Biomedical subjects

Kozo Kaibuchi

Publications and source records attributed to Kozo Kaibuchi.

At least 19 recordsLinked to original sources

PubMed2007

DISC1 regulates neurotrophin-induced axon elongation via interaction with Grb2.

Disrupted-in-Schizophrenia-1 (DISC1) is a candidate gene for susceptibility of schizophrenia. In the accompanying paper (Taya et al., 2006), we report that DISC1 acts as a linker between Kinesin-1 and DISC1-interacting molecules, such as NudE-like, lissencephaly-1, and 14-3-3epsilon. Here we identified growth factor receptor bound protein 2 (Grb2) as a novel DISC1-interacting molecule. Grb2 acts as an adaptor molecule that links receptor tyrosine kinases and the Ras-extracellular signal-regulated kinase (ERK) pathway. DISC1 formed a ternary complex with Grb2 and kinesin heavy chain KIF5A of Kinesin-1. In cultured rat hippocampal neurons, both DISC1 and Grb2 partially colocalized at the distal part of axons. Knockdown of DISC1 or kinesin light chains of Kinesin-1 by RNA interference inhibited the accumulation of Grb2 from the distal part of axons. Knockdown of DISC1 also inhibited the neurotrophin-3 (NT-3)-induced phosphorylation of ERK-1/2 at the distal part of axons and inhibited NT-3-induced axon elongation. These results suggest that DISC1 is required for NT-3-induced axon elongation and ERK activation at the distal part of axons by recruiting Grb2 to axonal tips.

Animals↗

PubMed2007

DISC1 regulates the transport of the NUDEL/LIS1/14-3-3epsilon complex through kinesin-1.

Disrupted-In-Schizophrenia 1 (DISC1) is a candidate gene for susceptibility to schizophrenia. DISC1 is reported to interact with NudE-like (NUDEL), which forms a complex with lissencephaly-1 (LIS1) and 14-3-3epsilon. 14-3-3epsilon is involved in the proper localization of NUDEL and LIS1 in axons. Although the functional significance of this complex in neuronal development has been reported, the transport mechanism of the complex into axons and their functions in axon formation remain essentially unknown. Here we report that Kinesin-1, a motor protein of anterograde axonal transport, was identified as a novel DISC1-interacting molecule. DISC1 directly interacted with kinesin heavy chain of Kinesin-1. Kinesin-1 interacted with the NUDEL/LIS1/14-3-3epsilon complex through DISC1, and these molecules localized mainly at cell bodies and partially in the distal part of the axons. DISC1 partially colocalized with Kinesin family member 5A, NUDEL, LIS1, and 14-3-3epsilon in the growth cones. The knockdown of DISC1 by RNA interference or the dominant-negative form of DISC1 inhibited the accumulation of NUDEL, LIS1, and 14-3-3epsilon at the axons and axon elongation. The knockdown or the dominant-negative form of Kinesin-1 inhibited the accumulation of DISC1 at the axons and axon elongation. Furthermore, the knockdown of NUDEL or LIS1 inhibited axon elongation. Together, these results indicate that DISC1 regulates the localization of NUDEL/LIS1/14-3-3epsilon complex into the axons as a cargo receptor for axon elongation.

14-3-3 Proteins↗

PubMed2006

Essential roles for GSK-3s and GSK-3-primed substrates in neurotrophin-induced and hippocampal axon growth.

Glycogen synthase kinase-3beta (GSK-3beta) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, we found no gross morphological deficits in nervous system development in GSK-3beta null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3alpha and beta markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.

Animals↗

PubMed2006

Design and synthesis of rho kinase inhibitors (III).

The structure-activity relationship of Rho kinase inhibitors bearing an isoquinoline scaffold was studied. N-(1-Benzyl-3-pyrrolidyl)-N-(5-isoquinolyl)amine analogues were optimized with respect to their inhibitory potencies for the enzyme and for chemotaxis. The potent analogues were further evaluated by an ex vivo test in which the selected compounds were orally administered to rats, and the Rho kinase inhibitory potency observed in the rat serum was evaluated 3h after the administration. Compound 23g showed a high level of Rho kinase inhibitory activity in the rat serum and was stable in an in vitro metabolic test using a microsomal cytochrome preparation. The (R)-isomer of 23g displayed a higher level of inhibitory potency than the (S)-isomer in a cell-free kinase assay and in the cell migration assay (IC(50)(ENZ)=25 nM and IC(50)(MCP)=1 microM). The (R)-isomer successfully inhibited the phosphorylation of MBS (myosin-binding subunit) in cells.

Alkylation↗

PubMed2006

Signaling networks in neuronal polarization.

A mature neuron is typically polarized both structurally and functionally, with a single long axon and several dendrites. Neuronal polarity is essential for unidirectional signal flow from somata or dendrites to axons. The initial event in establishing a polarized neuron is the specification of a single axon. Early in neuronal development, one immature neurite becomes differentiated from other neurites to form an axon. Although studies in the past two decades have yielded a catalog of structural, molecular, and functional differences between axons and dendrites, we are only now beginning to understand the molecular mechanisms involved in the establishment of neuronal polarity. In the last few years, neuronal polarity-regulating molecules have been revealed. There are two major signaling cascades in neuronal polarization. Several groups, including ours, reported that the phosphatidylinositol 3-kinase (PI3-kinase)/Akt/glycogen synthase kinase-3beta (GSK-3beta)/collapsin response mediator protein-2 pathway is important for axon specification and elongation. Recent studies have revealed that the positive feedback loop composed of Rho family small GTPases and the Par3/Par6/atypical protein kinase C complex plays a role in the initial events of neuronal polarization downstream of PI3-kinase. Here, we discuss the roles of signaling molecules for axon specification.

Animals↗

PubMed2006

Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication.

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

Animals↗

PubMed2006

Hypoxia-induced upregulation of endothelial small G protein RhoA and Rho-kinase/ROCK2 inhibits eNOS expression.

The small G protein RhoA and its downstream effector Rho-kinase/ROCK2 play an important role in regulation of various vasculature cellular functions. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is an important mediator of vascular homeostasis and cerebral blood flow. Using the human endothelial cell line HUVEC, the present study investigated the role of RhoA and Rho-kinase in endothelial eNOS protein expression under hypoxic conditions as an in vitro model of ischemia. RhoA protein levels in HUVEC were low under normoxic conditions, but were significantly increased after 5h of hypoxia. Endothelial Rho-kinase expression was not detected until after 3h of hypoxia; such expression remained significantly increased after 5h. On the other hand, endothelial eNOS expression was similar after 3h of hypoxia, but was significantly decreased after 5h. The hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of RhoA and significantly inhibited by suppression of RhoA expression by small interfering RNA. The hypoxia-induced decrease in eNOS expression was significantly inhibited when endogenous Rho-kinase activation was inhibited by Rho-binding domain expression. Furthermore, the hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of Rho-kinase. Since expression and activation of RhoA and Rho-kinase inhibit eNOS expression in endothelial cells, attempts to down-regulate RhoA and Rho-kinase by multiple drugs, such as statins or Rho-kinase inhibitors, might provide endothelial and cardiovascular benefits through upregulation of eNOS.

Cell Hypoxia↗

PubMed2006

Sustained elevation of serum cortisol level causes sensitization of coronary vasoconstricting responses in pigs in vivo: a possible link between stress and coronary vasospasm.

Vasospastic angina is induced by stress, for which cortisol secreted by activated hypothalamic/pituitary/adrenal axis may play an important role. However, direct evidence for this notion is still lacking. In this study, we examined whether sustained elevation of serum cortisol level sensitizes coronary vasoconstricting responses in pigs in vivo and, if so, whether Rho-kinase, which we found is a key molecule of coronary vasospasm, is involved. Oral administration of cortisol (20 mg/kg per day) increased its serum level to that seen in restraint stress in pigs. Thus, we examined coronary vasomotor responses in the following 4 groups: (1) control (without cortisol); (2) cortisol (20 mg/kg per day, p.o.) for 9 days; (3) cortisol plus RU38486 (a glucocorticoids receptor antagonist, 10 mg/kg per day, p.o.) for 9 days; and (4) cortisol for 9 days followed by 6-week withdrawal. Coronary angiography showed that intracoronary serotonin caused coronary hyperconstriction and reduction in coronary blood flow associated with ischemic ECG changes (coronary vasospasm) in only the cortisol group. All of these responses were abolished by hydroxyfasudil, a specific Rho-kinase inhibitor, in vivo. Organ chamber experiments demonstrated that serotonin concentration-dependently caused hypercontractions of coronary vascular smooth muscle associated with Rho-kinase activation (as evidenced by the enhanced phosphorylation of myosin binding subunit, a substrate of Rho-kinase) in only the cortisol group. All of these responses were again inhibited by hydroxyfasudil in vitro. These results indicate that sustained elevation of serum cortisol level sensitizes coronary vasoconstricting responses through Rho-kinase activation, suggesting the link between stress and coronary vasospasm.

Administration, Oral↗

PubMed2006

Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632.

Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability.

Amides↗

PubMed2006

Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse.

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). Here we characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppressed R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressed the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocked the growth cone collapse. The R-Ras GAP activity was necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and was also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induced dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. We conclude that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse.

Animals↗

PubMed2006

Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism.

Myelin-associated glycoprotein (MAG) and Nogo are potent inhibitors of neurite outgrowth from a variety of neurons, and they have been identified as possible components of the central nervous system myelin that prevents axonal regeneration in the adult vertebrate central nervous system. The activation of RhoA and Rho-kinase is reported to be an essential part of the signaling mechanism of these proteins. Here, we report that the collapsing response mediator protein-2 (CRMP-2) is phosphorylated by a Rho-kinase-dependent mechanism downstream of MAG or Nogo-66. The overexpression of the nonphosphorylated form of CRMP-2 at threonine 555, which is the phosphorylation site for Rho-kinase, counteracts the inhibitory effect of MAG on the postnatal cerebellar neurons. Additionally, the expression of the dominant negative form of CRMP-2 or knockdown of the gene using small interference RNA (siRNA) mimics the effect of MAG in vitro. Consistent with the function of CRMP-2, which promotes microtubule assembly, microtubule levels are down-regulated in the cerebellar neurons that are stimulated with MAG in vitro. Reduction in the density of microtubules is also observed in the injured axons following the spinal cord injury, and this effect depends on the Rho-kinase activity. Our data suggest the important roles of CRMP-2 and microtubules in the inhibition of the axon regeneration by the myelin-derived inhibitors.

Animals↗

PubMed2006

Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase.

Rho-associated coiled-coil protein kinase (ROCK) is an effector for the small GTPase Rho and plays a pivotal role in diverse cellular activities, including cell adhesion, cytokinesis, and gene expression, primarily through an alteration of actin cytoskeleton dynamics. Here, we show that ROCK2 is localized in the nucleus and associates with p300 acetyltransferase both in vitro and in cells. Nuclear ROCK2 is present in a large protein complex and partially cofractionates with p300 by gel filtration analysis. By immunofluorescence, ROCK2 partially colocalizes with p300 in distinct insoluble nuclear structures. ROCK2 phosphorylates p300 in vitro, and nuclear-restricted expression of constitutively active ROCK2 induces p300 phosphorylation in cells. p300 acetyltransferase activity is dependent on its phosphorylation status in cells, and p300 phosphorylation by ROCK2 results in an increase in its acetyltransferase activity in vitro. These observations suggest that nucleus-localized ROCK2 targets p300 for phosphorylation to regulate its acetyltransferase activity.

Animals↗

PubMed2006

Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE).

The blood-brain barrier (BBB) is compromised during progressive HIV-1 infection, but how this occurs is incompletely understood. We studied the integrity of tight junctions (TJs) of brain microvascular endothelial cells (BMVECs) in an in vitro BBB system and in human brain tissues with HIV-1 encephalitis (HIVE). A downregulation of TJ proteins, claudin-5 and occludin, paralleled monocyte migration into the brain during HIVE. Because small G proteins (such as Rho) can play a role in BMVEC TJ assembly, an artificial BBB system explored the relationship among TJs, Rho/Rho kinase (RhoK) activation, and transendothelial monocyte migration. Coculture of monocytes with endothelial cells led to Rho activation and phosphorylation of TJ proteins. Rho and RhoK inhibitors blocked migration of infected and uninfected monocytes. The RhoK inhibitor protected BBB integrity and reversed occludin/claudin-5 phosphorylation associated with monocyte migration. BMVEC transfection with a constitutively active mutant of RhoK led to dislocation of occludin from the membrane and loss of BMVEC cell contacts. When dominant-negative RhoK-transfected BMVECs were used in BBB constructs, monocyte migration was reduced by 84%. Thus, loss of TJ integrity was associated with Rho activation caused by monocyte brain migration, suggesting that Rho/RhoK activation in BMVECs could be an underlying cause of BBB impairment during HIVE.

Blood-Brain Barrier↗

PubMed2006

Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart.

Characterization of cardiac MYPT2 (an isoform of the smooth muscle phosphatase [MP] target subunit, MYPT1) is described. Several features of MYPT2 and MYPT1 were similar, including: a specific interaction with the catalytic subunit of type 1 phosphatase, delta isoform (PP1cdelta); interaction of MYPT2 with the small heart-specific MP subunit; interaction of the C-terminal region of MYPT2 with the active form of RhoA; phosphorylation by Rho-kinase at an inhibitory site, Thr646 and thiophosphorylation at Thr646 inhibited activity of the MYPT2-PP1cdelta complex. MYPT2 activated PP1cdelta activity, using light chains from smooth and cardiac muscle, by reducing K(m) and increasing k(cat). The extent of activation (k(cat)) was greater than for MYPT1 and could reflect distinct N-terminal sequences in the two MYPT isoforms. Adenovirus-mediated gene transfer of MYPT2 and PP1cdelta reduced the phosphorylation level of cardiac light chains following stimulation with A23187. Overexpression of MYPT2 and PP1cdelta blocked the angiotensin II-induced sarcomere organization in cultured cardiomyocytes. Electron microscopy indicated locations of MYPTs, at, or close to, the Z-line, the A band and mitochondria. Similarity of the two MYPT isoforms suggests common enzymatic mechanisms and regulation. Cardiac myosin is a substrate for the MYPT2 holoenzyme, but the Z-line location raises the possibility of other substrates.

Angiotensin II↗

PubMed2006

Evidence for a role of Collapsin response mediator protein-2 in signaling pathways that regulate the proliferation of non-neuronal cells.

Collapsin response mediator protein-2 or Crmp-2 plays a critical role in the establishment of neuronal polarity. In this study, we present evidence that apart from its functions in neurodevelopment, Crmp-2 is also involved in pathways that regulate the proliferation of non-neuronal cells through its phosphorylation by regulatory proteins. We show that Crmp-2 undergoes dynamic phosphorylation changes in response to contact inhibition-induced quiescence and that hyperphosphorylation of Crmp-2 occurs in a tumor. We further suggest that de-regulation of Crmp-2 phosphorylation levels at certain amino acid residues may lead to aberrant cell proliferation and consequently, tumorigenesis.

Animals↗

PubMed2006

Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2.

Numb has been implicated in cortical neurogenesis during nervous system development, as a result of its asymmetric partitioning and antagonizing Notch signaling. Recent studies have revealed that Numb functions in clathrin-dependent endocytosis by binding to the AP-2 complex. Numb is also expressed in postmitotic neurons and plays a role in axonal growth. However, the functions of Numb in later stages of neuronal development remain unknown. Here, we report that Numb specifically localizes to dendritic spines in cultured hippocampal neurons and is implicated in dendritic spine morphogenesis, partially through the direct interaction with intersectin, a Cdc42 guanine nucleotide exchange factor (GEF). Intersectin functions as a multidomain adaptor for proteins involved in endocytosis and cytoskeletal regulation. Numb enhanced the GEF activity of intersectin toward Cdc42 in vivo. Expression of Numb or intersectin caused the elongation of spine neck, whereas knockdown of Numb and Numb-like decreased the protrusion density and its length. Furthermore, Numb formed a complex with EphB2 receptor-type tyrosine kinase and NMDA-type glutamate receptors. Knockdown of Numb suppressed the ephrin-B1-induced spine development and maturation. These results highlight a role of Numb for dendritic spine development and synaptic functions with intersectin and EphB2.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Molecular mechanism for the regulation of rho-kinase by dimerization and its inhibition by fasudil.

Rho-kinase is a key regulator of cytoskeletal events and a promising drug target in the treatment of vascular diseases and neurological disorders. Unlike other protein kinases, Rho-kinase requires both N- and C-terminal extension segments outside the kinase domain for activity, although the details of this requirement have been elusive. The crystal structure of an active Rho-kinase fragment containing the kinase domain and both the extensions revealed a head-to-head homodimer through the N-terminal extension forming a helix bundle that structurally integrates the C-terminal extension. This structural organization enables binding of the C-terminal hydrophobic motif to the N-terminal lobe, which defines the correct disposition of helix alphaC that is important for the catalytic activity. The bound inhibitor fasudil significantly alters the conformation and, consequently, the mode of interaction with the catalytic cleft that contains local structural changes. Thus, both kinase and drug conformational pliability and stability confer selectivity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2006

Long-term inhibition of Rho-kinase ameliorates hypoxia-induced pulmonary hypertension in mice.

Pulmonary hypertension (PH) is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells, and migration of inflammatory cells for which no satisfactory treatment has yet been developed. It has been recently demonstrated that Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis and that long-term inhibition of Rho-kinase markedly ameliorates monocrotaline-induced PH in rats. However, it remains to be examined whether direct inhibition of Rho-kinase also ameliorates PH with a different etiology and whether endothelial nitric oxide synthase (eNOS) is involved in the beneficial effects of Rho-kinase inhibition. This study was designed to address those 2 important issues in a hypoxia-induced PH model using wild-type (WT) and eNOS-deficient (eNOS) mice. Long-term blockade of Rho-kinase with fasudil (100 mg/kg/d) for 3 weeks markedly improved PH and right ventricular hypertrophy in WT mice with a lesser but significant inhibition noted in eNOS mice. Fasudil upregulated eNOS with increased Akt phosphorylation in WT but not in eNOS mice. These results suggest that long-term inhibition of Rho-kinase also ameliorates hypoxia-induced PH in mice, for which eNOS activation may partially be involved.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗