Biomedical subjects

K Kaibuchi

Publications and source records attributed to K Kaibuchi.

At least 109 records · Page 6Linked to original sources

PubMed1998

Domain- and site-specific phosphorylation of bovine NF-L by Rho-associated kinase.

Rho-associated kinase (Rho-kinase), the putative target of the small GTP-binding protein Rho, phosphorylated neurofilament protein (NF-L) in vitro with approximately 1 mole phosphate per mole NF-L. Phosphorylated NF-L no longer formed the 10 nm filaments, and NF-L filaments were phosphorylated with a result of nearly complete disassembly. NF-L phosphorylated by Rho-kinase was digested with trypsin, and digested fragments were assigned by MALDI/TOF. Unique phosphorylation sites were found at Ser-26 and Ser-57 in the head domain of NF-L. These results indicate that domain- and site-specific phosphorylation by Rho-kinase may regulate the assembly-disassembly of NF-L filaments.

Amino Acid Sequence↗

PubMed1998

Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue.

The 2.4-A resolution crystal structure of a dominantly active form of the small guanosine triphosphatase (GTPase) RhoA, RhoAV14, complexed with the nonhydrolyzable GTP analogue, guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), reveals a fold similar to RhoA-GDP, which has been recently reported (Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto, R. K., Somlyo, A. V., Somlyo, A. P., and Derewenda, Z. S. (1997) Nat. Struct. Biol. 4, 699-703), but shows large conformational differences localized in switch I and switch II. These changes produce hydrophobic patches on the molecular surface of switch I, which has been suggested to be involved in its effector binding. Compared with H-Ras and other GTPases bound to GTP or GTP analogues, the significant conformational differences are located in regions involving switches I and II and part of the antiparallel beta-sheet between switches I and II. Key residues that produce these conformational differences were identified. In addition to these differences, RhoA contains four insertion or deletion sites with an extra helical subdomain that seems to be characteristic of members of the Rho family, including Rac1, but with several variations in details. These sites also display large displacements from those of H-Ras. The ADP-ribosylation residue, Asn41, by C3-like exoenzymes stacks on the indole ring of Trp58 with a hydrogen bond to the main chain of Glu40. The recognition of the guanosine moiety of GTPgammaS by the GTPase contains water-mediated hydrogen bonds, which seem to be common in the Rho family. These structural differences provide an insight into specific interaction sites with the effectors, as well as with modulators such as guanine nucleotide exchange factor (GEF) and guanine nucleotide dissociation inhibitor (GDI).

Amino Acid Sequence↗

PubMed1998

A new isoform of human myosin phosphatase targeting/regulatory subunit (MYPT2): cDNA cloning, tissue expression, and chromosomal mapping.

Myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin phosphatase, plays a pivotal role in the regulation of myosin phosphatase activity. Here we have cloned a novel isoform of MYPT1, termed MYPT2, from a human brain cDNA library screened with a cDNA fragment of rat MYPT1. Overlapping clones indicated an open reading frame of 3763 nucleotides and a predicted polypeptide of mass 110,398. Ankyrin repeats and leucine zipper motifs were identified for the sequences 57-316 and 956-982, respectively. Overall, the deduced amino acid sequence of MYPT2 was 61% identical to MYPT1. MYPT2 gene is transcribed abundantly in heart and skeletal muscle, while Western blots using an antibody specific for MYPT2 showed exclusive expression of MYPT2 in heart and brain. A recombinant of the N-terminal two-thirds of MYPT2 bound to the catalytic subunit of type 1 phosphatase (delta isoform) and increased activity toward phosphorylated myosin light chain. In situ hybridization localized the human MYPT2 gene on chromosome 1q32.1, compared to the chromosomal location 12q15-q21-2 for MYPT1. It is suggested that the products of the two gene families of myosin phosphatase target subunit may be localized differently among various tissues.

Amino Acid Sequence↗

PubMed1998

Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-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. Here we purified MBS-interacting proteins, identified them as adducin, and found that MBS specifically interacted with adducin in vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. We also found that Rho-kinase phosphorylated alpha-adducin in vitro and in vivo and that the phosphorylation of alpha-adducin by Rho-kinase enhanced the interaction of alpha-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward alpha-adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of adducin downstream of Rho and that the increased phosphorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments.

Actins↗

PubMed1998

Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association.

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and approximately 30 and approximately 100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase-dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.

3T3 Cells↗

PubMed1998

p140Sra-1 (specifically Rac1-associated protein) is a novel specific target for Rac1 small GTPase.

Rac1 small GTPase plays pivotal roles in various cell functions such as cell morphology, cell polarity, and cell proliferation. We have previously identified IQGAP1 from bovine brain cytosol as a target for Rac1 by an affinity purification method. By using the same method, we purified a specifically Rac1-associated protein with a molecular mass of about 140 kDa (p140) from bovine brain cytosol. This protein interacted with guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).glutathione S-transferase (GST)-Rac1 but not with the GDP.GST-Rac1, GTPgammaS.GST-Cdc42, or GTPgammaS.GST-RhoA. The amino acid sequences of this protein revealed that p140 is identified as a product of KIAA0068 gene. We denoted this protein as Sra-1 (Specifically Rac1-associated protein). Recombinant Sra-1 interacted with GTPgammaS.GST-Rac1 and weakly with GDP.Rac1 but not with GST-Cdc42 or GST-RhoA. The N-terminal domain of Sra-1 (1-407 amino acids) was responsible for the interaction with Rac1. Myc-tagged Sra-1 and the deletion mutant capable of interacting with Rac1, but not the mutants unable to bind Rac1, were colocalized with dominant active Rac1(Val-12) and cortical actin filament at the Rac1(Val-12)-induced membrane ruffling area in KB cells. Sra-1 was cosedimented with filamentous actin (F-actin), indicating that Sra-1 directly interacts with F-actin. These results suggest that Sra-1 is a novel and specific target for Rac1.

Amino Acid Sequence↗

PubMed1998

Myosin II activation promotes neurite retraction during the action of Rho and Rho-kinase.

BACKGROUND: The Rho small GTPase regulates myosin II activity through the phosphorylation of the myosin light chain (MLC) by activating Rho-kinase, which is a target of Rho. Several lines of evidence point to an important role of Rho in the action of lysophosphatidic acid (LPA) and thrombin in provoking neurite retraction in N1E-115 neuroblastoma cells. RESULTS: Here we examined whether Rho-kinase and myosin II are involved in neurite retraction in N1E-115 cells. We showed that the expression of constitutively active forms of Rho-kinase induced neurite retraction in NIE-115 cells and MLC phosphorylation in NIH 3T3 cells, whereas the expression of dominant negative forms of Rho-kinase inhibited the LPA-induced neurite retraction in N1E-115 cells and the serum-induced MLC phosphorylation in NIH 3T3 cells. The expression of mutant MLCT18D,S19D (substitution of Thr and Ser by Asp), which is known to lead to the activation of myosin ATPase and a conformational change of myosin II when reconstituted with myosin heavy chains in vitro, also promoted neurite retraction. CONCLUSION: These results indicate that Rho-kinase is involved in the LPA-induced neurite retraction downstream of Rho, and that myosin II activation promotes neurite retraction downstream of Rho and Rho-kinase.

3T3 Cells↗

PubMed1998

Microinjection of activated phosphatidylinositol-3 kinase induces process outgrowth in rat PC12 cells through the Rac-JNK signal transduction pathway.

We have previously shown that sustained phosphatidylinositol (PI)-3 kinase activity is necessary for neurite outgrowth of PC12 cells induced by nerve growth factor (NGF). Microinjection of a constitutively active mutant of PI-3 kinase induced process formation suggesting that PI-3 kinase is indeed involved in the neurite outgrowth. However, the processes appeared to be incomplete neurites as they had very poor organization of F-actin and GAP43 antigen. The microtubule network was enhanced in the process-bearing cells and process formation was inhibited by colchicine suggesting that microtubules play an important role in process formation downstream of PI-3 kinase. These cell responses were inhibited by dominant-negative mutants of Rac and Sek1/SAPK but not by a dominant-negative mutant Ras and PD98059, a MAP kinase kinase (MEK) inhibitor, suggesting that not the Ras-MAP kinase pathway but the Rac-Jun N-terminal kinase (JNK) pathway is involved in process formation.

Animals↗

PubMed1997

Regulation of cross-linking of actin filament by IQGAP1, a target for Cdc42.

We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rac1 small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1(val12) (Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 23363-23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to beta- and gamma-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1-216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).GST-Cdc42 but not by GDP.GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTPgammaS.GST-Cdc42 but not GDP.GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization.

Actins↗

PubMed1997

Regulation of cell-cell adhesion of MDCK cells by Cdc42 and Rac1 small GTPases.

Rac1, a member of the Rho small GTPases family, has recently been shown to be involved in the regulation of cell-cell adhesion mediated by cadherin. Here we showed that Cdc42, another member of Rho family, accumulated at cell-cell contact sites. Microinjection of Rho GDI, a negative regulator of the Rho family members, into Madin-Darby canine kidney (MDCK) cells resulted in perturbation of epithelial cell morphology and of cell-cell and cell-substratum adhesions, and comicroinjection of dominant active Cdc42 or Rac1 reversed the action of Rho GDI, suggesting that the active form of Cdc42 or Rac1 is required for maintaining the cell-cell and cell-substratum adhesions. These observations suggest that Cdc42, in addition to Rac1, can regulate the cell-cell adhesion.

Animals↗

PubMed1997

Regulation of myosin phosphatase through phosphorylation of the myosin-binding subunit in platelet activation.

Human platelets were found to contain myosin phosphatase consisting of a 38-kD catalytic subunit of protein phosphatase type 1delta, a 130-kD myosin-binding subunit (MBS) and a 20-kD subunit, all of which cross-reacted with antibodies against these subunits of smooth muscle myosin phosphatase. Anti-MBS antibody coimmunoprecipitated RhoA and Rho-kinase of human platelets. Platelets MBS is a substrate for Rho-kinase and phosphorylation of MBS decreases the activity of myosin phosphatase. Treatment of intact platelets with 9, 11-epithio-11,12-methano-thromboxane A2 led to a dramatic increase in phosphorylation of MBS and a significant decrease in the activity of myosin phosphatase. These findings suggest a putative mechanism for agonist-induced regulation of myosin phosphatase activity in platelets.

Binding Sites↗

PubMed1997

The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells.

The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell-cell contacts and found that AF-6 accumulated at the cell-cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

Animals↗

PubMed1997

Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase.

The small GTPase Rho is implicated in cytoskeletal rearrangements including stress fiber and focal adhesion formation and in the transcriptional activation of c-fos serum response element. In vitro, Rho-kinase, which is activated by Rho, phosphorylates not only myosin light chain (MLC) (thereby activating myosin ATPase) but also myosin phosphatase, thus inactivating myosin phosphatase. Rho-kinase is involved in the formation of stress fibers and focal adhesions in fibroblasts. Here we show that the expression of constitutively active Rho-kinase increased the level of MLC phosphorylation. The activity of Rho-kinase was necessary for maintaining the vinculin-containing focal adhesions, whereas organized actin stress fibers were not necessary for this. The microinjection of constitutively active Rho-kinase into fibroblasts induced the formation of focal adhesions to some extent under the conditions where organized actin stress fibers were disrupted. The expression of constitutively active Rho-kinase also stimulated the transcriptional activity of c-fos serum response element. These results suggest that Rho-kinase has distinct roles in divergent pathways downstream of Rho, which include MLC phosphorylation leading to stress fiber formation, focal adhesion formation, and gene expression.

3T3 Cells↗

PubMed1997

Chimeric MLL products with a Ras binding cytoplasmic protein AF6 involved in t(6;11) (q27;q23) leukemia localize in the nucleus.

In infantile leukemias and therapy-related leukemias, the MLL gene is frequently found to be disrupted and fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL as a result of 11q23 translocations. We previously showed that the N-terminal portion common to various chimeric MLL products, as well as to MLL-LTG9 and MLL-LTG19, localizes in the nuclei, and therefore suggested that it might play an important role in leukemogenesis. In the present study, MLL-AF6 chimeric products found in the t(6;11)(q27;q23) translocation were analysed since AF6, a Ras-binding protein, exhibits a different subcellular localization from that of LTG9/AF9 and LTG19/ENL. Immunofluorescence staining data and cell fractionation analyses demonstrated that MLL-AF6 chimeric products localize in the nuclei despite the fact that AF6 itself localizes in the cytoplasm, confirming the importance of the nuclear localization of chimeric MLL products. The region in the N-terminal portion of MLL responsible for this nuclear localization was examined and found to be a region containing AT-hook motifs.

Animals↗

PubMed1997

Localization of the gene coding for myosin phosphatase, target subunit 1 (MYPT1) to human chromosome 12q15-q21.

Myosin phosphatase target subunit 1 (MYPT1), which is also called the myosin-binding subunit of myosin phosphatase, is one of the subunits of myosin phosphatase. Myosin phosphatase regulates the interaction of actin and myosin downstream of the guanosine triphosphatase Rho, as previously shown (K. Kimura, et al., 1996, Science 273:245-248). To understand the role of MYPT1 in the regulation of the cytoskeleton in human diseases, we have cloned a 4855-bp cDNA for human MYPT1 using the rat MYPT1 cDNA as probe. Sequencing analysis has revealed that human MYPT1 contains 1030 amino acid residues with a calculated molecular mass of approximately 115 kDa. Fluorescence in situ hybridization analysis placed the human MYPT1 gene on chromosome 12q15-q21.2. Radiation hybrid mapping has shown that the human MYPT1 gene is located very near the highly polymorphic marker CHLC.GATA65A12, which lies between D12S350 and D12S106.

Amino Acid Sequence↗

PubMed1997

Expression of a constitutively active phosphatidylinositol 3-kinase induces process formation in rat PC12 cells. Use of Cre/loxP recombination system.

It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.

Animals↗

PubMed1997

Domain-specific phosphorylation of vimentin and glial fibrillary acidic protein by PKN.

PKN is a serine/threonine protein kinase with a catalytic domain homologous to the protein kinase C family and unique N-terminal leucine zipper-like sequences. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that the regulatory domain of PKN interacted with vimentin. We then examined whether PKN would phosphorylate vimentin in vitro. Vimentin proved to be an excellent substrate for PKN, and the phosphorylation of vimentin by PKN occurred in the head domain with the result of a nearly complete inhibition of its filament formation in vitro. Similar results were also obtained with another type III intermediate filament protein, glial fibrillary acidic protein (GFAP). These results raise the possibility that PKN may regulate filament structures of vimentin and GFAP by domain-specific phosphorylation.

Animals↗

PubMed1997

Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation.

Small GTPase Rho plays pivotal roles in the Ca2+ sensitization of smooth muscle. However, the GTP-bound active form of Rho failed to exert Ca2+-sensitizing effects in extensively Triton X-100-permeabilized smooth muscle preparations, due to the loss of the important diffusible cofactor (Gong, M. C., Iizuka, K., Nixon, G. , Browne, J. P., Hall, A., Eccleston, J. F., Sugai, M., Kobayashi, S. , Somlyo, A. V., and Somlyo, A. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1340-1345). Here we demonstrate the contractile effects of Rho-associated kinase (Rho-kinase), recently identified as a putative target of Rho, on the Triton X-100-permeabilized smooth muscle of rabbit portal vein. Introduction of the constitutively active form of Rho-kinase into the cytosol of Triton X-100-permeabilized smooth muscle provoked a contraction and a proportional increase in levels of monophosphorylation of myosin light chain in both the presence and the absence of cytosolic Ca2+. These effects of constitutively active Rho-kinase were wortmannin (a potent myosin light chain kinase inhibitor)-insensitive. Immunoblot analysis revealed that the amount of native Rho-kinase was markedly lower in Triton X-100-permeabilized tissue than in intact tissue. Our results demonstrate that Rho-kinase directly modulates smooth muscle contraction through myosin light chain phosphorylation, independently of the Ca2+-calmodulin-dependent myosin light chain kinase pathway.

Androstadienes↗