Biomedical subjects

K Kaibuchi

Publications and source records attributed to K Kaibuchi.

At least 91 records · Page 5Linked to original sources

PubMed1999

Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases.

Reports in the past two years have shown that Cdc42, Rac1, and Rho - belonging to the Rho small GTPase family - participate in the regulation of cadherin-mediated cell-cell adhesion. IQGAP1, an effector of Cdc42 and Rac1, interacts with cadherin and beta-catenin and induces the dissociation of alpha-catenin from the cadherin-catenins complex leading to disruption of cell-cell adhesion: activated Cdc42 and Rac1 counteract the effect of IQGAP1. Thus, Cdc42 and Rac1 appear to regulate cadherin-mediated cell-cell adhesion acting through IQGAP1.

Animals↗

PubMed1999

The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of PKN/PRK1.

The small G protein Rho has emerged as a key regulator of cellular events involving cytoskeletal reorganization. Here we report the 2.2 A crystal structure of RhoA bound to an effector domain of protein kinase PKN/PRK1. The structure reveals the antiparallel coiled-coil finger (ACC finger) fold of the effector domain that binds to the Rho specificity-determining regions containing switch I, beta strands B2 and B3, and the C-terminal alpha helix A5, predominantly by specific hydrogen bonds. The ACC finger fold is distinct from those for other small G proteins and provides evidence for the diverse ways of effector recognition. Sequence analysis based on the structure suggests that the ACC finger fold is widespread in Rho effector proteins.

Amino Acid Motifs↗

PubMed1999

The deubiquitinating enzyme Fam interacts with and stabilizes beta-catenin.

BACKGROUND: In the ubiquitin-proteasome pathway, the ubiquitinated substrates either undergo degradation by the proteasome or stabilization through the action of the deubiquitinating enzyme. We have previously found that the deubiquitinating enzyme Fam is colocalized with AF-6, one of the effectors of the Ras small GTPase, at cell-cell contact sites in epithelial cells and interacts with AF-6 in vivo and in vitro. Fam has deubiquitinating activity in vitro and prevents the ubiquitination of AF-6 in intact cells. The degradation of beta-catenin, which accumulates at the cell-cell contact sites as a cadherin/catenin complex, is thought to be regulated by the ubiquitin-proteasome pathway. These observations prompted us to examine the possible Fam regulation of the stabilization of beta-catenin. RESULTS: We found that Fam interacted with beta-catenin both in vivo and in vitro. The Fam-binding site of beta-catenin mapped to the region close to the APC or Axin-binding site of beta-catenin. Over-expression of Fam in mouse L cells resulted in an elevation of beta-catenin levels and in an elongation of the half-life of beta-catenin. In these L cells, Fam was colocalized with beta-catenin at the dot-like structures in the cytoplasm. CONCLUSION: These results indicate that Fam interacts with and stabilizes beta-catenin in vivo, presumably through the deubiquitination of beta-catenin.

Animals↗

PubMed1999

Ras-induced transformation and signaling pathway.

Ras is a signal-transducing, guanine nucleotide-binding protein for various membrane receptors including tyrosine kinase receptors. Ras participates in the regulation of cell proliferation, differentiation, and morphology. Activated ras oncogenes have been identified in various forms of human cancer including epithelial carcinomas of the lung, colon, and pancreas. The cells of these cancers, as well as those that have been experimentally transformed by the activated ras gene, exhibit abnormal growth, morphological changes and alterations of cell adhesions. Although the main effector protein has been thought to be Raf serine/threonine kinase, research has revealed that the Ras-induced signaling pathway is mediated by multiple effector proteins and has the crosstalk with various factors containing other small GTPases. In this review, we summarize the involvement of each effector protein for Ras and the crosstalk with other small GTPases in Ras-induced transformation.

Animals↗

PubMed1999

Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells.

Members of the Rho family of small Ras-like GTPases--including RhoA, -B, and -C, Rac1 and -2, and Cdc42--exhibit guanine nucleotide-binding activity and function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. The Rho family GTPases participate in regulation of the actin cytoskeleton and cell adhesion through specific targets. Identification and characterization of these targets have begun to clarify how the Rho family GTPases act to regulate cytoskeletal structure and cell-cell and cell-substratum contacts in mammalian cells. The Rho family GTPases are also involved in regulation of smooth muscle contraction, cell morphology, cell motility, neurite retraction, and cytokinesis. However, the molecular mechanisms by which the Rho family GTPases participate in the regulation of such processes are not well established.

Cell Adhesion↗

PubMed1999

Transcription factors Mash-1 and Prox-1 delineate early steps in differentiation of neural stem cells in the developing central nervous system.

Like other tissues and organs in vertebrates, multipotential stem cells serve as the origin of diverse cell types during genesis of the mammalian central nervous system (CNS). During early development, stem cells self-renew and increase their total cell numbers without overt differentiation. At later stages, the cells withdraw from this self-renewal mode, and are fated to differentiate into neurons and glia in a spatially and temporally regulated manner. However, the molecular mechanisms underlying this important step in cell differentiation remain poorly understood. In this study, we present evidence that the expression and function of the neural-specific transcription factors Mash-1 and Prox-1 are involved in this process. In vivo, Mash-1- and Prox-1-expressing cells were defined as a transient proliferating population that was molecularly distinct from self-renewing stem cells. By taking advantage of in vitro culture systems, we showed that induction of Mash-1 and Prox-1 coincided with an initial step of differentiation of stem cells. Furthermore, forced expression of Mash-1 led to the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells, and up-regulation of Prox-1, suggesting that Mash-1 positively regulates cell differentiation. In support of these observations in vitro, we found specific defects in cellular differentiation and loss of expression of Prox-1 in the developing brain of Mash-1 mutant mice in vivo. Thus, we propose that induction of Mash-1 and Prox-1 is one of the critical molecular events that control early development of the CNS.

Animals↗

PubMed1999

Cell adhesion and Rho small GTPases.

The Rho small GTPases, Cdc42, Rac1 and Rho, are implicated in regulation of integrin-mediated cell-substratum adhesion and cadherin-mediated cell-cell adhesion. Identification and characterization of effectors of these GTPases have provided insights into their modes of action. Rho-kinase, an effector of Rho, regulates integrin-mediated cell-substratum adhesion (focal adhesion) by regulating the phosphorylation state of myosin light chain (MLC): it directly phosphorylates MLC and also inactivates myosin phosphatase. IQGAP1, an effector of Cdc42 and Rac1, regulates cadherin-mediated cell-cell adhesion by interacting with (beta)-catenin and dissociating (alpha)-catenin from the cadherin-catenins complex. Activated Cdc42 and Rac1 inhibit IQGAP1, thereby stabilizing the cadherin-catenins complex. Cdc42/Rac1 and IQGAP1 thus appear to constitute a switch that regulates cadherin-mediated cell-cell adhesion.

Cadherins↗

PubMed1998

Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates moesin at Thr558 in vitro. Here, using a site- and phosphorylation state-specific antibody, we found that the expression of dominant active RhoA in COS7 cells induced moesin phosphorylation and the formation of microvilli-like structures at apical membranes where the Thr558-phosphorylated moesin accumulated, whereas the expression of dominant negative Rho-kinase inhibited both of these processes. The expression of dominant active Rho-kinase also induced moesin phosphorylation. When COS7 cells expressing moesin or moesinT558A (substitution of Thr by Ala) were cultured under serum-depleted conditions, there were few microvilli-like structures, whereas microvilli-like structures remained in the cells expressing moesinT558D (substitution of Thr by Asp). The expression of moesinT558A inhibited the dominant active RhoA-induced formation of microvilli-like structures. These results indicate that Rho-kinase regulates moesin phosphorylation downstream of Rho in vivo and that the phosphorylation of moesin by Rho-kinase plays a crucial role in the formation of microvilli-like structures.

Animals↗

PubMed1998

Rho-associated kinase phosphorylates desmin, the myogenic intermediate filament protein, at unique amino-terminal sites.

We obtained evidence that Rho-associated kinase (Rho-kinase) phosphorylates desmin, the myogenic intermediate filament protein, with approximately 2 mol phosphate per mole of desmin in vitro. Desmin phosphorylated by Rho-kinase lost the potential to form 10-nm filaments. Thr-16, Thr-75, and Thr-76 on desmin proved to be the major phosphorylation sites for Rho-kinase. All these sites are located within the head domain and are different from the reported phosphorylation sites of protein kinase. A, protein kinase C, and cdc2 kinase. We are entertaining the notion that Rho-kinase may regulate filament structures of desmin by site-specific phosphorylation.

Amino Acid Sequence↗

PubMed1998

Possible involvement of the inactivation of the Rho-Rho-kinase pathway in oncogenic Ras-induced transformation.

Recent evidence has strongly suggested the involvement of Rho family small guanosine triphosphatases (GTPases) in Ras-induced transformation. To further clarify the role of Rho family GTPases in Ras-induced transformation, we examined the effects of dominant active or dominant negative forms of Rho family GTPases on the morphological changes induced by oncogenic Ras (RasV12) in Rat1 fibroblasts. The cells expressing RasV12 showed the severe disruption of actin stress fibers and cell adhesions. The coexpression of dominant active form of Rho (RhoV14) reverted not only the formation of stress fibers and focal adhesions but also cell-cell adhesions in Ras-transformed Rat1 cells. In addition, the coexpression of constitutively activated Rho-kinase, a downstream effector of Rho, restored the assembly of stress fibers and focal adhesions. Treatment of Ratl cells with lysophosphatidic acid, which is known to activate the Rho-Rho-kinase pathway, enhanced the stress fiber formation, whereas it failed to induce the stress fiber formation in the cells expressing RasV12. These results suggest that the Rho-Rho-kinase pathway may be inactivated in the cells expressing RasV12, and this may contribute to oncogenic Ras-induced transformation.

Actins↗

PubMed1998

Roles of Rho-associated kinase in cytokinesis; mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.

Amino Acid Sequence↗

PubMed1998

Pasteurella multocida toxin increases endothelial permeability via Rho kinase and myosin light chain phosphatase.

Pasteurella multocida toxin (PMT) has been shown to induce actin reorganization through activation of the GTPase Rho. Here we investigated the involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial permeability and the underlying actin reorganization of endothelial cells. Stimulation of endothelial layers with PMT enhanced transendothelial permeability > 10-fold, and this was abolished by pretreatment with the specific Rho inactivator C3 transferase from Clostridium botulinum. The PMT-induced increase in endothelial permeability was associated with 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphorylation, and 3) endothelial cell retraction and actin stress fiber formation. PMT-stimulated actin reorganization could be prevented by 1) pretreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase. Together, these results suggest that PMT activates Rho/Rho kinase, which inactivates MLC phosphatase. The resulting increase in MLC phosphorylation causes endothelial cell retraction and a rise in endothelial permeability.

Bacterial Proteins↗

PubMed1998

The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.

The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

Animals↗

PubMed1998

Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells.

The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.

ADP Ribose Transferases↗

PubMed1998

PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.

3T3 Cells↗

PubMed1998

Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin- mediated cell-cell adhesion.

The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target of Cdc42 and Rac1, was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.

Animals↗

PubMed1998

Phosphorylation of vimentin by Rho-associated kinase at a unique amino-terminal site that is specifically phosphorylated during cytokinesis.

We found that vimentin, the most widely expressed intermediate filament protein, served as an excellent substrate for Rho-associated kinase (Rho-kinase) and that vimentin phosphorylated by Rho-kinase lost its ability to form filaments in vitro. Two amino-terminal sites on vimentin, Ser38 and Ser71, were identified as the major phosphorylation sites for Rho-kinase, and Ser71 was the most favored and unique phosphorylation site for Rho-kinase in vitro. To analyze the vimentin phosphorylation by Rho-kinase in vivo, we prepared an antibody GK71 that specifically recognizes the phosphorylation of vimentin-Ser71. Ectopic expression of constitutively active Rho-kinase in COS-7 cells induced phosphorylation of vimentin at Ser71, followed by the reorganization of vimentin filament networks. During the cell cycle, the phosphorylation of vimentin-Ser71 occurred only at the cleavage furrow in late mitotic cells but not in interphase or early mitotic cells. This cleavage furrow-specific phosphorylation of vimentin-Ser71 was observed in the various types of cells we examined. All these accumulating observations increase the possibility that Rho-kinase may have a definite role in governing regulatory processes in assembly-disassembly and turnover of vimentin filaments at the cleavage furrow during cytokinesis.

Amino Acid Sequence↗

PubMed1998

Association of the myosin-binding subunit of myosin phosphatase and moesin: dual regulation of moesin phosphorylation by Rho-associated kinase and myosin phosphatase.

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. We found that in MDCK epithelial cells, MBS accumulated at the tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling area, where moesin, a member of the ERM (ezrin, radixin, and moesin) family, was localized. Neither membrane ruffling nor an accumulation of moesin and MBS at the free-end plasma membrane was induced when MDCK cells were stimulated with TPA after the microinjection of C3, which ADP-ribosylates and inactivates Rho. MBS was colocalized with moesin at the cell-cell contact sites in MDCK cells. We also found that moesin was coimmunoprecipitated with MBS from MDCK cells. Recombinant MBS interacted with the amino-terminal domains of moesin and ezrin. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward moesin, which was phosphorylated by Rho-kinase. The phosphatase activity was inhibited when MBS was phosphorylated by Rho-kinase. These results suggest that MBS is recruited with moesin to the plasma membrane and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of moesin downstream of Rho.

ADP Ribose Transferases↗