue of Drosophila connector enhancer of KSR (CNK), is known to interact with PSD-95 and has a short isoform, MAGUIN-2. The Densin-180 PDZ domain binds to the COOH-terminal PDZ domain-binding motif of MAGUIN-1. Densin-180 co-immunoprecipitates with MAGUIN-1 as well as with PSD-95 from the rat brain. In dissociated hippocampal neurones Densin-180 co-localizes with MAGUINs and PSD-95, mainly at neuritic spines. In transfected cells, Densin-180 forms a ternary complex with MAGUIN-1 and PSD-95, whereas no association was detected between Densin-180 and PSD-95 in the absence of MAGUIN-1. MAGUIN-1 forms a dimer or multimer via the COOH-terminal leucine-rich region which is present in MAGUIN-1 but not in MAGUIN-2. Among the PDZ domains of PSD-95, the first is sufficient for interaction with MAGUIN-1. These results suggest that the potential to dimerize or multimerize allows MAGUIN-1 to bind simultaneously to both Densin-180 and PSD-95, leading to the ternary complex assembly of these proteins at the postsynaptic membrane (Ohtakara, 2002).
scribble, discs large and lethal giant larvae encode proteins that regulate cell polarity and have been identified as neoplastic tumour suppressor genes in Drosophila melanogaster. The Drosophila model system was used to provide the first functional evidence that human Scribble (hScrib) can act as a tumour suppressor. hScrib protein displays highly polarized localization in mammalian epithelial cells and colocalizes with mammalian Dlg, similar to D. melanogaster Scribble (DmScrib) distribution in Drosophila epithelium. Furthermore, hScrib can rescue the polarity and tumorous overgrowth defects of scrib mutant Drosophila. hScrib therefore can act as an effective tumour suppressor in vivo, regulating both apical-basal polarity and cellular proliferation in a manner similar to that of DmScrib in Drosophila. These data demonstrate that hScrib is a functional homologue of DmScrib and therefore predict an important role for hScrib in the suppression of mammalian tumorigenesis (Dow, 2003).
In mammals, an example of planar cell polarity (PCP) is the uniform orientation of the hair cell stereociliary bundles within the cochlea. The PCP pathway of Drosophila refers to a conserved signalling pathway that regulates the coordinated orientation of cells or structures within the plane of an epithelium. A mutation in Vangl2, a mammalian homolog of the Drosophila PCP gene Strabismus/Van Gogh, results in significant disruptions in the polarization of stereociliary bundles in mouse cochlea as a result of defects in the direction of movement and/or anchoring of the kinocilium within each hair cell. Similar, but less severe, defects are observed in animals containing a mutation in the LAP protein family gene Scrb1 (homologous with Drosophila scribbled). Polarization defects in animals heterozygous for Vangl2 and Scrb1 are comparable to Vangl2 homozygotes, demonstrating genetic interactions between these genes in the regulation of PCP in mammals. These results demonstrate a role for the PCP pathway in planar polarization in mammals, and identify Scrb1 as a PCP gene (Montcouquiol, 2003).
Circletail is one of only two mouse mutants that exhibit the most severe form of neural tube defect (NTD), termed craniorachischisis. In this disorder, almost the entire brain and spinal cord is affected, owing to a failure to initiate neural tube closure. Craniorachischisis is a significant cause of lethality in humans, yet the molecular mechanisms involved remain poorly understood. This study reports the identification of the gene mutated in circletail (Crc), using a positional cloning approach. This gene, Scrb1, encodes a member of the LAP protein family related to Drosophila scribble, with 16 leucine rich repeats and four PDZ domains. The Crc mutant contains a single base insertion that creates a frame shift and leads to premature termination of the Scrb1 protein. Scrb1 expression closely mirrors the phenotypic defects observed in Crc/Crc mutants. In addition, circletail genetically interacts with the loop-tail mutant, and overlapping expression is demonstrated of Scrb1 with Vangl2, the gene mutated in loop-tail. The identification of the Crc gene further defines the nature of the genetic pathway required for the initiation of neural tube closure and provides an important new candidate that may be implicated in the aetiology of human NTDs (Murdoch, 2003).
The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP [LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1)] proteins, which play key roles in cell polarity, reach their target membrane, a structure-function study of three LAP proteins was carried out: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, since the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. These data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells (Legouis, 2003).
Among the cellular properties that are essential for the organization of tissues during animal development, the importance of cell polarity in the plane of epithelial sheets has become increasingly clear in the past decades. Planar cell polarity (PCP) signaling in vertebrates has indispensable roles in many aspects of their development, in particular, controlling alignment of various types of epithelial cells. Disrupted PCP has been linked to developmental defects in animals and to human pathology. Neural tube closure defects (NTD) and disorganization of the mechanosensory cells of the organ of Corti are commonly known consequences of disturbed PCP signaling in mammals. A typical PCP phenotype exists in a mouse mutant for the Sec24b gene, including the severe NTD craniorachischisis, abnormal arrangement of outflow tract vessels and disturbed development of the cochlea. In addition, genetic interaction was observed between Sec24b and the known PCP gene, scribble. Sec24b is a component of the COPII coat protein complex that is part of the endoplasmic reticulum (ER)-derived transport vesicles. Sec24 isoforms are thought to be directly involved in cargo selection, and evidence is presented that Sec24b deficiency specifically affects transport of the PCP core protein Vangl2, based on experiments in embryos and in cultured primary cells (Wansleeben, 2010).
Drosophila Scribble is implicated in the development of normal synapse structure and epithelial tissues, but it remains unclear how it plays a role and which process it controls. The mammalian homolog of Scribble, hScrib, has a primary structure and subcellular localization similar to that of its fly homolog, but its function remains unknown. Tandem mass spectrometry was used to identify major components of the hScrib network. It includes ßPIX (also called Cool-1), a guanine nucleotide exchange factor (GEF), and its partner GIT1 (also called p95-APP1), a GTPase activating protein (GAP). ßPIX directly binds to the hScrib PDZ domains, and the hScrib/ßPIX complex is efficiently recovered in epithelial and neuronal cells and tissues. In cerebellar granule cell cultures, hScrib and ßPIX are both partially localized at neuronal presynaptic compartments. Furthermore, hScrib is required to anchor ßPIX at the cell cortex and dominant-negative ßPIX or hScrib proteins can each inhibit Ca2+-dependent exocytosis in neuroendocrine PC12 cells, demonstrating a functional relationship between these proteins. These data reveal the existence of a tight hScrib/ßPIX interaction and suggest that this complex potentially plays a role in neuronal transmission (Audebert, 2004).
Although genetic studies have brought about significant insights into the function of Scribble in Drosophila, it remains unclear how Scribble controls epithelial and neuronal morphogenesis at the molecular level. This study has identified the hScrib-ßPIX-GIT1 complex in a variety of tissues, including brain and intestine, and demonstrated that expression of ßPIX promotes regulated exocytosis and that both GEF activity and membrane localization of ßPIX mediated by hScrib are needed in this process. How might the hScrib-ßPIX-GIT1 complex regulate exocytosis? Like hScrib, ßPIX and GIT1 are multidomain proteins engaged in multiple protein-protein interactions. For example, ßPIX is a known partner for Rac1 and Cdc42, two small GTPases that belong to the Rho protein family and are involved in dynamic reorganization of the cytoskeleton. Furthermore, ßPIX has a GEF activity toward these proteins. Interestingly, ßPIX displaying impaired GEF activity did not exhibit exocytotic activity in PC12 cells, suggesting a role of Rac1 or Cdc42 in this process. Indeed, Rac1 has recently been implicated in the control of fusion competence, and Cdc42 has been implicated in promoting the actin structure at the plasma membrane, two steps in exocytosis. Using GST-hScrib PDZ domains, it was possible to pull down a substantial fraction of Rac1 from high KCl-stimulated PC12 cell extracts. This interaction is specific because Cdc42 was not present in the ßPIX-GIT1 complex bound to the hScrib PDZ domains. In light of these data, it is believed that hScrib acts as a membrane anchor for ßPIX, which can then recruit Rac1 to form a functional complex potentially regulating exocytosis (Audebert, 2004).
Major components of the signaling network associated with hScrib have been characterized and a major role has been demonstrated of the PDZ domains in assembling the hScrib-ßPIX-GIT1 complex. Interestingly, the importance of the hScrib PDZ domains was recently highlighted by the characterization of circletail mice harboring an hScrib gene point mutation that leads to a premature termination of the protein after the second PDZ domain. These mice exhibit a profound neural-tube defect (craniorachischisis) and die soon after birth. Removal of two out of four PDZ domains in hScrib may potentially decrease interaction with the ßPIX-GIT1 complex or affect the association of hScrib with additional effectors such as Vangl2, a protein required for planar-cell polarity (Audebert, 2004).
PIX proteins have been conserved throughout evolution, and recent data have shed light on the role of the PIX homolog (dPIX or rtGEF) in neurons in Drosophila (Parnas, 2001). dpix mutants display abnormal presynaptic vesicle accumulation and morphology in neuromuscular junctions (NMJ), and these findings evoke a potential role for this exchange factor in the regulation of exocytosis in the presynaptic compartment. Interestingly, a similar hypothesis was proposed upon observation of NMJ defects in scribble mutants (Roche, 2002). Together, these data suggest that dpix and scribble might participate in a common pathway devoted to vesicle trafficking at presynaptic sites in flies. Future genetic and biochemical experiments will have to evaluate a potential functional interaction among Scribble, PIX, and GIT1 at the NMJ of flies. Drosophila Scribble plays a key function during epithelial polarization. Conservation of this LAP protein throughout evolution suggests a similar function in vertebrates. The presence of an hScrib-ßPIX complex at epithelial cell-cell junctions implies that it may also function during epithelial polarization (Audebert, 2004).
At sites of cell adhesion, proteins exist that not only perform structural tasks but also have a signaling function. The Lipoma Preferred Partner (LPP) protein is localized at sites of cell adhesion such as focal adhesions and cell-cell contacts, and shuttles to the nucleus where it has transcriptional activation capacity. LPP is a member of the zyxin family of proteins, which contains five members: ajuba, LIMD1, LPP, TRIP6 and zyxin. LPP has three LIM domains (zinc-finger protein interaction domains) at its carboxy-terminus, which are preceded by a proline-rich pre-LIM region containing a number of protein interaction domains. To catch the role of LPP at sites of cell adhesion, an effort was made to identify binding partners of LPP. The tumor suppressor protein Scrib, which is a component of cell-cell contacts, has been identified as interaction partner of LPP. Human Scrib, which is a functional homologue of Drosophila scribble, is a member of the leucine-rich repeat and PDZ (LAP) family of proteins that is involved in the regulation of cell adhesion, cell shape and polarity. In addition, Scrib displays tumor suppressor activity. The binding between Scrib and LPP is mediated by the PDZ domains of Scrib and the carboxy-terminus of LPP. Both proteins localize in cell-cell contacts. Whereas LPP is also localized in focal adhesions and in the nucleus, Scrib could not be detected at these locations in MDCKII and CV-1 cells. Furthermore, Scrib is dispensable for targeting LPP to focal adhesions and to cell-cell contacts, and LPP is not necessary for localizing Scrib in cell-cell contacts. All four PDZ domains of Scrib are dispensable for localizing this protein in cell-cell contacts. In conclusion, an interaction has been described between one of zyxin's family members, LPP, and the tumor suppressor protein Scrib. Both proteins localize in cell-cell contacts. This interaction links Scrib to a communication pathway between cell-cell contacts and the nucleus, and implicates LPP in Scrib-associated functions (Petit, 2005).
Densin-180, a protein purified from the postsynaptic density fraction of the rat forebrain, is the founding member of a newly described family of proteins termed the LAP [leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains] family that plays essential roles in establishment of cell polarity. To identify Densin-180-binding proteins, a yeast two-hybrid library was screened using the carboxyl-terminal fragment of Densin-180 containing PDZ domain as bait, and delta-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) was isolated as a Densin-180-interacting protein. delta-catenin/NPRAP, a member of the armadillo repeat family, is a nervous system-specific adherens junction protein originally discovered as an interactor with presenilin-1, a protein involved in Alzheimer's disease. Densin-180 PDZ domain binds the COOH terminus of delta-catenin/NPRAP containing the PDZ domain-binding sequence. Endogenous Densin-180 was co-immunoprecipitated with delta-catenin/NPRAP and N-cadherin. Although Densin-180 is reported to be a transmembrane protein, Densin-180 is not accessible to surface biotinylation in dissociated hippocampal neurons; hence Densin-180 may be a cytosolic protein. Densin-180 co-localizes with delta-catenin/NPRAP at synapses in delta-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with the delta-catenin/NPRAP-N-cadherin complex (Izawa, 2002a).
ERBIN, an ErbB2 receptor-interacting protein, belongs to a recently described family of proteins termed the LAP [leucine-rich repeats and PSD-95/dLg-A/ZO-1 (PDZ) domains] family, which has essential roles in establishment of cell polarity. To identify new ERBIN-binding proteins, a yeast two-hybrid library was screened, using the carboxyl-terminal fragment of ERBIN containing PDZ domain as the bait, and p0071 (also called plakophilin-4) was isolated as an ERBIN-interacting protein. p0071 is a member of the p120 catenin family, defined as proteins with 10 armadillo repeats; these proteins localize along the cell-cell border. The ERBIN PDZ domain binds the COOH-terminus of p0071 containing the PDZ domain-binding sequence. Endogenous ERBIN co-immunoprecipitates with p0071. In fully polarized Madin-Darby canine kidney (MDCK) cells, ERBIN co-localizes largely with beta-catenin and partly with desmoplakin along the lateral plasma membrane domain. At these cell-cell contact regions, ERBIN co-localizes with p0071. Over-expression of the dominant active forms of Cdc42, Rac1 or RhoA, Rho family small GTPases, results in a marked accumulation of ERBIN at the cell-cell contacts of MDCK and HeLa cells. These results show that ERBIN interacts in vivo with p0071 and that it may be involved in the organization of adherens junctions and the desmosomes of epithelia. In addition, the subcellular localization of ERBIN might be regulated by Rho family small GTPases (Izawa, 2002b).
Integrity of epithelial tissues relies on the proper apical-basolateral polarity of epithelial cells. Members of the LAP (LRR and PDZ) protein family such as LET-413 and Scribble are involved in maintaining epithelial cell polarity in Caenorhabditis elegans and Drosophila melanogaster, respectively. Erbin is a mammalian LET-413 homologue interacting with ERBB2/HER2, an epidermal growth factor receptor family member. Erbin and ERBB2/HER2 are located in the basolateral membranes of epithelial cells. Erbin interacts with p0071 (also called plakophilin-4), an armadillo repeat protein linked to the cytoskeleton. Erbin binds to p0071 in vitro and in vivo in a PDZ domain-dependent manner, and both proteins colocalize in desmosomes of epithelial cells. Using a dominant negative approach, it was found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. It is proposed that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis (Jaulin-Bastard, 2002).
Erbin is a member of the LAP (leucine-rich repeat and PDZ domain) protein family. A C-terminally displayed phage peptide library was used to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bind with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resemble the C-terminal PDZ domain-binding motifs of three p120-related catenins: delta-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or delta-catenin also demonstrates specific high affinity binding. The interactions between the Erbin PDZ domain and both ARVCF and delta-catenin were characterized in vitro and in vivo. The Erbin PDZ domain co-localizes and coprecipitates with ARVCF or delta-catenin complexed with beta-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments show that the association of Erbin with the cadherin-catenin complex is mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and delta-catenin. Finally, endogenous delta-catenin and Erbin were shown to co-localize in and co-immunoprecipitate from neurons. These results suggest that delta-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains (Laura, 2002).
How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel provide a unique experimental paradigm for defining the role and control of single-cell orientation for tissue-level morphogenesis in vivo. This study shows that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. This study shows that Scribble is required for oriented cell division, and its function in this process is independent of canonical apicobasal and planar polarity pathways. A role is identified for Scribble in controlling clustering of β-catenin foci in dividing progenitors. Loss of either Scrib or N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions, and similar neural tube defects. It is proposed that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, the data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis (Zigman, 2011).
In the developing vertebrate hindbrain, the characteristic trajectory of the facial (nVII) motor nerve is generated by caudal migration of the nVII motor neurons. The nVII motor neurons originate in rhombomere (r) 4, and migrate caudally into r6 to form the facial motor nucleus. Using a transgenic zebrafish line that expresses green fluorescent protein (GFP) in the cranial motor neurons, two novel mutants, designated landlocked (llk) and off-road (ord), have been isolated that both show highly specific defects in the caudal migration of the nVII motor neurons. The landlocked locus contains the gene scribble1 (scrb1), and its zygotic expression is required for migration of the nVII motor neurons mainly in a non cell-autonomous manner. Taking advantage of the viability of the llk mutant embryos, it was found that maternal expression of scrb1 is required for convergent extension (CE) movements during gastrulation. Furthermore, a genetic interaction is seen between scrb1 and trilobite(tri)/strabismus(stbm) in CE. The dual roles of the scrb1 gene in both neuronal migration and CE provide a novel insight into the underlying mechanisms of cell movement in vertebrate development (Wada, 2005).
Although the results suggest that there is a genetic interaction between scrb1 and stbm, it is not known whether the PDZ domains of Scrb directly interact with the PDZ-binding domain of Stbm. In Drosophila, the second PDZ domain of Scrb interacts with Dlg via GUKH (guanylate kinase holder protein) to form a scaffolding complex at synaptic junctions. Furthermore, Dlg interacts with Stbm and this complex is required for plasma membrane formation in epithelial cells. These results suggest that Scrb, Stbm and Dlg may constitute a functional complex during the formation of membrane structures. If Tri/Stbm and Llk/Scrb1 form a functional complex, this complex would probably have two sites that associate with membranes: the transmembrane domain of Tri/Stbm and the LRR domain of Llk/Scrb1. Knock-down of Tri/Stbm with overexpression of Llk/Scrb1 leads to the most severe impairment of CE. These results indicate that Tri/Stbm may be required for localization of Llk/Scrb1 protein to the specific site of the membrane where they are anchored and function together. Release of membrane-associated Llk/Scrb1 from such positional constraint in the absence of Stbm may have more markedly perturbed the functional protein complexes controlling CE than simple overexpression of Scrb1 in the presence of Stbm (Wada, 2005).
The Scrb1rw16 protein, which has a single amino acid substitution in the first PDZ domain, has lower activity than the wild-type protein to rescue migration of the nVII motor neurons in the llk mutation. Similarly, overexpression of Scrb1rw16 induces CE defects to a lesser extent than that of wild-type Scrb1 protein. These results indicate that the first PDZ domain is also essential for Scrb1 activity. The first PDZ domain of Llk/Scrb1 may interact with another, as yet unidentified, component to establish a multi-protein complex required for its function (Wada, 2005).
Scribble (Scrib) is a conserved polarity protein required in Drosophila for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. Knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to ßPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration (Qin, 2005).
During epithelial morphogenesis, a complex comprising the βPIX (PAK-interacting exchange factor β) and class I PAKs (p21-activated kinases) is recruited to adherens junctions. Scrib, the mammalian ortholog of the Drosophila polarity determinant and tumor suppressor Scribble, binds βPIX directly. Scrib is also targeted to adherens junctions by E-cadherin, where Scrib strengthens cadherin-mediated cell-cell adhesion. Although a role for the Scrib-βPIX-PAK signaling complex in promoting membrane protrusion at wound edges has been elucidated, a function for this complex at adherens junctions remains unknown. This study establish, in cultured mammalian cells, that Scrib targets βPIX and PAK2 to adherens junctions where a βPIX-PAK2 complex counterbalances apoptotic stimuli transduced by Scrib and elicited by cadherin-mediated cell-cell adhesion. Moreover, it was shown that this signaling pathway regulates cell survival in response to osmotic stress. Finally, it was determined that in suspension cultures, the Scrib-βPIX-PAK2 complex functions to regulate anoikis elicited by cadherin engagement, with Scrib promoting and the βPIX-PAK2 complex suppressing anoikis, respectively. These findings demonstrate that the Scrib-βPIX-PAK2 signaling complex functions as an essential modulator of cell survival when localized to adherens junctions of polarized epithelia. The activity of this complex at adherens junctions is thereby essential for normal epithelial morphogenesis and tolerance of physiological stress. Furthermore, when localized to adherens junctions, the Scrib-βPIX-PAK2 signaling complex serves as a key determinant of anoikis sensitivity, a pivotal mechanism in tumor suppression. Thus, this work also reveals the need to expand the definition of anoikis to include a central role for adherens junctions (Frank, 2012).
This study has established that differentiated epithelial cells rely on a survival signaling network associated with adherens junctions that is distinct from that used at cell-matrix adhesions. Specifically, it was demonstrated that epithelial cells are critically dependent on Scrib-mediated localization of a βPIX-PAK2 complex to adherens junctions to counterbalance the apoptosis-promoting effects of E-cadherin engagement. As a result, disruption of βPIX-PAK2 signaling results in a near complete loss of epithelial viability at confluent density. Consistent with an established role in phosphorylating cellular targets involved in apoptosis, a functional kinase domain is required for PAK2-dependent survival signaling. In subconfluent cultures, where the βPIX-PAK2 complex localizes to focal adhesions, the complex is dispensable for cell survival. Thus, as epithelial cells form cell-cell junctions and polarize, they become critically dependent on a prosurvival signal provided by the βPIX-PAK2 complex at adherens junctions (Frank, 2012).
To date, the only direct evidence for the function of PAKs at adherens junctions is in the regulation of cadherin adhesiveness and actin-dependent cell contractility. While invoked by prior findings, the current results demonstrate for the first time that Scrib via its association with βPIX promotes targeting of PAK2 to adherens junctions. In cultured human keratinocytes, PAK1 has been reported to augment cadherin adhesiveness in response to activated Rac. PAK1 and PAK2 have also been reported to enhance and suppress, respectively, loss of cell-cell contacts in response to hepatocyte growth factor. The current results suggest that neither PAK1 nor PAK2 play a major role in steady state junctional integrity in MDCK cells. However, because MDCK cells express both PAK1 and PAK2, the possibility cannot be excluded that they may function redundantly in regulation of E-cadherin adhesiveness and/or junctional remodeling (Frank, 2012).
βPIX and PAKs have been most extensively characterized for their role in focal adhesion dynamics, where they play a coordinated role in regulating turnover of these integrin attachment sites and promoting directional motility. PAK1 and PAK2 appear to have nonredundant functions in cell invasion and motility. Moreover, there is evidence to suggest that when localized to focal adhesions, the βPIX-PAK complex promotes mitogenic signaling and that redistribution of the complex from focal adhesions to adherens junctions contributes to the cessation of epithelial proliferation and establishment of contact inhibition. Scrib also shuttles between adherens junctions and protrusive membrane structures where it promotes βPIX-PAK complex-dependent cell motility. It will be interesting to determine whether Scrib will play a role in redistribution of the βPIX-PAK2 complex from focal adhesions to adherens junctions, which occurs as cells undergo contact inhibition. In brief, taken together with the present work, these studies suggest that PAKs have distinct roles when localized to focal adhesions and adherens junctions. When localized to focal adhesions, PAKs participate in signaling pathways that control proliferation and motility, whereas at adherens junctions PAKs regulate adhesiveness and survival. As such, the translocation of the βPIX-PAK complex from to lateral membranes upon formation of stable adherens junctions likely plays a fundamental role in the transition from motile and mitogenic states to a nonmotile and quiescent state (Frank, 2012).
E-cadherin is a potent inhibitor of multiple signaling pathways and plays a fundamental role in suppression of motility and proliferation upon establishment of cell-cell contact. The ability of Scrib to promote E-cadherin adhesiveness may underlie some of its tumor suppressive potential in mammalian cells. However, increasing evidence suggests that Scrib also plays E-cadherin-independent roles in the regulation of signaling pathways, such as inhibition of ERK and AKT, as well as activation of Hippo signaling. These Scrib-dependent effects would be predicted to sensitize cells to apoptosis; a prediction supported by the present results, which furthermore establish that Scrib-mediated apoptosis is counterbalanced by its recruitment of active PAK2. Taken together, these findings demonstrate that Scrib transduces both pro- and anti-apoptotic stimuli (Frank, 2012).
Metastasis requires that cells tolerate the loss of matrix adhesion, i.e., that they are protected against anoikis. However, in spite of seminal work suggesting a role for cell-cell adhesion in modulating anoikis, the role for epithelial architecture in anoikis remains unappreciated and poorly characterized. Nevertheless, there is growing evidence indicating that loss of E-cadherin function suffices to abrogate anoikis. The current results support a key role of adherens junctions in modulating anoikis. In simple polarized epithelia, which likely rely on the summation of survival signals emanating from both focal adhesions and adherens junctions, apoptotic stimuli emanating from E-cadherin will sensitize cells to anoikis. In contrast, suprabasal cells in stratified epithelia depend exclusively on survival signals from adherens junctions. PAK2 is highly expressed in suprabasal keratinocytes, which may suggest that upregulation of PAK2-dependent survival signals emanating from adherens junctions are required to offset loss of integrin-mediated survival signaling. In summary, the current results indicate that E-cadherin does not function simply as a prosurvival or proapoptotic factor but rather as a regulatory node to coordinate death and survival signaling. The balance between pro- and antiapoptotic signaling emanating from adherens junctions plays a key role in epithelial cell viability and is likely essential in regulating diverse processes, including epithelial morphogenesis, wound healing, physiological stress, and metastasis (Frank, 2012).
Wnt signaling effectors direct the development and adult remodeling of the female reproductive tract (FRT); however, the role of non-canonical Wnt signaling has not been explored in this tissue. The non-canonical Wnt signaling protein van gogh-like 2 is mutated in loop-tail (Lp) mutant mice (Vangl2Lp), which display defects in multiple tissues. Vangl2Lp mutant uterine epithelium displays altered cell polarity, concommitant with changes in cytoskeletal actin and scribble (scribbled, Scrb1) localization. The postnatal mutant phenotype is an exacerbation of that seen at birth, exhibiting more smooth muscle and reduced stromal mesenchyme. These data suggest that early changes in cell polarity have lasting consequences for FRT development. Furthermore, Vangl2 is required to restrict Scrb1 protein to the basolateral epithelial membrane in the neonatal uterus, and an accumulation of fibrillar-like structures observed by electron microscopy in Vangl2Lp mutant epithelium suggests that mislocalization of Scrb1 in mutants alters the composition of the apical face of the epithelium. Heterozygous and homozygous Vangl2Lp mutant postnatal tissues exhibit similar phenotypes and polarity defects and display a 50% reduction in Wnt7a levels, suggesting that the Vangl2Lp mutation acts dominantly in the FRT. These studies demonstrate that the establishment and maintenance of cell polarity through non-canonical Wnt signaling are required for FRT development (Vandenberg, 2009).
Components of the planar cell polarity (PCP) pathway are required for the caudal tangential migration of facial branchiomotor (FBM) neurons, but how PCP signaling regulates this migration is not understood. In a forward genetic screen, a new gene was identified, nhsl1b, that is required for FBM neuron migration. nhsl1b encodes a WAVE-homology domain-containing protein related to human Nance-Horan syndrome (NHS) protein and Drosophila GUK-holder (Gukh), which have been shown to interact with components of the WAVE regulatory complex that controls cytoskeletal dynamics and with the polarity protein Scribble, respectively. Nhsl1b localizes to FBM neuron membrane protrusions and interacts physically and genetically with Scrib to control FBM neuron migration. Using chimeric analysis, it was shown that FBM neurons have two modes of migration: one involving interactions between the neurons and their planar-polarized environment, and an alternative, collective mode involving interactions between the neurons themselves. The first mode of migration requires the cell-autonomous functions of Nhsl1b and the PCP components Scrib and Vangl2 in addition to the non-autonomous functions of Scrib and Vangl2, which serve to polarize the epithelial cells in the environment of the migrating neurons. These results define a role for Nhsl1b as a neuronal effector of PCP signaling and indicate that proper FBM neuron migration is directly controlled by PCP signaling between the epithelium and the migrating neurons (Walsh, 2011).
The appropriate trafficking of glutamate receptors to synapses is crucial for basic synaptic function and synaptic plasticity. It is now accepted that NMDA receptors (NMDARs; see Drosophila NMDA receptors) internalize and are recycled at the plasma membrane but also exchange between synaptic and extrasynaptic pools; these NMDAR properties are also key to governing synaptic plasticity. Scribble1 is a large PDZ protein required for synaptogenesis and synaptic plasticity. This study shows that the level of Scribble1 is regulated in an activity-dependent manner and that Scribble1 controls the number of NMDARs at the plasma membrane. Notably, Scribble1 prevents GluN2A subunits from undergoing lysosomal trafficking and degradation by increasing their recycling to the plasma membrane following NMDAR activation. Finally, it was shown that a specific YxxR motif on Scribble1 controls these mechanisms through a direct interaction with AP2 (see Drosophila AP2). Altogether, these findings define a molecular mechanism to control the levels of synaptic NMDARs via Scribble1 complex signaling (Piguel, 2014).
Human scribble (hScrib), human homolog of the Drosophila tumor suppressor Scribble, has been identified a substrate of human papillomavirus E6 oncoproteins for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP. Human Scribble, classified as a LAP protein containing leucine-rich repeats and PDZ domains, interacts with E6 through its PDZ domains and C-terminal PDZ domain-binding motif of E6 protein. Interaction between human Discs Large (hDlg), a substrate of E6 for the ubiquitin-mediated degradation, and adenomatous polyposis coli (APC) has been shown. This study investigated whether hScrib and APC interact with each other in vitro and in vivo. Interaction between hScrib and APC is mediated by the PDZ domains 1 and 4 of hScrib and C-terminal PDZ domain-binding motif of APC. Human Scribble co-localizes with APC at the synaptic sites of hippocampal neuron and at the tip of membrane protrusion in the epithelial cell line. Interference of the interaction between hScrib and APC causes disruption of adherens junction. Knockdown of hScrib expression by RNAi disrupts localization of APC at the adherens junction. These data suggest that hScrib may participate in the hDlg-APC complex through its PDZ domains and regulate cell cycle and neural function by associating with APC (Takizawa, 2006).
Drosophila tumor suppressor Scribble has been identified as an apical-basolateral polarity determinant in epithelia. A human homolog of Drosophila Scribble, human Scribble (hScrib), has been identified as a protein targeted by human papillomavirus E6 for the ubiquitin-mediated degradation dependent on E6AP, a cellular ubiquitin-protein ligase. Human Scribble is classified as a LAP protein, having leucine-rich repeats (LRRs) and PDZ domains. Whether hScrib, which is thought to have a role in polarity determination based on the data of its Drosophila homolog, is involved in cell-cycle regulation and proliferation control of epithelia was investigated. Transfection of hScrib inhibits cell-cycle progression from G1 to S phase, and it up- and down-regulates expression of adenomatous polyposis coli and cyclins A and D1, respectively. Knockdown of hScrib expression by siRNA leads to cell-cycle progression from G1 to S phase. Functional domain mapping was explored to reveal which domains of hScrib are critical for its cellular proliferation control and localization at the basolateral membrane. LRRs and PDZ domain 1 were found to be indispensable for hScrib to inhibit cell growth by blocking cell-cycle progression and to keep its proper localization. These data indicate that basolateral membrane localization of hScrib is closely related to its proliferation control. These findings suggest the possibility that hScrib is involved in signal transduction to negatively regulate cell proliferation by localizing at the basolateral membrane of epithelial cells through LRRs and PDZ domains (Nagasaka, 2006).
This study asked if the mouse homolog of Drosophila Scribbled is required for establishment and/or maintenance of epithelial identity in vivo. To do so, Scrib was conditionally deleted in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting epithelial-mesenchymal transition (EMT) was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of alphaSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling (Yamben, 2013).
Scribble (SCRIB) localizes to cell-cell junctions and regulates establishment of epithelial cell polarity. Loss of expression of SCRIB functions as a tumor suppressor in Drosophila and mammals, conversely, overexpression of SCRIB promotes epithelial differentiation in mammals. This study reports that SCRIB is frequently amplified, mRNA over-expressed and protein is mislocalized from cell-cell junctions in human breast cancers. High levels of SCRIB mRNA are associated with poor clinical prognosis identifying an unexpected role for SCRIB in breast cancer. Transgenic mice expressing a SCRIB mutant (Pro 305 to Leu (P305L)) that fails to localize to cell-cell junctions, under the control of the mouse mammary tumor virus long terminal repeat promoter, develop multifocal hyperplasia that progresses to highly pleomorphic and poorly differentiated tumors with basal characteristics. SCRIB interacts with PTEN and the expression of P305L, but not wild-type SCRIB, promotes an increase in PTEN levels in the cytosol. Overexpression of P305L, but not wild type SCRIB, activates the Akt/mTOR/S6K signaling pathway. Human breast tumors overexpressing SCRIB have high levels of S6K but do not harbor mutations in PTEN or PIK3CA, identifying SCRIB amplification as a mechanism of activating PI3K signaling in tumors without mutations in PIK3CA or PTEN. Thus, this study has demonstrated that high levels of mislocalized SCRIB functions as a neomorph to promote mammary tumorigenesis by affecting subcellular localization of PTEN and activating an Akt/mTOR/S6kinase signaling pathway (Feigin, 2014).
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