Presenilins interact with armadillo proteins including neural-specific plakophilin-related protein and beta-catenin

Abstract : Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early-onset familial Alzheimer's disease. We have used yeast-two-hybrid and coimmunoprecipitation methods to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including β-catenin, p0071, and a novel neuronal-specific armadillo protein—neural plakophilin-related armadillo protein (NPRAP). The PS1 : NPRAP interaction occurs between the arm repeats of NPRAP and residues 372-399 at the C-terminal end of the large cytoplasmic loop of PS1. The latter residues contain a single arm -like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.     

PAPIN. A novel multiple PSD-95/Dlg-A/ZO-1 protein interacting with neural plakophilin-related armadillo repeat protein/delta-catenin and p0071

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.     

The p120 family of cell adhesion molecules

p120 is the prototypic member of the p120 subfamily of armadillo-related proteins that includes p0071, delta-catenin/NPRAP, ARVCF and the more distantly related plakophilins 1-3. Like armadillo, beta-catenin and plakoglobin these proteins are involved in mediating cell-cell adhesion. Besides their junctional localization they also reveal a cytoplasmic and nuclear localization. Non-cadherin-associated, cytoplasmic p120 functions in Rho signaling and regulation of cytoskeletal organization and actin dynamics. The nuclear function remains largely unsolved. Some characteristics seem to be shared by the various members of the family but it seems unlikely that p120-related proteins have solely redundant functions and compete for interactions with identical binding partners. Stabilization of cadherins at the membrane seems a common function of p120, p0071, delta-catenin and ARVCF but it is not yet known if and how these proteins confer distinct properties to cellular junctions. Moreover, p0071, NPRAP and ARVCF have a C-terminal PDZ-binding motif that is lacking in p120 pointing to distinct roles of these proteins. PDZ domains are found in a series of proteins involved in establishing cell polarity in epithelial cells. Thus, p120 proteins may not only be master regulators of cadherin abundance and activity but play additional roles in regulating cell polarity. This review focuses on the putative roles of p120 proteins in cell polarity.     

The Armadillo family protein p0071 is a VE-cadherin- and desmoplakin-binding protein

p0071, a member of the armadillo protein family, localizes to both adherens junctions and desmosomes in epithelial cells and exhibits homology to the adherens junction protein p120 and the desmosomal protein plakophilin-1. p0071 is also present at dermal microvascular endothelial intercellular junctions and colocalizes with VE-cadherin, an endothelium-specific cadherin that associates with both actin and intermediate filament networks. To define the role of p0071 in junction assembly, p0071 was tested for interactions with other components of the endothelial junctional complex. In transient expression assays, p0071 colocalized with and formed complexes with both VE-cadherin and desmoplakin. Deletion analysis using the yeast two-hybrid system revealed that the armadillo repeat domain of p0071 bound directly to VE-cadherin. Site-directed mutagenesis experiments demonstrated that p0071 and p120 bound to the same region on the cytoplasmic tail of VE-cadherin and that overexpression of p0071 could displace p120 from intercellular junctions. In contrast to VE-cadherin, desmoplakin was found to associate with the non-armadillo head domain of p0071. Cotransfections and triple-label immunofluorescence analysis revealed that VE-cadherin colocalization with desmoplakin in transfected COS cells required p0071, suggesting that p0071 may couple VE-cadherin to desmoplakin. Based on previous findings that both VE-cadherin and desmoplakin play central roles in vasculogenesis, these new results suggest that p0071 may play an important role in endothelial junction assembly and in the morphogenic events associated with vascular remodeling.     

Interaction between Erbin and a Catenin-related protein in epithelial cells.

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. We previously described Erbin as 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. We show here that 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 colocalized in desmosomes of epithelial cells. Using a dominant negative approach, we found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. We propose that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis.     

Folliculin,the Product of the Birt-Hogg-Dube Tumor Suppressor Gene,Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd^flox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.     

A myristoylated calcium-binding protein that preferentially interacts with the Alzheimer's disease presenilin 2 protein.

It is well established that mutations in the presenilin 1 and 2 genes cause the majority of early onset Alzheimer's disease (AD). However, our understanding of the cellular functions of the proteins they encode remains rudimentary. Knowledge of proteins with which the presenilins interact should lead to a better understanding of presenilin function in normal and disease states. We report here the identification of a calcium-binding protein, calmyrin, that interacts preferentially with presenilin 2 (PS2). Calmyrin is myristoylated, membrane-associated, and colocalizes with PS2 when the two proteins are overexpressed in HeLa cells. Yeast two-hybrid liquid assays, affinity chromatography, and coimmunoprecipitation experiments confirm binding between PS2 and calmyrin. Functionally, calmyrin and PS2 increase cell death when cotransfected into HeLa cells. These results allude to several provocative possibilities for a dynamic role of calmyrin in signaling, cell death, and AD.     

The armadillo protein p0071 regulates Rho signalling during cytokinesis

Cytokinesis requires the spatio-temporal coordination of cell-cycle control and cytoskeletal reorganization. Members of the Rho-family of GTPases are crucial regulators of this process and assembly of the contractile ring depends on local activation of Rho signalling. Here, we show that the armadillo protein p0071, unlike its relative p120(ctn), is localized at the midbody during cytokinesis and is essential for cell division. Both knockdown and overexpression of p0071 interfered with normal cell growth and survival due to cytokinesis defects with formation of multinucleated cells and induction of apoptosis. This failure of cytokinesis seemingly correlated with the deregulation of Rho activity in response to altered p0071 expression. The function of p0071 in regulating Rho activity occurred through an association of p0071 with RhoA, as well as the physical and functional interaction of p0071 with Ect2, the one Rho guanine-nucleotide exchange factor (GEF) essential for cytokinesis. These findings support an essential role for p0071 in spatially regulating restricted Rho signalling during cytokinesis.     

Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.     

Corrupted ER‐mitochondrial calcium homeostasis promotes the collapse of proteostasis

Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.     

Presenilins are required for the formation of comma- and S-shaped bodies during nephrogenesis

Mammalian presenilins consist of two highly homologous proteins, PSEN1 and PSEN2, which share redundant activities in Notch processing and signaling. To bypass the early lethality of the Psen1- and Psen2-double (PSEN) null embryos, we used a human PSEN1 transgene to rescue the somite patterning defects in PSEN-null animals and to allow a determination of the function of presenilins in late embryogenesis. We report here that expression of the human PSEN1 transgene supported the survival of PSEN-null embryos to the perinatal stage. However, presenilin deficiency in the kidney led to severe nephrogenesis defects and virtually no comma- or S-shaped bodies, or mature glomeruli were formed. We document that the mesenchyme was induced which could further progress to renal vesicles in the PSEN-null kidney, indicating that the presenilins are not essential for the inductive interactions and mesenchyme to epithelium transition. However, renal vesicles failed to pattern to form proximal tubules and glomerular epithelium. A presenilin-dependent, signaling-competent form of Notch1 was detected in mesenchymal derivatives but not in the ureteric buds of wild-type mice. Consistent with an obligatory role of presenilins in Notch processing and activation, the active form of Notch1 and its downstream target Hesr1 were absent in the PSEN-null kidney. Importantly, sustained Notch1 signaling was required for the maintenance of Notch ligand Jag1 expression. These results identify presenilins as one determinant of renal vesicle patterning in the developing mouse kidney, and we hypothesize that they act through the Notch signaling pathway.     

The presenilins

The presenilins are evolutionarily conserved transmembrane proteins that regulate cleavage of certain other proteins in their transmembrane domains. The clinical significance of this regulation is shown by the contribution of presenilin mutations to 20-50% of early-onset cases of inherited Alzheimer's disease. Although the precise molecular mechanism underlying presenilin function or dysfunction remains elusive, presenilins are thought to be part of a complex of proteins that has 'γ-secretase cleavage' activity, which is clearly central in the pathogenesis of Alzheimer's disease. Mutations in presenilins increase the production of the longer isoforms of amyloid β peptide, which are neurotoxic and prone to self-aggregation. Biochemical studies indicate that the presenilins do not act alone but operate within large heteromeric protein complexes, whose components and enzymatic core are the subject of much study and controversy; one essential component is nicastrin. The presenilin primary sequence is remarkably well conserved in eukaryotes, suggesting some functional conservation; indeed, defects caused by mutations in the nemotode presenilin homolog can be rescued by human presenilin.     

Plakophilins: Multifunctional proteins or just regulators of desmosomal adhesion?

Plakophilins 1-3 are members of the p120(ctn) family of armadillo-related proteins. The plakophilins have been characterized as desmosomal proteins, whereas p120(ctn) and the closely related delta-catenin, ARVCF and p0071 associate with adherens junctions and play essential roles in stabilizing cadherin mediated adhesion. Recent evidence suggests that plakophilins are essential components of the desmosomal plaque where they interact with desmosomal cadherins as well as the cytoskeletal linker protein desmoplakin. Plakophilins stabilize desmosomal proteins at the plasma membrane and therefore may function in a manner similar to p120(ctn) in the adherens junctions. The three plakophilins reveal distinct expression patterns, and although partially redundant in their function, mediate distinct effects on desmosomal adhesion. Besides a structural role, a function in signaling has been postulated in analogy to other armadillo proteins such as beta-catenin. At least plakophilins 1 and 2 are also localized in the nucleus, and all three proteins occur in a cytoplasmic pool. This review aims to summarize the current knowledge of plakophilin function in the context of cell adhesion, signaling and their putative role in diseases.     

The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.     

Presenilins promote the cellular uptake of copper and zinc and maintain copper chaperone of SOD1-dependent copper/zinc superoxide dismutase activity

Dyshomeostasis of extracellular zinc and copper has been implicated in β-amyloid aggregation, the major pathology associated with Alzheimer disease. Presenilin mediates the proteolytic cleavage of the β-amyloid precursor protein to release β-amyloid, and mutations in presenilin can cause familial Alzheimer disease. We tested whether presenilin expression affects copper and zinc transport. Studying murine embryonic fibroblasts (MEFs) from presenilin knock-out mice or RNA interference of presenilin expression in HEK293T cells, we observed a marked decrease in saturable uptake of radiolabeled copper and zinc. Measurement of basal metal levels in 6-month-old presenilin 1 heterozygous knock-out (PS1(+/-)) mice revealed significant deficiencies of copper and zinc in several tissues, including brain. Copper/zinc superoxide dismutase (SOD1) activity was significantly decreased in both presenilin knock-out MEFs and brain tissue of presenilin 1 heterozygous knock-out mice. In the MEFs and PS1(+/-) brains, copper chaperone of SOD1 (CCS) levels were decreased. Zinc-dependent alkaline phosphatase activity was not decreased in the PS null MEFs. These data indicate that presenilins are important for cellular copper and zinc turnover, influencing SOD1 activity, and having the potential to indirectly impact β-amyloid aggregation through metal ion clearance.     

Identification of a novel family of putative methyltransferases that interact with human and Drosophila presenilins.

Mutations in the presenilin genes have been shown to cause the majority of cases of early-onset familial Alzheimer's disease (AD). In addition to their role in AD, presenilins are also known to function during development by interacting with the Notch pathway. To determine if presenilins have additional functions during development and AD we have used a yeast two-hybrid approach to search for proteins that can bind to presenilins. Here, we show the identification and characterization of a novel putative methyltransferase (Metl) that interacts with the loop region of Drosophila presenilin as well as human presenilin-1 and presenilin-2, suggesting that this interaction is evolutionarily conserved and functionally important. Metl appears to be a member of a conserved family of methyltransferases that share homology with, but are distinct from, the UbiE family of methyltransferases involved in ubiquinone and menaquinone biosynthesis. In Drosophila, the metl gene gives rise to two major isoforms by alternative splicing that are broadly expressed throughout development and found in the central nervous system in an overlapping pattern with Drosophila presenilin. Finally, we show that two independent dominant adult phenotypes produced by overexpression of presenilin can be enhanced by overexpression of metl in the same tissue. Taken together, these results suggest that presenilin and Metl functionally and genetically interact during development.     

A role for calsenilin and related proteins in multiple aspects of neuronal function

The protein variously called calsenilin, DREAM, or KChIP3 has been independently discovered three times, accounting for the three different names currently in use. Calsenilin appears to have three, perhaps independent, roles. Calsenilin binds and modulates some of the effects of the Alzheimer disease-related protein presenilin, while presenilin can modulate some of the effects of calsenilin. Calsenilin binds the dynorphin response element and regulates dynorphin expression, hence regulating nociception. Calsenilin binds Kv channels and modulates potassium conductance, playing a role in long-term potentiation as well as in other important plastic pathways. Other members of the calsenilin family share at least some of the roles. For example, KChIP1, KChIP2, and CALP can all bind presenilins and can all modulate A-type potassium channels. Further functional dissection of this family of proteins will provide insight into numerous aspects of neuronal function and will illuminate the role of the calsenilin family of proteins in disease.