Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C

BACKGROUND: Asymmetric cell division in the Caenorhabditis elegans embryos requires products of par (partitioning defective) genes 1-6 and atypical protein kinase C (aPKC), whereas Cdc42 and Rac, members of the Rho family GTPases, play an essential role in cell polarity establishment in yeast and mammalian cells. However, little is known about a link between PAR proteins and the GTPases in cell polarization. RESULTS: Here we have cloned cDNAs for three human homologues of PAR6, designated PAR6alpha, beta and gamma, comprising 345, 372 and 376 amino acids, respectively. The PAR6 proteins harbour a PDZ domain and a CRIB-like motif, and directly interact with GTP-bound Rac and Cdc42 via this motif and with the aPKC isoforms PKCiota/lambda and PKCzeta via the N-terminal head-to-head association. These interactions are not mutually exclusive, thereby allowing the PAR6 proteins to form a ternary complex with the GTPases and aPKC, both in vitro and in vivo. When PAR6 and aPKC are expressed with a constitutively active form of Rac in HeLa or COS-7 cells, these proteins co-localize to membrane ruffles, which are known to occur at the leading edge of polarized cells during cell movement. CONCLUSION: Human PAR6 homologues most likely play an important role in the cell polarization of mammalian cells, by functioning as an adaptor protein that links activated Rac and Cdc42 to aPKC signalling.     

PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex

BACKGROUND: PAR-6, aPKC and PAR-3 are polarity proteins that co-operate in the establishment of cell polarity in Caenorhabditis elegans and Drosophila embryos. We have recently shown that mammalian aPKC is required for the formation of the epithelia-specific cell-cell junctional structure. We have also revealed that a mammalian PAR-6 forms a ternary complex with aPKC and ASIP/PAR-3, and localizes at the most apical end of the junctional complex in epithelial cells. RESULTS: The ternary complex formation and junctional co-localization of PAR-6 with aPKC and ASIP/PAR-3 are observed during the early stage of epithelial cell polarization. In addition, over-expression of the PAR-6 mutant with CRIB/PDZ domain in MDCK cells disturbs the cell-cell contact-induced junctional localization of tight junction proteins, as well as inhibiting TER development. Furthermore, the binding of Cdc42:GTP to the CRIB/PDZ domain of PAR-6 enhances the kinase activity of PAR-6-bound aPKC. Detailed analyses suggest that the binding of PAR-6 to aPKC has the intrinsic potential to activate aPKC, which is only released when Cdc42:GTP binds to the CRIB/PDZ domain. CONCLUSION: The results indicate the involvement of PAR-6 in the aPKC function which is required for the cell-cell adhesion-induced formation of epithelial junctional structures, possibly through the cooperative regulation of aPKC activity with Cdc42.     

Establishment of cell polarity by afadin during the formation of embryoid bodies

Afadin directly links nectin, an immunoglobulin-like cell–cell adhesion molecule, to actin filaments (F-actin) at adherens junctions (AJs). The nectin–afadin complex is important for the formation of not only AJs but also tight junctions (TJs) in epithelial cells. Studies using afadin-knockout mice have revealed that afadin is indispensable for embryonic development by organizing the formation of cell–cell junctions. However, the molecular mechanism of cell–cell junction disorganization during embryonic development in afadin-knockout mice is poorly understood. To address this, we took advantage of embryoid bodies (EBs) as a model system. The formation of cell–cell junctions including AJs and TJs was impaired in afadin-null EBs. The proper accumulation of the Par complex and the activation of Cdc42 and atypical PKC (aPKC), which are crucial for the formation of cell polarity, were also inhibited by knockout of afadin. In addition, the disruption of afadin caused the abnormal deposition of laminin and the dislocalization of its receptors integrin α₆ and integrin β₁. These results indicate that afadin organizes the formation of cell–cell junctions by regulating cell polarization in early embryonic development.     

Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes

Motile lymphocytes adopt a polarized morphology with different adhesion molecules (ICAM, CD43 and CD44) and ERM actin-binding proteins concentrated on the uropod, a slender posterior appendage with important functions in cell-cell interactions and lymphocyte recruitment. We have studied the role of Rho family of GTPases (Rho, Rac and Cdc42) in the control of lymphocyte polarity and migration by analyzing the effects of exogenously introduced Rho GTPase mutants. Transfection of T cell lines that constitutively display a polarized motile morphology with activated mutants of RhoA, Rac1 and Cdc42 impaired cell polarization. A guanosine nucleotide exchange factor for Rac, Tiam-1, induced the same effect as activated Rac1. Conversely, dominant negative forms of the three GTP-binding proteins induced a polarized phenotype in constitutively round-shaped T cells with redistribution of ICAM-3 and moesin to the uropod in an integrin-dependent manner. On the other hand, overexpression of dominant negative Cdc42 and activated mutants of all three Rho GTPases significantly inhibited SDF-1alpha-induced T cell chemotaxis. Together, these data demonstrate that Rho GTPases regulate lymphocyte polarization and chemokine-induced migration, and underscore the key role of Cdc42 in lymphocyte directional migration.     

Regulated protein-protein interaction between aPKC and PAR-3 plays an essential role in the polarization of epithelial cells

BACKGROUND: Recent studies have revealed that aPKC (atypical protein kinase C), PAR-3 and PAR-6 play indispensable roles in the regulation of various cell polarization events, from worms to mammals, suggesting that they comprise an evolutionarily conserved protein machinery which is essential for cell polarization. The three proteins interact with each other to form a ternary complex and thus mutually regulate their functionality and localization. Here, we investigated the biochemical nature of the aPKC-PAR-3 interaction in detail to clarify its functional importance in cell polarity. RESULTS: The highly conserved 26 amino acid sequence 816-841, in PAR-3 was found to be necessary and sufficient for the tight association with aPKC. Among several conserved serine/threonine residues within the region, aPKC preferentially phosphorylates serine-827 in vitro, and this phosphorylation reduces the stability of the PAR-3-aPKC interaction. Several analyses using a phospho-serine 827 specific antibody have established that this phosphorylation by aPKC occurs in vivo. Over-expression of a point mutant of PAR-3 (S827A), which is predicted to form a stable complex with aPKC, causes defects in the cell-cell contact-induced cell polarization of epithelial MDCK cells, similarly to a dominant negative mutant of aPKC. CONCLUSIONS: These results imply that serine 827 in the aPKC binding site of PAR-3 is a target of aPKC and that the regulated interaction between a protein kinase, aPKC, and its substrate, PAR-3, plays an essential role in the establishment of cell polarity.     

Over-expression of PAR-3 suppresses contact-mediated inhibition of cell migration in MDCK cells

BACKGROUND: PAR-3 is one of the PAR proteins, previously named ASIP, which are indispensable for the establishment of cell polarity in the embryo as well as differentiated epithelial cells. In mammalian epithelial cells, it forms a ternary complex with aPKC and PAR-6, and is localized to the tight junction that has been suggested as being important for creating cell polarity. RESULTS: To gain insights into the mode of PAR-3 function in mammalian epithelial cells, we examined the effect of PAR-3 over-expression in MDCK cells. Although exogenous PAR-3-expression does not affect the epithelial polarity of confluent cells, it drastically transforms the morphology of cells at low density into a fibroblastic form with developed membrane protrusions. Time-lapse observations have revealed that PAR-3 over-expressing cells show intense motility, even after they have assembled into loose colonies, suggesting that the contact-mediated inhibition of cell migration (CIM) is suppressed. The expressions of E-cadherin and vimentin do not change with PAR-3 over-expression, suggesting that exogenous PAR-3 only disturbs the endogenous equilibrium of cellular states between a fundamental fibroblastic structure and an epithelial one. The co-expression of a dominant negative mutant of Rac1 and the addition of nocodazole strongly antagonize the effect of PAR-3 over-expression, suggesting the involvement of Rac1 activation and microtubule polymerizations. CONCLUSIONS: : The data presented here suggest an intriguing link between the contact-mediated inhibition of cell migration and the regulation of cell polarity. The putative PAR-3 activities demonstrated here may function endogenously in the epithelial cell polarization process by being sequestered from the cytosol to the cell-cell junctional regions with aPKC and PAR-6 upon cell-cell adhesion.     

Cdc42 and Rac small G proteins activated by trans-interactions of nectins are involved in activation of c-Jun N-terminal kinase,but not in association of nectins and cadherin to form adherens junctions,in fibroblasts

BACKGROUND: Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules which associate with cadherins to form adherens junctions (AJs) in epithelial cells and fibroblasts. Nectin-1 and -3 are members of the nectin family which most strongly trans-interact, causing cell-cell adhesion. The trans-interaction between nectin-1 and -3 induces the activation of both Cdc42 and Rac small G proteins in epithelial cells. We studied the roles of Cdc42 and Rac activated in this way in L fibroblasts stably expressing both nectin-1 and E-cadherin (nectin-1-EL cells). RESULTS: The trans-interaction between nectin-1 and -3 induced the activation of Cdc42 and Rac in nectin-1-EL cells. Cdc42, and presumably Rac, activated in this way, induced the activation of c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK). Cdc42 or Rac was not essential for the association of nectin-1 and E-cadherin to form AJs. Reorganization of the actin cytoskeleton was not required for the association of nectin-1 and E-cadherin. CONCLUSION: These results indicate that Cdc42 and Rac activated by the trans-interaction of nectins selectively induce the activation of JNK, but are not essential for the association of nectins and cadherin to form AJs in fibroblasts.     

Importance of spatial activation of Cdc42 and rac small G proteins by frabin for microspike formation in MDCK cells

BACKGROUND: Frabin is an actin filament (F-actin)-binding protein that shows GDP/GTP exchange activity for Cdc42 small G protein (Cdc42). Frabin furthermore induces indirect activation of Rac small G protein (Rac) in intact cells. We have recently shown that in nonepithelial cells, frabin induces the formation of both filopodia- and lamellipodia-like processes through the activation of Cdc42 and Rac, respectively. In epithelial cells such as MDCK cells, Cdc42 and Rac regulate cell-cell adherens junctions (AJs) via the accumulation of F-actin and E-cadherin, although neither Cdc42 nor Rac induces the formation of filopodia or lamellipodia. In this study, we have examined the effects of frabin on the reorganization of the actin cytoskeleton in MDCK cells. RESULTS: Frabin induces the formation of microspikes at the basal area of the lateral membranes through the activation of Cdc42 and Rac in MDCK cells, although a dominant active mutant of Cdc42 or Rac alone, or both, did not induce the formation of microspikes. Furthermore, frabin weakly increased the accumulation of F-actin and E-cadherin at cell-cell AJs and the formation of stress fibres through the activation of Cdc42 and Rac, under conditions where the dominant active mutant of Cdc42 or Rac markedly showed these effects. The Cdc42- and Rac-induced formation of stress fibres was dependent on the activation of Rho small G protein. CONCLUSION: These results indicate that the frabin-dependent spatial activation of Cdc42 and Rac is important for the formation of microspikes.     

Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1(V12) or Cdc42(V12) could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA(L63) decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.     

Cdc42 controls progenitor cell differentiation and beta-catenin turnover in skin

Differentiation of skin stem cells into hair follicles (HFs) requires the inhibition of beta-catenin degradation, which is controlled by a complex containing axin and the protein kinase GSK3beta. Using conditional gene targeting in mice, we show now that the small GTPase Cdc42 is crucial for differentiation of skin progenitor cells into HF lineage and that it regulates the turnover of beta-catenin. In the absence of Cdc42, degradation of beta-catenin was increased corresponding to a decreased phosphorylation of GSK3beta at Ser 9 and an increased phosphorylation of axin, which is known to be required for binding of beta-catenin to the degradation machinery. Cdc42-mediated regulation of beta-catenin turnover was completely dependent on PKCzeta, which associated with Cdc42, Par6, and Par3. These data suggest that Cdc42 regulation of beta-catenin turnover is important for terminal differentiation of HF progenitor cells in vivo.     

Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of?a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as?a central mechanism that couples Ag extraction to Ag processing and presentation.     

Cytoskeletal remodeling in lymphocyte activation

The formerly distinct fields of lymphocyte signal transduction and cytoskeletal remodeling have recently become linked, as proteins involved in transducing signals downstream of lymphocyte antigen receptors have also been implicated in actin cytoskeleton remodeling, microtubule dynamics and regulation of cell polarity. These discoveries have fuelled interest in understanding both the role of the actin cytoskeleton as an integral component of lymphocyte activation and the interplay between lymphoid cell-cell contact sites (immunological synapse), retractile pole structures (uropod, distal pole complex), and Rho-family GTPases (Rac, Rho, Cdc42), their upstream activators (Dbl-family guanine nucleotide exchange factors) and their downstream effectors (WASp, Arp2/3, ADAP). To understand how these complex regulatory networks are wired, a new breed of computational biologists uses mathematical language to reproduce and simulate signaling circuits 'in silico'.     

ERBIN associates with p0071, an armadillo protein,at cell-cell junctions of epithelial cells

BACKGROUND: 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. RESULTS: To identify new ERBIN-binding proteins, we screened a yeast two-hybrid library, using the carboxyl-terminal fragment of ERBIN containing PDZ domain as the bait, and we isolated p0071 (also called plakophilin-4) as an ERBIN-interacting protein. p0071 is a member of the p120 catenin family, which are defined as proteins with 10 armadillo repeats, and localizes along the cell-cell border. The ERBIN PDZ domain binds the COOH-terminus of p0071 containing the PDZ domain-binding sequence. Endogenous ERBIN was co-immunoprecipitated with p0071. In fully polarized Madin-Darby canine kidney (MDCK) cells, ERBIN co-localized 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, resulted in a marked accumulation of ERBIN at the cell-cell contacts of MDCK and HeLa cells. CONCLUSION: 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, we demonstrated that the subcellular localization of ERBIN might be regulated by Rho family small GTPases.     

N-Acylhomoserine lactones are potent neutrophil chemoattractants that act via calcium mobilization and actin remodeling

In gram-negative bacteria, cell-cell communication based on HSL QS molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior. Using a Transwell migration assay, we found that human primary neutrophils are strongly stimulated by 3O-C(12)-HSL and -C(10)-HSL but not C(4)-HSL in a concentration-dependent manner. Moreover, 3O-C(12)-HSL and -C(10)-HSL activate PLCγ1 but not -γ2, mobilize intracellular calcium, and up-regulate IP(3)R. These changes were paralleled by F-actin accumulation, primarily in the leading edge of neutrophils, as evidenced by phalloidin staining and confocal microscopy. F- and G-actin isolation and quantification by immunoblotting revealed that the F/G-actin ratio was increased significantly after treatment with all three HSLs. Furthemore, 3O-C(12)-HSL- and 3O-C(10)-HSL treatment resulted in phosphorylation of Rac1 and Cdc42. In contrast, C(4)-HSL had negligible influence on the phosphorylation status of PLC and Rac1/Cdc42 and failed to attract neutrophils and induce calcium release. The calcium inhibitor thapsigargin, which blocks ER calcium uptake, strongly prevented neutrophil migration toward 3O-C(12)-HSL and -C(10)-HSL. These findings show that the bacterial QS molecules 3O-C(12)-HSL and -C(10)-HSL may attract human neutrophils to the sites of bacterial infection and developing biofilms. Indeed, recognition of HSL QS signals by neutrophils may play a critical role in their recruitment during infections.     

Rho家族小GTP酶在黑素细胞树突形成和黏附中的作用

黑素细胞通过其树突将合成的黑素转运至角质形成细胞，进而发挥生理功能。黑素细胞树突形成是黑素转运过程中的重要环节，黑素转运必须在黑素细胞和角质形成细胞紧密接触后才能实现。黑素细胞形态学改变包括胞体大小和树突变化等细胞骨架的改变，细胞骨架变化主要与肌动蛋白和微管结构的重排有关。Rho家族小GTP酶包括20种成员，其中RhoA,Rac1和Cdc42对黑素细胞细胞骨架的变化和细胞黏附的调节起重要作用。     

Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cells

BACKGROUND: Invadopodia are membrane protrusions into the extracellular matrix by aggressive tumour cells. These structures are associated with sites of matrix degradation and invasiveness of malignant tumour cells in an in vitro fibronectin degradation/invasion assay. The Rho family small G proteins, consisting of the Rho, Rac and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, adhesion, and motility, through reorganization of the actin cytoskeleton. We studied the roles of the Rho family small G proteins in invadopodia formation. RESULTS: We first demonstrated that invadopodia of RPMI7951 human melanoma cells extended into the matrix substratum on a vertical view using a laser scanning confocal microscope system. We confirmed that invadopodia were rich in actin filaments (F-actin) and visualized clearly with F-actin staining on a vertical view as well as on a horizontal view. We then studied the roles of Rho, Rac, and Cdc42 in invasiveness of the same cell line. In the in vitro fibronectin degradation/invasion assay, a dominant active mutant of Cdc42 enhanced dot-like degradation, whereas a dominant active mutant of Rac enhanced diffuse-type degradation. Furthermore, frabin, a GDP/GTP exchange protein for Cdc42 with F-actin-binding activity, enhanced both dot-like and diffuse-type degradation. However, a dominant active mutant of Rho did not affect the fibronectin degradation. Moreover, inhibition of phosphatidylinositol-3 kinase (PI3K) disrupted the Rac and Cdc42-dependent actin structures and blocked the fibronectin degradation. CONCLUSION: These results suggest that Cdc42 and Rac play important roles in fibronectin degradation and invasiveness in a coordinate manner through the frabin-Cdc42/Rac-PI3K signalling pathway.     

PECAM-1 affects GSK-3beta-mediated beta-catenin phosphorylation and degradation

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates a variety of endothelial and immune cell biological responses. PECAM-1-null mice exhibit prolonged and increased permeability after inflammatory insults. We observed that in PECAM-1-null endothelial cells (ECs), beta-catenin remained tyrosine phosphorylated, coinciding with a sustained increase in permeability. Src homology 2 domain containing phosphatase 2 (SHP-2) association with beta-catenin was diminished in PECAM-1-null ECs, suggesting that lack of PECAM-1 inhibits the ability of this adherens junction component to become dephosphorylated, promoting a sustained increase in permeability. beta-Catenin/Glycogen synthase kinase 3 (GSK-3beta) association and beta-catenin serine phosphorylation levels were increased and beta-catenin expression levels were reduced in PECAM-1-null ECs. Glycogen synthase kinase 3 (GSK-3beta) serine phosphorylation (inactivation) was blunted in PECAM-1-null ECs after histamine treatment or shear stress. Our data suggest that PECAM-1 serves as a critical dynamic regulator of endothelial barrier permeability. On stimulation by a vasoactive substance or shear stress, PECAM-1 became tyrosine phosphorylated, enabling recruitment of SHP-2 and tyrosine-phosphorylated beta-catenin to its cytoplasmic domain, facilitating dephosphorylation of beta-catenin, and allowing reconstitution of adherens junctions. In addition, PECAM-1 modulated the levels of beta-catenin by regulating the activity of GSK-3beta, which in turn affected the serine phosphorylation of beta-catenin and its proteosomal degradation, affecting the ability of the cell to reform adherens junctions in a timely fashion.     

Cdc42 and the actin-related protein/neural Wiskott-Aldrich syndrome protein network mediate cellular invasion by Cryptosporidium parvum

Cryptosporidium parvum invasion of epithelial cells involves host cell membrane alterations which require a remodeling of the host cell actin cytoskeleton. In addition, an actin plaque, possibly associated with the dense-band region, forms within the host cytoplasm at the host-parasite interface. Here we show that Cdc42 and RhoA, but not Rac1, members of the Rho family of GTPases, are recruited to the host-parasite interface in an in vitro model of human biliary cryptosporidiosis. Interestingly, activation of Cdc42, but not RhoA, was detected in the infected cells. Neural Wiskott-Aldrich syndrome protein (N-WASP) and p34-Arc, actin-regulating downstream effectors of Cdc42, were also recruited to the host-parasite interface. Whereas cellular expression of a constitutively active mutant of Cdc42 promoted C. parvum invasion, overexpression of a dominant negative mutant of Cdc42, or depletion of Cdc42 mRNA by short interfering RNA-mediated gene silencing, inhibited C. parvum invasion. Expression of the WA fragment of N-WASP to block associated actin polymerization also inhibited C. parvum invasion. Moreover, inhibition of host cell Cdc42 activation by dominant negative mutation inhibited C. parvum-associated actin remodeling, membrane protrusion, and dense-band formation. In contrast, treatment of cells with a Rho inhibitor, exoenzyme C3, or cellular overexpression of dominant negative mutants of RhoA and Rac1 had no effect on C. parvum invasion. These data suggest that C. parvum invasion of target epithelia results from the organism's ability to activate a host cell Cdc42 GTPase signaling pathway to induce host cell actin remodeling at the attachment site.