Kazuko Fujisawa - Academia.edu (original) (raw)

Papers by Kazuko Fujisawa

Research paper thumbnail of Additional file 1 of Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans

Research paper thumbnail of ADP-ribosylation of the rho gene product: I, Protein tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase by lysophosphatidic acid

Japanese Journal of Pharmacology, 1994

Research paper thumbnail of Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans

Molecular Neurodegeneration, 2021

Background Parkinson’s disease is a disabling neurodegenerative movement disorder characterized b... more Background Parkinson’s disease is a disabling neurodegenerative movement disorder characterized by dopaminergic neuron loss induced by α-synuclein oligomers. There is an urgent need for disease-modifying therapies for Parkinson’s disease, but drug discovery is challenged by lack of in vivo models that recapitulate early stages of neurodegeneration. Invertebrate organisms, such as the nematode worm Caenorhabditis elegans, provide in vivo models of human disease processes that can be instrumental for initial pharmacological studies. Methods To identify early motor impairment of animals expressing α-synuclein in dopaminergic neurons, we first used a custom-built tracking microscope that captures locomotion of single C. elegans with high spatial and temporal resolution. Next, we devised a method for semi-automated and blinded quantification of motor impairment for a population of simultaneously recorded animals with multi-worm tracking and custom image processing. We then used genetic a...

Research paper thumbnail of ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells

Journal of Biological Chemistry, 1993

Botulinum C3 exoenzyme was used to specifically ADP-ribosylate and inactivate rho p21, and the ef... more Botulinum C3 exoenzyme was used to specifically ADP-ribosylate and inactivate rho p21, and the effects of rho p21 inactivation on lysophosphatidic acid (LPA)-induced tyrosine phosphorylation were examined in cultured Swiss 3T3 cells. LPA induced a rapid increase in the tyrosine phosphorylation of a number of proteins. Pretreatment of the cells with the C3 exoenzyme caused ADP-ribosylation of rho p21 in the cells and selectively attenuated the phosphorylation of several proteins, including p43 mitogen-activated protein kinase, p125 focal adhesion kinase, and two proteins of 72 and 88 kDa. C3 exoenzyme pretreatment did not block the initial phosphorylation and activation of mitogen-activated protein kinase but suppressed its subsequent rise. In contrast, the enzyme treatment inhibited the induction of phosphorylation of the 72-and 88-kDa proteins and suppressed the basal and LPA-induced tyrosine phosphorylation of p125 focal adhesion kinase. In addition, immunoprecipitation of cell lysates with an antibody directed against the 85-kDa subunit of phosphatidylinositol 3-kinase (PI 3-kinase) co-precipitated a tyrosinephosphorylated band of 180 kDa. C3 exoenzyme pretreatment suppressed both the phosphorylation of this band and PI 3-kinase activation associated with LPA stimulation. These findings suggest that rho p21 works as a link between the LPA receptor signal and the subsequent tyrosine phosphorylation and PI 3-kinase activation in these cells.

Research paper thumbnail of Hormone-induced apoptosis by Fas-nuclear receptor fusion proteins: novel tools for controlling apoptosis in vivo

Japanese Journal of Pharmacology, 1995

Suppose M and N are PL manifolds and f: M->N is a proper PL map. Triangulate M and N so that / is... more Suppose M and N are PL manifolds and f: M->N is a proper PL map. Triangulate M and N so that / is simplical and let X be the dual complex in N. Then for each open simplex σ in X, f'Ko) is a PL submanifold of M, so the stratification of N by the open simpliceβ of X pulls back to a stratification of M. In other words, any such PL map can be regarded as a map of combinatorially stratified sets in which each ^-stratum of therange is a disjoint union of copies of R n. Here we prove the analogous theorem for a smooth map f:M->N between smooth manifolds.

Research paper thumbnail of Identification of a putative Rho target molecule as a serine/threonine kinase, p160ROCK

Japanese Journal of Pharmacology, 1996

To elucidate the possible involvement of actin filaments in exocytosis, we examined the effects o... more To elucidate the possible involvement of actin filaments in exocytosis, we examined the effects of a novel actin filament stabilizing agent, Jasplakinolide (JAS), on secretory fu nction. Competitive binding study using rhodaminephalloidin showed that JAS binds to F-actin in living acinar cells in a dose dependent manner. At concentrations higher than 10-8 M, JAS inhibited agonist induced amylase release from isolated acini up to 75% at 10-5 M. In the time course of secretion, JAS inhibited the sustained release after 5 min without affecting the initial response .The latter phase was inhibited completely at effective concentrations to cause stabilization of F-actin. Histological examination could not be detected any alteration with JAS treatment. These results suggest that reorganization of actin filaments is essential to the sustained release in succession to the initial secretory response and that actin filaments act as a barrier which restrict docking of granules with apical membrane.

Research paper thumbnail of The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase

The EMBO Journal, 1996

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhes... more The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure-600 amino acids long, and a cysteine-rich zinc fingerlike motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part ofcoiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Research paper thumbnail of Integrin-dependent translocation of p160ROCK to cytoskeletal complex in thrombin-stimulated human platelets

Biochemical Journal, 1997

p160ROCK is a protein serine/threonine kinase that binds to GTP-Rho and is activated by this bind... more p160ROCK is a protein serine/threonine kinase that binds to GTP-Rho and is activated by this binding. We have recently found that the expression of p160ROCK induces focal adhesions and stress fibres in HeLa cells, whereas a dominant-negative form of this kinase suppresses Rho-induced formation of these structures, suggesting that this kinase is a downstream target of Rho in this process [Ishizaki, Naito, Fujisawa, Maekawa, Watanabe, Saito and Narumiya (1997) FEBS Lett. 404, 118-124]. To find out the mode of action of p160ROCK, we developed immunoblotting with an anti-p160ROCK antibody and investigated the subcellular localization of p160ROCK during platelet aggregation. In resting human platelets, more than 90% of p160ROCK was present in the Triton X-100-soluble fraction. When platelets were stimulated with thrombin, approx. 10% of p160ROCK was translocated to the Triton X-100-insoluble fraction. This translocation was detected as early as 20 s after stimulation and reached a maximu...

Research paper thumbnail of Role of citron kinase as a target of the small GTPase Rho in cytokinesis

Nature, Jan 30, 1998

During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of anim... more During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures, is known to be essential. Protein kinases are also thought to participate in cytokinesis. We now show that a splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK14 and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. We find that overexpression of citron mutants results in the production of multinucleate cells and that a...

Research paper thumbnail of Protein Kinase N (PKN) and PKN-Related Protein Rhophilin as Targets of Small GTPase Rho

Science, 1996

The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosp... more The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

Research paper thumbnail of Citron, a Rho-target, interacts with PSD-95 at glutamatergic synapses in the thalamus

Neuroscience Research, 1998

Research paper thumbnail of C. elegans Slit Acts in Midline, Dorsal-Ventral, and Anterior-Posterior Guidance via the SAX-3/Robo Receptor

Neuron, 2001

tors shares a common transmembrane structure with five extracellular immunoglobulin (IG) domains,... more tors shares a common transmembrane structure with five extracellular immunoglobulin (IG) domains, three extracellular fibronectin type 3 (FN3) domains, and several conserved motifs in a long intracellular domain (Kidd et al., 1998; Zallen et al., 1998). Mammalian Robo recep

Research paper thumbnail of Different Regions of Rho Determine Rho-selective Binding of Different Classes of Rho Target Molecules

Journal of Biological Chemistry, 1998

Based on their Rho binding motifs several Rho target molecules can be classified into three group... more Based on their Rho binding motifs several Rho target molecules can be classified into three groups; class I includes the protein kinase PKN, rhophilin, and rhotekin, class II includes the protein kinases, Rho-associated coiled-coil containing protein kinases, ROCK-I and ROCK-II, and class III includes citron. Taking advantage of the selectivity in recognition by these targets between Rho and Rac, we examined the regions in Rho required for selective binding of each class of Rho target molecules. Yeast two-hybrid assays were performed using Rho/Rac chimeras and either rhophilin, ROCK-I, or citron. This study showed the existence of at least two distinct regions in Rho (amino acids 23-40 and 75-92) that are critical for the selective binding of these targets. The former was required for binding to citron, whereas the latter was necessary for binding to rhophilin. On the other hand, either region showed affinity to ROCK-I. This was further confirmed by ligand overlay assay using both recombinant ROCK-I and ROCK-II proteins. Consistently, Rho/Rac chimeras containing either region can induce stress fibers in transfected HeLa cells, and this induction is suppressed by treatment with Y-27632, a specific inhibitor of ROCK kinases. These results suggest that the selective binding of different classes of Rho targets to Rho is determined by interaction between distinct Rho-binding motifs of the targets and different regions of Rho.

Research paper thumbnail of Rhotekin, a New Putative Target for Rho Bearing Homology to a Serine/Threonine Kinase, PKN, and Rhophilin in the Rho-binding Domain

Journal of Biological Chemistry, 1996

Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners f... more Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35 S-labeled guanosine 5-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPaseactivating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares ϳ30% identity with the Rho-binding domains of rhophilin and a serine/ threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G.

Research paper thumbnail of Identification of the Rho-binding Domain of p160 , a Rho-associated Coiled-coil Containing Protein Kinase

Journal of Biological Chemistry, 1996

Research paper thumbnail of p160ROCK , a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions

FEBS Letters, 1997

pl60 is a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by t... more pl60 is a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by this binding. To identify its function, we transfected HeLa cells with wild type and mutants of pl60 ROCK and examined morphology of the transfected cells. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induced focal adhesions and stress fibers, while no induction was observed with a kinase-defective mutant or a mutant containing only the kinase domain. Furthermore, Rho-induced formation of focal adhesions and stress fibers was inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induced an increase in F-actin content in these cells. These results suggest that pl60 ROCK works downstream of Rho to induce formation of focal adhesions and that Rho-induced actin polymerization is mediated by other effector(s).

Research paper thumbnail of ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice

FEBS Letters, 1996

We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target o... more We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target of the small GTPase Rho. Using the human ROCK cDNA as a probe, we isolated cDNA of two distinct, highly related sequences from mouse libraries. One encoded a mouse counterpart of human ROCK (ROCK-I), and the other encoded a novel ROCK-related kinase (ROCK-II). Like ROCK/ROCK-I, ROCK-II also bound to GTP-Rho selectively. ROCK-I mRNA was ubiquitously expressed except in the brain and muscle, whereas ROCK-II mRNA was expressed abundantly in the brain, muscle, heart, lung and placenta. These results suggest that at least two ROCK isoforms are present in a single species and play distinct roles in Rho-mediated signalling pathways.

Research paper thumbnail of Lysophosphatidic acid induces tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase in cultured Swiss 3T3 cells

FEBS Letters, 1993

Lysophosphatidic acid (LPA) added to serum-starved Swiss 3T3 cells induced, m a time-and concentr... more Lysophosphatidic acid (LPA) added to serum-starved Swiss 3T3 cells induced, m a time-and concentration-dependent manner. tyrosine phosphorylatton of multiple proteins. includmg proteins of 43, 64, 88 kDa and a group of proteins between 1 IO and 130 kDa. Among them, two proteins, p43 and ~120. were identified as mttogen-activated protein kmase (MAP-kinase) and focal adhesion kinase (FAK), respectively, by immunoprectpitation and immunoblot analysis. Tyrosine phosphorylation of p64 peaked at 1 mm and declined rapidly, whereas that of MAP-kinase and FAK peaked at 5 and 10 min after the addition of LPA, respectively. The activity of MAP-kmase determined as phosphorylation of myelm basic protein Increased transiently about 3-fold at 5 min, and correlated with tyrosine phosphorylation. These results indicate that tyrosine phosphorylation of these proteins is a part of the signal transduction by LPA and may be involved in its mitogemc responses.

Research paper thumbnail of Lysophosphatidic acid-induced activation of protein Ser/Thr kinases in cultured rat 3Y1 fibroblasts Possible involvement in rho p21-mediated signalling

FEBS Letters, 1995

Renaturation kinase assay was used to detect protein kinases activated by lysophosphatidic acid (... more Renaturation kinase assay was used to detect protein kinases activated by lysophosphatidic acid (LPA) in cultured rat 3YI fibroblasts. LPA activated several Ser/Thr protein kinases with apparent molecular weights of 145K, 85K, 64-65K (a doublet), and 60K (each named p145, p85, p64/65 and/760, respectively) in addition to p43 mitogen activated protein (MAP)kinase. Experiments using pertussis toxin and botulinum C3 exoenzyme showed that p145, p85, and p64165 kinases were activated by a pertussis toxin-insensitive rho p21-dependent pathway and that the activation of MAP-kinase was mediated by both the pertnssis toxin-sensitive rho p21-independent and the pertussis toxin-insensitive rho p21-dependent pathways.

Research paper thumbnail of The slit receptor EVA-1 coactivates a SAX-3/robo-mediated guidance signal in C. elegans

Science, 2007

The SAX-3/roundabout (Robo) receptor has SLT-1/Slit-dependent and -independent functions in guidi... more The SAX-3/roundabout (Robo) receptor has SLT-1/Slit-dependent and -independent functions in guiding cell and axon migrations. We identified enhancer of ventral-axon guidance defects of unc-40 mutants (EVA-1) as a Caenorhabditis elegans transmembrane receptor for SLT-1. EVA-1 has two predicted galactose-binding ectodomains, acts cell-autonomously for SLT-1/Slit-dependent axon migration functions of SAX-3/Robo, binds to SLT-1 and SAX-3, colocalizes with SAX-3 on cells, and provides cell specificity to the activation of SAX-3 signaling by SLT-1. Double mutants of eva-1 or slt-1 with sax-3 mutations suggest that SAX-3 can (when slt-1 or eva-1 function is reduced) inhibit a parallel-acting guidance mechanism, which involves UNC-40/deleted in colorectal cancer.

Research paper thumbnail of Additional file 1 of Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans

Research paper thumbnail of ADP-ribosylation of the rho gene product: I, Protein tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase by lysophosphatidic acid

Japanese Journal of Pharmacology, 1994

Research paper thumbnail of Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans

Molecular Neurodegeneration, 2021

Background Parkinson’s disease is a disabling neurodegenerative movement disorder characterized b... more Background Parkinson’s disease is a disabling neurodegenerative movement disorder characterized by dopaminergic neuron loss induced by α-synuclein oligomers. There is an urgent need for disease-modifying therapies for Parkinson’s disease, but drug discovery is challenged by lack of in vivo models that recapitulate early stages of neurodegeneration. Invertebrate organisms, such as the nematode worm Caenorhabditis elegans, provide in vivo models of human disease processes that can be instrumental for initial pharmacological studies. Methods To identify early motor impairment of animals expressing α-synuclein in dopaminergic neurons, we first used a custom-built tracking microscope that captures locomotion of single C. elegans with high spatial and temporal resolution. Next, we devised a method for semi-automated and blinded quantification of motor impairment for a population of simultaneously recorded animals with multi-worm tracking and custom image processing. We then used genetic a...

Research paper thumbnail of ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells

Journal of Biological Chemistry, 1993

Botulinum C3 exoenzyme was used to specifically ADP-ribosylate and inactivate rho p21, and the ef... more Botulinum C3 exoenzyme was used to specifically ADP-ribosylate and inactivate rho p21, and the effects of rho p21 inactivation on lysophosphatidic acid (LPA)-induced tyrosine phosphorylation were examined in cultured Swiss 3T3 cells. LPA induced a rapid increase in the tyrosine phosphorylation of a number of proteins. Pretreatment of the cells with the C3 exoenzyme caused ADP-ribosylation of rho p21 in the cells and selectively attenuated the phosphorylation of several proteins, including p43 mitogen-activated protein kinase, p125 focal adhesion kinase, and two proteins of 72 and 88 kDa. C3 exoenzyme pretreatment did not block the initial phosphorylation and activation of mitogen-activated protein kinase but suppressed its subsequent rise. In contrast, the enzyme treatment inhibited the induction of phosphorylation of the 72-and 88-kDa proteins and suppressed the basal and LPA-induced tyrosine phosphorylation of p125 focal adhesion kinase. In addition, immunoprecipitation of cell lysates with an antibody directed against the 85-kDa subunit of phosphatidylinositol 3-kinase (PI 3-kinase) co-precipitated a tyrosinephosphorylated band of 180 kDa. C3 exoenzyme pretreatment suppressed both the phosphorylation of this band and PI 3-kinase activation associated with LPA stimulation. These findings suggest that rho p21 works as a link between the LPA receptor signal and the subsequent tyrosine phosphorylation and PI 3-kinase activation in these cells.

Research paper thumbnail of Hormone-induced apoptosis by Fas-nuclear receptor fusion proteins: novel tools for controlling apoptosis in vivo

Japanese Journal of Pharmacology, 1995

Suppose M and N are PL manifolds and f: M->N is a proper PL map. Triangulate M and N so that / is... more Suppose M and N are PL manifolds and f: M->N is a proper PL map. Triangulate M and N so that / is simplical and let X be the dual complex in N. Then for each open simplex σ in X, f'Ko) is a PL submanifold of M, so the stratification of N by the open simpliceβ of X pulls back to a stratification of M. In other words, any such PL map can be regarded as a map of combinatorially stratified sets in which each ^-stratum of therange is a disjoint union of copies of R n. Here we prove the analogous theorem for a smooth map f:M->N between smooth manifolds.

Research paper thumbnail of Identification of a putative Rho target molecule as a serine/threonine kinase, p160ROCK

Japanese Journal of Pharmacology, 1996

To elucidate the possible involvement of actin filaments in exocytosis, we examined the effects o... more To elucidate the possible involvement of actin filaments in exocytosis, we examined the effects of a novel actin filament stabilizing agent, Jasplakinolide (JAS), on secretory fu nction. Competitive binding study using rhodaminephalloidin showed that JAS binds to F-actin in living acinar cells in a dose dependent manner. At concentrations higher than 10-8 M, JAS inhibited agonist induced amylase release from isolated acini up to 75% at 10-5 M. In the time course of secretion, JAS inhibited the sustained release after 5 min without affecting the initial response .The latter phase was inhibited completely at effective concentrations to cause stabilization of F-actin. Histological examination could not be detected any alteration with JAS treatment. These results suggest that reorganization of actin filaments is essential to the sustained release in succession to the initial secretory response and that actin filaments act as a barrier which restrict docking of granules with apical membrane.

Research paper thumbnail of The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase

The EMBO Journal, 1996

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhes... more The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure-600 amino acids long, and a cysteine-rich zinc fingerlike motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part ofcoiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Research paper thumbnail of Integrin-dependent translocation of p160ROCK to cytoskeletal complex in thrombin-stimulated human platelets

Biochemical Journal, 1997

p160ROCK is a protein serine/threonine kinase that binds to GTP-Rho and is activated by this bind... more p160ROCK is a protein serine/threonine kinase that binds to GTP-Rho and is activated by this binding. We have recently found that the expression of p160ROCK induces focal adhesions and stress fibres in HeLa cells, whereas a dominant-negative form of this kinase suppresses Rho-induced formation of these structures, suggesting that this kinase is a downstream target of Rho in this process [Ishizaki, Naito, Fujisawa, Maekawa, Watanabe, Saito and Narumiya (1997) FEBS Lett. 404, 118-124]. To find out the mode of action of p160ROCK, we developed immunoblotting with an anti-p160ROCK antibody and investigated the subcellular localization of p160ROCK during platelet aggregation. In resting human platelets, more than 90% of p160ROCK was present in the Triton X-100-soluble fraction. When platelets were stimulated with thrombin, approx. 10% of p160ROCK was translocated to the Triton X-100-insoluble fraction. This translocation was detected as early as 20 s after stimulation and reached a maximu...

Research paper thumbnail of Role of citron kinase as a target of the small GTPase Rho in cytokinesis

Nature, Jan 30, 1998

During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of anim... more During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures, is known to be essential. Protein kinases are also thought to participate in cytokinesis. We now show that a splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK14 and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. We find that overexpression of citron mutants results in the production of multinucleate cells and that a...

Research paper thumbnail of Protein Kinase N (PKN) and PKN-Related Protein Rhophilin as Targets of Small GTPase Rho

Science, 1996

The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosp... more The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

Research paper thumbnail of Citron, a Rho-target, interacts with PSD-95 at glutamatergic synapses in the thalamus

Neuroscience Research, 1998

Research paper thumbnail of C. elegans Slit Acts in Midline, Dorsal-Ventral, and Anterior-Posterior Guidance via the SAX-3/Robo Receptor

Neuron, 2001

tors shares a common transmembrane structure with five extracellular immunoglobulin (IG) domains,... more tors shares a common transmembrane structure with five extracellular immunoglobulin (IG) domains, three extracellular fibronectin type 3 (FN3) domains, and several conserved motifs in a long intracellular domain (Kidd et al., 1998; Zallen et al., 1998). Mammalian Robo recep

Research paper thumbnail of Different Regions of Rho Determine Rho-selective Binding of Different Classes of Rho Target Molecules

Journal of Biological Chemistry, 1998

Based on their Rho binding motifs several Rho target molecules can be classified into three group... more Based on their Rho binding motifs several Rho target molecules can be classified into three groups; class I includes the protein kinase PKN, rhophilin, and rhotekin, class II includes the protein kinases, Rho-associated coiled-coil containing protein kinases, ROCK-I and ROCK-II, and class III includes citron. Taking advantage of the selectivity in recognition by these targets between Rho and Rac, we examined the regions in Rho required for selective binding of each class of Rho target molecules. Yeast two-hybrid assays were performed using Rho/Rac chimeras and either rhophilin, ROCK-I, or citron. This study showed the existence of at least two distinct regions in Rho (amino acids 23-40 and 75-92) that are critical for the selective binding of these targets. The former was required for binding to citron, whereas the latter was necessary for binding to rhophilin. On the other hand, either region showed affinity to ROCK-I. This was further confirmed by ligand overlay assay using both recombinant ROCK-I and ROCK-II proteins. Consistently, Rho/Rac chimeras containing either region can induce stress fibers in transfected HeLa cells, and this induction is suppressed by treatment with Y-27632, a specific inhibitor of ROCK kinases. These results suggest that the selective binding of different classes of Rho targets to Rho is determined by interaction between distinct Rho-binding motifs of the targets and different regions of Rho.

Research paper thumbnail of Rhotekin, a New Putative Target for Rho Bearing Homology to a Serine/Threonine Kinase, PKN, and Rhophilin in the Rho-binding Domain

Journal of Biological Chemistry, 1996

Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners f... more Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35 S-labeled guanosine 5-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPaseactivating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares ϳ30% identity with the Rho-binding domains of rhophilin and a serine/ threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G.

Research paper thumbnail of Identification of the Rho-binding Domain of p160 , a Rho-associated Coiled-coil Containing Protein Kinase

Journal of Biological Chemistry, 1996

Research paper thumbnail of p160ROCK , a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions

FEBS Letters, 1997

pl60 is a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by t... more pl60 is a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by this binding. To identify its function, we transfected HeLa cells with wild type and mutants of pl60 ROCK and examined morphology of the transfected cells. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induced focal adhesions and stress fibers, while no induction was observed with a kinase-defective mutant or a mutant containing only the kinase domain. Furthermore, Rho-induced formation of focal adhesions and stress fibers was inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induced an increase in F-actin content in these cells. These results suggest that pl60 ROCK works downstream of Rho to induce formation of focal adhesions and that Rho-induced actin polymerization is mediated by other effector(s).

Research paper thumbnail of ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice

FEBS Letters, 1996

We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target o... more We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target of the small GTPase Rho. Using the human ROCK cDNA as a probe, we isolated cDNA of two distinct, highly related sequences from mouse libraries. One encoded a mouse counterpart of human ROCK (ROCK-I), and the other encoded a novel ROCK-related kinase (ROCK-II). Like ROCK/ROCK-I, ROCK-II also bound to GTP-Rho selectively. ROCK-I mRNA was ubiquitously expressed except in the brain and muscle, whereas ROCK-II mRNA was expressed abundantly in the brain, muscle, heart, lung and placenta. These results suggest that at least two ROCK isoforms are present in a single species and play distinct roles in Rho-mediated signalling pathways.

Research paper thumbnail of Lysophosphatidic acid induces tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase in cultured Swiss 3T3 cells

FEBS Letters, 1993

Lysophosphatidic acid (LPA) added to serum-starved Swiss 3T3 cells induced, m a time-and concentr... more Lysophosphatidic acid (LPA) added to serum-starved Swiss 3T3 cells induced, m a time-and concentration-dependent manner. tyrosine phosphorylatton of multiple proteins. includmg proteins of 43, 64, 88 kDa and a group of proteins between 1 IO and 130 kDa. Among them, two proteins, p43 and ~120. were identified as mttogen-activated protein kmase (MAP-kinase) and focal adhesion kinase (FAK), respectively, by immunoprectpitation and immunoblot analysis. Tyrosine phosphorylation of p64 peaked at 1 mm and declined rapidly, whereas that of MAP-kinase and FAK peaked at 5 and 10 min after the addition of LPA, respectively. The activity of MAP-kmase determined as phosphorylation of myelm basic protein Increased transiently about 3-fold at 5 min, and correlated with tyrosine phosphorylation. These results indicate that tyrosine phosphorylation of these proteins is a part of the signal transduction by LPA and may be involved in its mitogemc responses.

Research paper thumbnail of Lysophosphatidic acid-induced activation of protein Ser/Thr kinases in cultured rat 3Y1 fibroblasts Possible involvement in rho p21-mediated signalling

FEBS Letters, 1995

Renaturation kinase assay was used to detect protein kinases activated by lysophosphatidic acid (... more Renaturation kinase assay was used to detect protein kinases activated by lysophosphatidic acid (LPA) in cultured rat 3YI fibroblasts. LPA activated several Ser/Thr protein kinases with apparent molecular weights of 145K, 85K, 64-65K (a doublet), and 60K (each named p145, p85, p64/65 and/760, respectively) in addition to p43 mitogen activated protein (MAP)kinase. Experiments using pertussis toxin and botulinum C3 exoenzyme showed that p145, p85, and p64165 kinases were activated by a pertussis toxin-insensitive rho p21-dependent pathway and that the activation of MAP-kinase was mediated by both the pertnssis toxin-sensitive rho p21-independent and the pertussis toxin-insensitive rho p21-dependent pathways.

Research paper thumbnail of The slit receptor EVA-1 coactivates a SAX-3/robo-mediated guidance signal in C. elegans

Science, 2007

The SAX-3/roundabout (Robo) receptor has SLT-1/Slit-dependent and -independent functions in guidi... more The SAX-3/roundabout (Robo) receptor has SLT-1/Slit-dependent and -independent functions in guiding cell and axon migrations. We identified enhancer of ventral-axon guidance defects of unc-40 mutants (EVA-1) as a Caenorhabditis elegans transmembrane receptor for SLT-1. EVA-1 has two predicted galactose-binding ectodomains, acts cell-autonomously for SLT-1/Slit-dependent axon migration functions of SAX-3/Robo, binds to SLT-1 and SAX-3, colocalizes with SAX-3 on cells, and provides cell specificity to the activation of SAX-3 signaling by SLT-1. Double mutants of eva-1 or slt-1 with sax-3 mutations suggest that SAX-3 can (when slt-1 or eva-1 function is reduced) inhibit a parallel-acting guidance mechanism, which involves UNC-40/deleted in colorectal cancer.