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Kozo Kaibuchi - One of the best experts on this subject based on the ideXlab platform.
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proteomic screening for Rho Kinase substrates by combining Kinase and phosphatase inhibitors with 14 3 3ζ affinity chromatography
Cell Structure and Function, 2012Co-Authors: Tomoki Nishioka, Mutsuki Amano, Masanori Nakayama, Kozo KaibuchiAbstract:The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-Kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-Kinase substrates have been reported, but the known substrates do not fully explain the Rho-Kinase functions. Herein, we describe the comprehensive screening for Rho-Kinase substrates by treating HeLa cells with Rho-Kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-Kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-Kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other Kinases.
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Rho Kinase rock a key regulator of the cytoskeleton and cell polarity
Cytoskeleton, 2010Co-Authors: Mutsuki Amano, Masanori Nakayama, Kozo KaibuchiAbstract:Rho-associated Kinase (Rho-Kinase/ROCK/ROK) is an effector of the small GTPase Rho and belongs to the AGC family of Kinases. Rho-Kinase has pleiotropic functions including the regulation of cellular contraction, motility, morphology, polarity, cell division, and gene expression. Pharmacological analyses have revealed that Rho-Kinase is involved in a wide range of diseases such as vasospasm, pulmonary hypertension, nerve injury, and glaucoma, and is therefore considered to be a potential therapeutic target. This review focuses on the structure, function, and modes of activation and action of Rho-Kinase.
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structural basis for induced fit binding of Rho Kinase to the inhibitor y 27632
Journal of Biochemistry, 2006Co-Authors: Hiroto Yamaguchi, Mutsuki Amano, Kozo Kaibuchi, Yukiko Miwa, Miyuki Kasa, Ken Kitano, Toshio HakoshimaAbstract: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.
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myelin associated glycoprotein inhibits microtubule assembly by a Rho Kinase dependent mechanism
Journal of Biological Chemistry, 2006Co-Authors: Fumiaki Mimura, Kozo Kaibuchi, Satoru Yamagishi, Masashi Fujitani, Nariko Arimura, Takekazu Kubo, Toshihide YamashitaAbstract:Abstract 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.
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long term treatment with a Rho Kinase inhibitor improves monocrotaline induced fatal pulmonary hypertension in rats
Circulation Research, 2004Co-Authors: Kohtaro Abe, Kozo Kaibuchi, Tsuyoshi Hattori, Keiko Morikawa, Toyokazu Uwatoku, Yasuharu Matsumoto, Yutaka Nakashima, Katsuo Sueishi, Akira TakeshitAbstract:Primary pulmonary hypertension is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells (VSMCs), and migration of inflammatory cells, for which no satisfactory treatment has yet been developed. We have recently demonstrated that intracellular signaling pathway mediated by Rho-Kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis. In the present study, we examined whether the Rho-Kinase-mediated pathway is also involved in the pathogenesis of fatal pulmonary hypertension in rats. Animals received a subcutaneous injection of monocrotaline, which resulted in the development of severe pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions in 3 weeks associated with subsequent high mortality rate. The long-term blockade of Rho-Kinase with fasudil, which is metabolized to a specific Rho-Kinase inhibitor hydroxyfasudil after oral administration, markedly improved survival when started concomitantly with monocrotaline and even when started after development of pulmonary hypertension. The fasudil treatment improved pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions with suppression of VSMC proliferation and macrophage infiltration, enhanced VSMC apoptosis, and amelioration of endothelial dysfunction and VSMC hypercontraction. These results indicate that Rho-Kinase-mediated pathway is substantially involved in the pathogenesis of pulmonary hypertension, suggesting that the molecule could be a novel therapeutic target for the fatal disorder.
Mutsuki Amano - One of the best experts on this subject based on the ideXlab platform.
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proteomic screening for Rho Kinase substrates by combining Kinase and phosphatase inhibitors with 14 3 3ζ affinity chromatography
Cell Structure and Function, 2012Co-Authors: Tomoki Nishioka, Mutsuki Amano, Masanori Nakayama, Kozo KaibuchiAbstract:The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-Kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-Kinase substrates have been reported, but the known substrates do not fully explain the Rho-Kinase functions. Herein, we describe the comprehensive screening for Rho-Kinase substrates by treating HeLa cells with Rho-Kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-Kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-Kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other Kinases.
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Rho Kinase rock a key regulator of the cytoskeleton and cell polarity
Cytoskeleton, 2010Co-Authors: Mutsuki Amano, Masanori Nakayama, Kozo KaibuchiAbstract:Rho-associated Kinase (Rho-Kinase/ROCK/ROK) is an effector of the small GTPase Rho and belongs to the AGC family of Kinases. Rho-Kinase has pleiotropic functions including the regulation of cellular contraction, motility, morphology, polarity, cell division, and gene expression. Pharmacological analyses have revealed that Rho-Kinase is involved in a wide range of diseases such as vasospasm, pulmonary hypertension, nerve injury, and glaucoma, and is therefore considered to be a potential therapeutic target. This review focuses on the structure, function, and modes of activation and action of Rho-Kinase.
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structural basis for induced fit binding of Rho Kinase to the inhibitor y 27632
Journal of Biochemistry, 2006Co-Authors: Hiroto Yamaguchi, Mutsuki Amano, Kozo Kaibuchi, Yukiko Miwa, Miyuki Kasa, Ken Kitano, Toshio HakoshimaAbstract: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.
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Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin a1 by Rho Kinase
Genes to Cells, 2005Co-Authors: Takako Kaneko, Yoji Kawano, Masanori Nakayama, Akio Maeda, Mikito Takefuji, Hiroki Aoyama, Saeko Kawabata, Akihiro Iwamatsu, Mutsuki AmanoAbstract:After binding of epidermal growth factor (EGF), the EGF receptor is activated, internalized by endocytosis, and subsequently degraded in the lysosomal pathway. Endocytotic trafficking of the activated EGF receptor is essential for controlling EGF signaling. Upon ligand-induced activation of EGF receptors, Cbl (ubiquitin ligase) binds to the activated receptor and leads to translocation of the CIN85 (Cbl-interacting protein of 85 kDa)/endophilin complex in the vicinity of the activated EGF receptors. Endophilin is known as a key regulator of clathrin-mediated endocytosis, and the translocation of endophilin in the vicinity of active EGF receptor is thought to promote receptor internalization. The constitutively active mutant of small GTPase Rho inhibits EGF receptor endocytosis. In this study, we found that this inhibitory effect was canceled by the dominant negative form of Rho-associated Kinase (Rho-Kinase), which is an effector of Rho. To clarify the molecular mechanisms of endocytosis downstream of Rho/Rho-Kinase signal, we searched for and identified endophilin A1 as a novel substrate of Rho-Kinase. We identified the phosphorylation site of endophilin A1 at Thr-14 and made endophilin T14D (substitution of Thr-14 by Asp), which is expected to mimic the phosphorylation state of endophilin A1. Endophilin T14D inhibited EGF receptor internalization. Furthermore, phosphorylation of endophilin by Rho-Kinase inhibited the binding to CIN85. Taken together, these results suggest that Rho-Kinase phosphorylates endophilin downstream of Rho and regulates EGF receptor endocytosis through the inhibition of binding between endophilin and CIN85.
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Rho Kinase mediated contraction of isolated stress fibers
Journal of Cell Biology, 2001Co-Authors: Kazuo Katoh, Mutsuki Amano, Kozo Kaibuchi, Yumiko Kano, Hirofumi Onishi, Keigi FujiwaraAbstract:It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC Kinase (MLCK) together with calmodulin and Ca2+. However, recent studies indicate that Rho-Kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-Kinase mediates MLC phosphorylation and their contraction in the absence of Ca2+. More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-Kinase. When the activity of Rho-Kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two Kinase systems: the Ca2+-dependent MLCK and the Rho-Kinase systems. We propose that Ca2+ is used to generate rapid contraction, whereas Rho-Kinase plays a major role in maintaining sustained contraction in cells.
Yuko Fukata - One of the best experts on this subject based on the ideXlab platform.
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Rho Rho Kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non muscle cells
Trends in Pharmacological Sciences, 2001Co-Authors: Yuko Fukata, Kozo Kaibuchi, Mutsuki AmanoAbstract:Abstract Hypercontraction or abnormal contraction of vascular smooth muscle is a major cause of diseases such as hypertension and vasospasm of the coronary and cerebral arteries. A better understanding of the mechanism of regulation of smooth muscle contraction should lead to improved treatments for such diseases. Recent studies have revealed important roles for the small GTPase Rho and its effector, Rho-associated Kinase (Rho Kinase) in Ca 2+ -independent regulation of smooth muscle contraction. The Rho–Rho-Kinase pathway modulates the level of phosphorylation of the myosin light chain of myosin II, mainly through inhibition of myosin phosphatase, and contributes to agonist-induced Ca 2+ -sensitization in smooth muscle contraction. Rho–Rho-Kinase mechanisms also participate in a variety of the cellular functions of non-muscle cells, such as stress-fibre formation, cytokinesis and cell migration. This review summarizes the role of the Rho–Rho-Kinase pathway in contractile processes of smooth muscle and in non-muscle cell functions, and the pathophysiological implications of this pathway.
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phosphorylation of myosin binding subunit mbs of myosin phosphatase by Rho Kinase in vivo
Journal of Cell Biology, 1999Co-Authors: Yoji Kawano, Mutsuki Amano, Yuko Fukata, Noriko Oshiro, Masaaki Ito, Masaki Inagaki, Fumio Matsumura, Toshikazu Nakamura, Kozo KaibuchiAbstract:Rho-associated Kinase (Rho-Kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-Kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-Kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-Kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-Kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-Kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-Kinase spatiotemporally regulate the phosphorylation state of Rho-Kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.
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Phosphorylation of Adducin by Rho-Kinase Plays a Crucial Role in Cell Motility
The Journal of cell biology, 1999Co-Authors: Yuko Fukata, Vann Bennett, Yoichiro Matsuoka, Yoshiharu Matsuura, Noriko Oshiro, Nagatoki Kinoshita, Yoji Kawano, Kozo KaibuchiAbstract:Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated Kinase (Rho- Kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-adducin and thereby enhances the F-actin–binding activity of α-adducin in vitro. Here we identified the sites of phosphorylation of α-adducin by Rho-Kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-Kinase phosphorylated α-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-Kinase, or α-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-Kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-Kinase phosphorylates α-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-Kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
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phosphorylation of moesin by Rho associated Kinase Rho Kinase plays a crucial role in the formation of microvilli like structures
Journal of Biological Chemistry, 1998Co-Authors: Noriko Oshiro, Yuko Fukata, Kozo KaibuchiAbstract: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.
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myosin ii activation promotes neurite retraction during the action of Rho and Rho Kinase
Genes to Cells, 1998Co-Authors: Mutsuki Amano, Yuko Fukata, Kazuyasu Chihara, Nao Nakamura, Takeo Yano, Masao Shibata, Mitsuo Ikebe, Kozo KaibuchiAbstract: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 N1E-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.
Shashi Bellam - One of the best experts on this subject based on the ideXlab platform.
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the Rhoa Rho Kinase pathway regulates nuclear localization of serum response factor
American Journal of Respiratory Cell and Molecular Biology, 2003Co-Authors: Andrew J Halayko, Darren J Fernandes, Gregory S Harmon, Joel Mccauley, Pawel Kocieniewski, John F Mcconville, Yiping Fu, Sean M Forsythe, Paul Kogut, Shashi BellamAbstract:RhoA and its downstream target Rho Kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression. We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus. Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho Kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization. Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter. Conversely, inhibition of the RhoA/Rho Kinase pathway by cotransfection with a plasmid expressing d...
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the Rhoa Rho Kinase pathway regulates nuclear localization of serum response factor
American Journal of Respiratory Cell and Molecular Biology, 2003Co-Authors: Hong Wei Liu, Andrew J Halayko, Darren J Fernandes, Gregory S Harmon, Joel Mccauley, Pawel Kocieniewski, John F Mcconville, Sean M Forsythe, Paul Kogut, Shashi BellamAbstract:RhoA and its downstream target Rho Kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression. We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus. Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho Kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization. Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter. Conversely, inhibition of the RhoA/Rho Kinase pathway by cotransfection with a plasmid expressing dominant negative RhoAN19, by cotransfection with a plasmid expressing Clostridial C3 toxin, or by incubation with the Rho Kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity. Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target, selectively reduced nuclear SRF protein content, and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry. Treatment of cultured airway myocytes with latrunculin B, which reduces actin polymerization, also caused partial redistribution of SRF into the cytoplasm. Together, these results demonstrate for the first time that the RhoA/Rho Kinase pathway controls smooth muscle gene transcription in differentiated smooth muscle cells, in part by regulating the subcellular localization of SRF. It is conceivable that the RhoA/Rho Kinase pathway influences SRF localization through its effect on actin polymerization dynamics.
David L. Epstein - One of the best experts on this subject based on the ideXlab platform.
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Modulation of Aqueous Humor Outflow Facility by the Rho Kinase–Specific Inhibitor Y-27632
2013Co-Authors: Vasantha P Rao, Janardan Kumar, Pei-feng Deng, David L. EpsteinAbstract:PURPOSE. The goal of this study was to investigate the role of Rho Kinase in the modulation of aqueous humor outflow facility. Rho Kinase, a critical downstream effector of Rho GTPase is recognized to control the formation of actin stress fibers, focal adhesions, and cellular contraction. METHODS. Expression of Rho GTPase, Rho Kinase, and other downstream targets of Rho GTPase were determined in human trabecular meshwork (HTM) and Schlemm’s canal (SC) primary cell cultures by Western blot analysis. The Rho Kinase–specific inhibitor (Y-27632)-induced changes in actin stress fibers, focal adhesions, and protein phosphotyrosine status were evaluated by staining with Rhodamine-phalloidin, anti-paxillin, and antiphosphotyrosine antibodies, respectively. Myosin light-chain phosphorylation was determined by Western blot analysis. Y-27632-induced changes in SC cell monolayer permeabilit
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modulation of aqueous humor outflow facility by the Rho Kinase specific inhibitor y 27632
Investigative Ophthalmology & Visual Science, 2001Co-Authors: Vasantha P Rao, Janardan Kumar, Pei-feng Deng, David L. EpsteinAbstract:PURPOSE The goal of this study was to investigate the role of Rho Kinase in the modulation of aqueous humor outflow facility. Rho Kinase, a critical downstream effector of Rho GTPase is recognized to control the formation of actin stress fibers, focal adhesions, and cellular contraction. METHODS Expression of Rho GTPase, Rho Kinase, and other downstream targets of Rho GTPase were determined in human trabecular meshwork (HTM) and Schlemm's canal (SC) primary cell cultures by Western blot analysis. The Rho Kinase-specific inhibitor (Y-27632)-induced changes in actin stress fibers, focal adhesions, and protein phosphotyrosine status were evaluated by staining with Rhodamine-phalloidin, anti-paxillin, and anti-phosphotyrosine antibodies, respectively. Myosin light-chain phosphorylation was determined by Western blot analysis. Y-27632-induced changes in SC cell monolayer permeability were quantitated using a colorimetric assay to evaluate horseradish peroxidase diffusion through SC cell monolayers grown in transwell chambers. Aqueous humor outflow facility was measured using enucleated porcine eyes and a constant-pressure perfusion system. RESULTS Treatment of HTM and SC cells with Y-27632 (10 microM) led to significant but reversible changes in cell shape and decreases in actin stress fibers, focal adhesions, and protein phosphotyrosine staining. SC cell monolayer permeability increased (by 80%) in response to Y-27632 (10 microM) treatment, whereas myosin light-chain phosphorylation was decreased in both HTM and SC cells. Aqueous humor outflow facility increased (40%-80%) in enucleated porcine eyes perfused with Y-27632 (10-100 microM), and this effect was associated with widening of the extracellular spaces, particularly the optically empty area of the juxtacanalicular tissue (JCT). The integrity of inner wall of aqueous plexi, however, was observed to be intact. CONCLUSIONS Based on the Rho Kinase inhibitor-induced changes in myosin light-chain phosphorylation and actomyosin organization, it is reasonable to conclude that cellular relaxation and loss of cell-substratum adhesions in HTM and SC cells could result in either increased paracellular fluid flow across Schlemm's canal or altered flow pathway through the JCT, thereby lowering resistance to outflow. This study also suggests Rho Kinase as a potential therapeutic target for the development of drugs to modulate intraocular pressure in glaucoma patients.
