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.     

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.     

Cell polarity protein Par3 complexes with DNA-PK via Ku70 and regulates DNA double-strand break repair

The author affiliations were mixed up in the previous published version. The third fund number of National Natural Science Foundation of China in the Acknowledgments was wrong, it should be ＂30270335＂. The Shanghai Municipal Council for Science and Technology （No.06DZ22032） was missed in the Acknowledgments. There are some labeling and production errors in Figure 2A, Figure 3B and 3C, Figure 5C, Figure 6B and 6E, Figure 7B and 7D.     

Proteome identification of binding-partners interacting with cell polarity protein Par3 in Jurkat cells

The evolutionarily conserved cell polarity protein Par3, a scaffold-like PDZreontaining protein, plays a critical role in the establishment and maintenance of epithelial cell polarity. Although the role of Par3 in establishing cell polarity in epithelial cells has been intensively explored, the function of Par3 in hematopoietic cells remains elusive. To address this issue, we generated GST-fusion proteins of Par3 PDZ domains. By combiningthe GST-pull-down approach with liquid chromatography-tandem mass spectrometry, we identified 10 potential novel binding proteins of PDZ domains of Par3 in Jurkat cells (a T-cell line). The interaction of Par3 with three proteins—nuclear transport protein importin-α4 and proteasome activators PA28β and PA28γ—was confirmed using in vitro binding assay, co-immunoprecipitation assay and immunofluorescence microscopy. Our results have the potential to uncover novel functions of the cell polarity protein Par3 in blood cells.     

The G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and Gialpha3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells

We addressed the role of the G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and G-proteins (Gialpha3) in the positioning of the spindle pole during mammalian cell division. Immunocytochemistry indicated that both LGN and Gialpha3 co-localized at the spindle pole and at the midbody and the cell cortex during the different phases of mitosis. In marked contrast to the positioning of the spindle pole at metaphase midway between the cell cortex and the metaphase plate, the spindle pole was juxtaposed with the cell cortex at metaphase following increased expression of Gialpha3 and LGN. This repositioning of the spindle pole required the interaction of LGN with Gialpha. The influence of LGN and Gialpha3 on the cortical positioning of the spindle pole likely reflects either stronger pulling forces on the spindle pole exerted from the cell cortex or increased pushing forces exerted on the spindle pole from the mitotic spindle indicating that these events are regulated by GPR motif-containing proteins and G-proteins independent of asymmetry.     

PAR3beta,a novel homologue of the cell polarity protein PAR3, localizes to tight junctions

The cell polarity protein PAR3, conserved from the nematode to the vertebrate, forms a complex with PAR6 and atypical protein kinase C (aPKC), and the protein complex occurs at the tight junctions in mammalian epithelial cells. Here we have cloned human cDNA for a novel PAR3 homologue, designated PAR3beta, whose messages are present in a variety of tissues and most abundantly expressed in the adult and fetal kidneys. The encoded protein of 1,205 amino acids contains a region homologous to the aPKC-binding domain of PAR3alpha, another human homologue previously identified, and three PDZ domains; the first PDZ domain of PAR3alpha is considered to interact with PAR6. Unexpectedly, in contrast to other PAR3s found in various species, PAR3beta is incapable of binding to any isotypes of PAR6 or aPKC. Nevertheless PAR3beta, expressed intrinsically or extrinsically, localizes to the tight junctions, indicating that the localization does not require the ternary complex formation.     

Cathepsin B links oxidative stress to the activation of NLRP3 inflammasome

Oxidative stress-mediated activation of NLRP3 inflammasome in microglia is critical in the development of neurodegerative diseases such as Alzheimer's disease (AD), Parkinson disease (PD). However, the mechanism underlying oxidative stress activates NLRP3 inflammasome remains exclusive. Here we demonstrated cathepsin B (CTSB) as a regulator of the activation of NLRP3 inflammasome by H₂O₂·H₂O₂ induced IL-1β secretion in NLRP3 inflammasome-dependent manner·H₂O₂ treatment increased CTSB activity, which in turn activated NLRP3 inflammasome, and subsequently processed pro-caspase-1 cleavage into caspase-1, resulting in IL-1 β secretion. Genetic inhibition or pharmacological inhibition of CTSB blocked the cleavage of pro-caspase-1 into caspase-1 and subsequent IL-1 β secretion induced by H₂O₂. Importantly, CTSB activity, IL-1β levels and malondialdehyde (MDA) were remarkably elevated in plasma of AD patients compared to healthy controls, while glutathione was significantly lower than healthy controls. Correlation analyses showed that CTSB activity was positively correlated with IL-1β and MDA levels, but negatively correlated with GSH levels in plasma of AD patients. Taken together, our results indicate that oxidative stress activates NLRP3 through upregulating CTSB activity. Our results identify an important biological function of CTSB in neuroinflammation, suggesting that CTSB is a potential target in AD therapy.     

P311 binds to the latency associated protein and downregulates the expression of TGF-beta1 and TGF-beta2

P311 is an 8-kDa protein originally found in neurons and muscle. We recently showed that expression of P311 in NIH 3T3 cells induced a myofibroblast phenotype with low TGF-beta1 expression. Here we demonstrate that P311 downregulates not only TGF-beta1, but also TGF-beta2, expression, with no effect on TGF-beta3. In addition, P311 interacts with TGF-beta2 in a yeast two-hybrid system through a sequence encompassing part of the TGF-beta latent associated protein (LAP) and part of mature TGF-beta2. Coimmunoprecipitations demonstrated interaction between P311 and TGF-beta1 and 2, but not TGF-beta3. Additional coimmunoprecipitations after introducing LAP or mature TGF-beta1 into cells demonstrated P311 binding to LAP, but not to mature TGF-beta. P311 has a conserved PEST domain, which generally serves as a rapid degradation signal. Deletion of the PEST domain reversed the effect of P311 on TGF-beta isoforms. Finally, Smad3 activity was decreased in P311-expressing cells, but was corrected by exogenous TGF-beta1 treatment, which also elevated TGF-beta1 mRNA level. This suggested that P311 downregulates TGF-beta1 and 2 in part by blocking TGF-beta autoinduction.     

The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains.

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH(2)-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.     

Identification of an active site on the laminin alpha4 chain globular domain that binds to alphavbeta3 integrin and promotes angiogenesis

Angiogenesis is important for wound healing, tumor growth, and metastasis. The laminin alpha4 chain, a component of laminin-8 and -9, is expressed in endothelial cell basement membranes. It mediates endothelial cell adhesion by binding with its receptors such as alphavbeta3 integrin and participates in new blood vessel formation. In this study, we found the recombinant laminin alpha4LG modules (rLG1-3, rLG1, and rLG2) mediate HUVECs adhesion. The attachment of HUVECs to the rLG2 was specifically inhibited by a function-blocking monoclonal antibody LM609 specific for alphavbeta3 integrin. Using deletion mutants of the alpha4LG2 revealed the HUVECs-adhesion site is located in amino acids 1121-1139. A synthetic G(1121-1139) peptide could be attached by HUVECs at same efficiency with the rLG2 and promoted angiogenesis in CAM. In conclusion, we have identified a new alphavbeta3 integrin-interacting peptide within laminin alpha4 G domain. This suggests that G(1121-1139) peptide-containing proteins may perform their biological functions by interacting with alphavbeta3 integrin.     

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.     

Identification of signalling pathways activated by Tyro3 that promote cell survival,proliferation and invasiveness in human cancer cells

Tyro3 is a member of the TAM subfamily of receptor tyrosine kinases alongside Axl and MerTK, which are activated by homologous ligands Gas6 and protein S. The TAMs activate signalling pathways that mediate diverse functions including cell survival, proliferation, phagocytosis and immune regulation, and defects in TAM-dependent processes are associated with the development of human autoimmune diseases and numerous cancers. In this study, we have focused on the signalling and functional roles of Tyro3, about which much remains unknown. For this purpose, we used cultured human cancer cell lines with different levels of TAM expression to reveal the relative significance of Tyro3 amongst the TAMs. Knockdown of Tyro3 expression by siRNA in MGH-U3 cells, which express Tyro3 as sole TAM, caused a reduction in cell viability, which could not be rescued by TAM ligand, demonstrating the dependence of these cells solely on Tyro3. In contrast, the reduced viability of SCC-25 cells upon Tyro3 knockdown could be rescued by Gas6 as these cells express both Tyro3 and Axl and hence Axl expression was preserved. An increase in the fraction of Tyro3 knockdown cells in the early apoptotic phase was observed in four different cell lines each with a different TAM expression profile, revealing a broad anti-apoptotic function of Tyro3. Furthermore, in the Tyro3-dependent cells, Tyro3 depletion caused a significant increase in cells in the G0/G1 phase of the cell cycle concomitant with decreases in the G2/M and S phases. In addition, a cancer pathway gene discovery array revealed distinct sets of genes that were altered in expression in cancer cells upon Tyro3 knockdown. Together, these results have elucidated further a role of Tyro3 in promoting multiple tumour-supporting pathways in human cancer cells, which differs in extent depending on the presence of other TAMs in the same cells.     

Activation of moesin, a protein that links actin cytoskeleton to the plasma membrane, occurs by phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequentially to two sites and releasing an autoinhibitory linker

Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the "POCKET" Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the "PATCH," Lys-253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the "FLAP." Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.     

Coronin7 forms a novel E3 ubiquitin ligase complex to promote the degradation of the anti-proliferative protein Tob

Tob belongs to the anti-proliferative Tob/BTG family. The level of Tob throughout the cell cycle is regulated by the SCF (Skp1/Cullin/F-box protein)^Skp2 ubiquitin ligase (E3) complex. Here, we show that Coronin7 (CRN7) is also involved in Tob degradation. We identified CRN7 as a Tob-interacting molecule. A sequence containing two of the six WD motifs in the middle of CRN7 was responsible for the interaction. CRN7 enhanced the polyubiquitination of Tob in vitro, and overexpression of CRN7 promoted proteasome-dependent degradation of Tob. Furthermore, CRN7 interacted with Cullin1 and Roc1 to form a novel SCF-like E3 complex, suggesting that Tob protein is regulated by multiple ubiquitination machineries.

Structured summary

Cullin1physically interacts with CRN7: shown by anti tag coimmunoprecipitation (view interaction)Roc1physically interacts with CRN7: shown by anti tag coimmunoprecipitation (view interaction)CRN7physically interacts with Tob1: shown by anti tag coimmunoprecipitation (view interaction)CDC34physically interacts with CRN7: shown by anti tag coimmunoprecipitation (view interaction)Tob1 and CRN7colocalize: shown by fluorescence microscopy (view interaction)Elongin Bphysically interacts with CRN7: shown by anti tag coimmunoprecipitation (view interaction)Elongin Cphysically interacts with CRN7: shown by anti tag coimmunoprecipitation (view interaction)Tob1physically interacts with CRN7: shown by two hybrid (view interaction)     

Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity

The pheromone signal in the yeastSaccharomyces cerevisiae is transmitted by the and subunits of the mating response G-protein. TheSTE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G mutation. The same genetic screen identifiedBEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designatedMDG1 (multicopy suppressor ofdefectiveG-protein). TheMDG1 gene was independently isolated in a search for multicopy suppressors of abem1 mutation. TheMDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein fromAequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion ofMDG1 causes sterility in cells in which the wild-type G has been replaced by partly defective G derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted forSTE20 is partially suppressed by multiple copies ofBEM1 andCDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels ofSTE20 andBEM1 are capable of suppressing a temperature-sensitive mutation inCDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.     

Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and intracellular localization of ORP3, ORP6, and ORP7

The human OSBP related protein (ORP) family consists of 12 members, which can be divided into six subfamilies based on the genomic organization and amino acid homology. Here we performed basic characterization of subfamily III, which consists of three members: ORP3, ORP6, and ORP7. According to cDNA hybridization, the three genes are expressed in a tissue-specific manner. While ORP3 mRNA is most abundant in kidney, lymph nodes, and thymus, ORP6 shows highest expression in brain and skeletal muscle, and ORP7 in the gastrointestinal tract. Using monospecific peptide antibodies, we confirmed the presence of the three proteins in human and mouse tissues. ORP6 gene expression was induced upon differentiation of F9 embryonic carcinoma cells into parietal endoderm, while ORP3 and ORP7 mRNA levels were unchanged. In the F9 cells, endogenous ORP6 associated predominantly with the nuclear envelope. When expressed from the cDNA in cultured cells, the three proteins were distributed between the cytosol and endoplasmic reticulum (ER) membranes, with a minor portion found at the plasma membrane. Experiments with truncated constructs showed that the N-terminal portion of the proteins, containing a pleckstrin homology (PH) domain, has markedly strong plasma membrane targeting specificity, while the C-terminal half remains largely cytosolic. The expression data demonstrates that ORP3, -6, and -7 are not merely redundant gene products but show marked quantitative differences in tissue expression, suggesting tissue-specific aspects in their function. The dual targeting of the proteins indicates a putative role in communication between the ER and the plasma membrane.This study was supported by the Clinical Research Fund of Helsinki University Central Hospital (J.T.), the Academy of Finland (grant 51883 to M.L.; grants 49987, 50641, and 54301 to V.M.O.), the Sigrid Juselius Foundation, and the Finnish Cultural Foundation     

The roles of MCP-1 and protein kinase Cδ activation in human eosinophilic leukemia EoL-1 cells

Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-α (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKCδ in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G_i/G_o protein, phospholipase C (PLC), PKCδ, p38 MAPK and NF-κB. MCP-1 activates p38 MAPK via G_i/G_o protein, PLC and PKCδ cascade. MCP-1 also induces NF-κB translocation and the activation is inhibited by PKCδ activation. The increase in the basal expression and activity of PKCδ in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKCδ is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKCδ functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.     

Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs

Summary. The actin-nucleating and -organizing Arp2/3 protein complex is well known to be conserved throughout the eukaryotic kingdom. For higher plants, however, only limited evidence is available for the presence of the Arp2/3 complex so far. Using heterologous antibodies against the Dictyostelium discoideum and Schizosaccharomyces pombe proteins and a bovine peptide, we found immunological evidence for the presence of Arp3 homologues in plants. First, proteins with a molecular mass of about 47–50 kDa were clearly recognized in extracts of both a dicotyledonous plant (tobacco) and a monocotyledonous plant (maize) in immunoblots with the anti-Arp3 antibodies. Second, immunolocalization with these Arp3 antibodies was performed on different plant cells, selected for their diverse actin organizations and functions. On isolated plasma membrane ghosts derived from tobacco leaf protoplasts, a putative Arp3 was localized along cortical actin filaments. In the inner cortex of maize roots, Arp3 was localized to actin-rich plasmodesmata and pit fields and to multivesicular bodies in the cytoplasm. During root hair formation, distinct site-specific localization was found at the protruding apical plasma membrane portions of these tip-growing cells.Correspondence and reprints: Department of Biology, Universitaire Instelling Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium.Received January 3, 2003; accepted February 7, 2003; published online August 26, 2003