The Experts below are selected from a list of 2706 Experts worldwide ranked by ideXlab platform
Kensaku Mizuno - One of the best experts on this subject based on the ideXlab platform.
-
Damnacanthal, an effective inhibitor of LIM-Kinase, inhibits cell migration and invasion.
2014Co-Authors: Kazumasa Ohashi, Kaori Sampei, Mami Nakagawa, Naoto Uchiumi, Tatsuya Amanuma, Setsuya Aiba, Masato Oikawa, Kensaku MizunoAbstract:LIM-Kinases (LIMKs) play crucial roles in various cell activities, including migration, division, and morphogenesis, by phosphorylating and inactivating cofilin. Using a bimolecular fluorescence complementation assay to detect the actin-cofilin interaction, we screened LIMK1 inhibitors and identified two effective inhibitors, damnacanthal (Dam) and MO-26 (a pyrazolopyrimidine derivative). These compounds have already been shown to inhibit Lck, a Src family tyrosine Kinase. However, in vitro Kinase assays revealed that Dam inhibited LIMK1 more effectively than Lck. Dam suppressed LIMK1-induced cofilin phosphorylation and deceleration of actin retrograde flow in lamellipodia in N1E-115 cells. Dam impaired CXCL12-induced chemotactic migration of Jurkat T lymphocytes and Jurkat-derived, Lck-deficient JCaM1.6 cells and also inhibited serum-induced migration and invasion of MDA-MB-231 breast carcinoma cells. These results suggest that Dam has the potential to suppress cell migration and invasion primarily through the inhibition of LIMK Kinase activity. Topical application of Dam also suppressed hapten-induced migration of epidermal Langerhans cells in mouse ears. Dam provides a useful tool for investigating cellular and physiological functions of LIMKs and holds promise for the development of agents against LIMK-related diseases. The bimolecular fluorescence complementation assay system used in this study will provide a useful method to screen for inhibitors of various protein Kinases.
-
camkiiβ mediated LIM Kinase activation plays a crucial role in bdnf induced neuritogenesis
2013Co-Authors: Akihiko Saito, Kazumasa Ohashi, Ken Miyajima, Junichi Akatsuka, Hiroshi Kondo, Toshiya Mashiko, Tai Kiuchi, Kensaku MizunoAbstract:LIM-Kinase 1 (LIMK1) regulates actin cytoskeletal reorganization by phosphorylating and inactivating actin-depolymerizing factor and cofilin. We examined the role of LIMK1 in brain-derived neurotrophic factor (BDNF)-induced neuritogenesis in primary-cultured rat cortical neurons. Knockdown of LIMK1 or expression of a Kinase-dead LIMK1 mutant suppressed BDNF-induced enhancement of primary neurite formation. By contrast, expression of an active form of LIMK1 promoted primary neuritogenesis in the absence of BDNF. BDNF-induced neuritogenesis was inhibited by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein Kinases (CaMKs), but not by STO-609, an inhibitor of CaMK-Kinase (CaMKK). CaMKK activity is required for the activation of CaMKI and CaMKIV, but not CaMKII, which suggests that CaMKII is principally involved in BDNF-induced enhancement of neuritogenesis. Knockdown of CaMKIIβ, but not CaMKIIα, suppressed BDNF-induced neuritogenesis. Active CaMKIIβ promoted neuritogenesis, and this promotion was inhibited by knockdown of LIMK1, indicating that CaMKIIβ is involved in BDNF-induced neuritogenesis via activation of LIMK1. Furthermore, in vitro Kinase assays revealed that CaMKIIβ phosphorylates LIMK1 at Thr-508 in the Kinase domain and activates the cofilin-phosphorylating activity of LIMK1. In summary, these results suggest that CaMKIIβ-mediated activation of LIMK1 plays a crucial role in BDNF-induced enhancement of primary neurite formation.
-
LIM Kinase has a dual role in regulating lamellipodium extension by decelerating the rate of actin retrograde flow and the rate of actin polymerization
2011Co-Authors: Kazumasa Ohashi, Hiroshi Kondo, Tai Kiuchi, Masaaki Sato, Sachiko Fujiwara, Takuya Watanabe, Kensaku MizunoAbstract:Abstract Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM-Kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. We examined the role of LIMK1 in lamellipodium extension by measuring the rates of actin polymerization, actin retrograde flow and lamellipodium extension, using time-lapse imaging of fluorescence recovery after photobleaching. In the non-extending lamellipodia of active Rac-expressing N1E-115 cells, LIMK1 expression decelerated and LIMK1 knockdown accelerated actin retrograde flow. In the extending lamellipodia of neuregulin- stimulated MCF-7 cells, LIMK1 knockdown accelerated both the rate of actin polymerization and the rate of actin retrograde flow, but the accelerating effect on retrograde flow was greater than the effect on polymerization, thus resulting in a decreased rate of lamellipodium extension. These results indicate that LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization and that in MCF-7 cells, endogenous LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow more effectively than decelerating actin polymerization.
-
LIM-Kinase is critical for the mesenchymal-to-amoeboid cell morphological transition in 3D matrices
2010Co-Authors: Toshiaki Mishima, Kazumasa Ohashi, Moyu Naotsuka, Yuji Horita, Masaaki Sato, Kensaku MizunoAbstract:Tumor cells can migrate in 3D matrices in either a mesenchymal-like or amoeboid mode. HT1080 fibrosarcoma cells cultured in 3D collagen gels change their morphology from mesenchymal-like (elongated) to amoeboid (round) following protease inhibitor (PI) treatment or active Rho or ROCK expression. In this study, we examined the role of LIM-Kinase 1 (LIMK1) in the PI- or Rho/ROCK-induced cell morphological change. We showed that LIMK1 was activated after PI treatment of HT1080 cells in 3D collagen gels and this activation was blocked by a ROCK inhibitor. While overexpression of LIMK1 induced cell rounding, knockdown of LIMK1 or the expression of Kinase-inactive LIMK1 suppressed PI- or Rho/ROCK-induced cell rounding. These results suggest that LIMK1 plays an essential role in the PI- or Rho/ROCK-induced mesenchymal-to-amoeboid cell morphological transition of HT1080 cells cultured in 3D collagen gels. Furthermore, LIMK1 knockdown suppressed the invasive activity of HT1080 cells in collagen gels with or without PIs, indicating that LIMK1 mediates both the mesenchymal and amoeboid modes of invasion of HT1080 cells.
-
ca2 calmodulin dependent protein Kinase iv mediated LIM Kinase activation is critical for calcium signal induced neurite outgrowth
2009Co-Authors: Miyohiko Takemura, Kazumasa Ohashi, Toshiaki Mishima, Yan Wang, Jiro Kasahara, Kohji Fukunaga, Kensaku MizunoAbstract:Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM Kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a Kinase-dead mutant of LIMK1 suppressed ionomycin-induced cofilin phosphorylation and neurite outgrowth in Neuro-2a cells. Ionomycin-induced cofilin phosphorylation and neurite outgrowth were also blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein Kinases (CaMKs), and STO-609, an inhibitor of CaMK Kinase. An active form of CaMKIV but not CaMKI enhanced Thr-508 phosphorylation of LIMK1 and increased the Kinase activity of LIMK1. Moreover, the active form of CaMKIV induced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV suppressed ionomycin-induced neurite outgrowth. Taken together, our results suggest that LIMK1-mediated cofilin phosphorylation is critical for ionomycin-induced neurite outgrowth and that CaMKIV mediates ionomycin-induced LIMK1 activation.
Kazumasa Ohashi - One of the best experts on this subject based on the ideXlab platform.
-
Damnacanthal, an effective inhibitor of LIM-Kinase, inhibits cell migration and invasion.
2014Co-Authors: Kazumasa Ohashi, Kaori Sampei, Mami Nakagawa, Naoto Uchiumi, Tatsuya Amanuma, Setsuya Aiba, Masato Oikawa, Kensaku MizunoAbstract:LIM-Kinases (LIMKs) play crucial roles in various cell activities, including migration, division, and morphogenesis, by phosphorylating and inactivating cofilin. Using a bimolecular fluorescence complementation assay to detect the actin-cofilin interaction, we screened LIMK1 inhibitors and identified two effective inhibitors, damnacanthal (Dam) and MO-26 (a pyrazolopyrimidine derivative). These compounds have already been shown to inhibit Lck, a Src family tyrosine Kinase. However, in vitro Kinase assays revealed that Dam inhibited LIMK1 more effectively than Lck. Dam suppressed LIMK1-induced cofilin phosphorylation and deceleration of actin retrograde flow in lamellipodia in N1E-115 cells. Dam impaired CXCL12-induced chemotactic migration of Jurkat T lymphocytes and Jurkat-derived, Lck-deficient JCaM1.6 cells and also inhibited serum-induced migration and invasion of MDA-MB-231 breast carcinoma cells. These results suggest that Dam has the potential to suppress cell migration and invasion primarily through the inhibition of LIMK Kinase activity. Topical application of Dam also suppressed hapten-induced migration of epidermal Langerhans cells in mouse ears. Dam provides a useful tool for investigating cellular and physiological functions of LIMKs and holds promise for the development of agents against LIMK-related diseases. The bimolecular fluorescence complementation assay system used in this study will provide a useful method to screen for inhibitors of various protein Kinases.
-
camkiiβ mediated LIM Kinase activation plays a crucial role in bdnf induced neuritogenesis
2013Co-Authors: Akihiko Saito, Kazumasa Ohashi, Ken Miyajima, Junichi Akatsuka, Hiroshi Kondo, Toshiya Mashiko, Tai Kiuchi, Kensaku MizunoAbstract:LIM-Kinase 1 (LIMK1) regulates actin cytoskeletal reorganization by phosphorylating and inactivating actin-depolymerizing factor and cofilin. We examined the role of LIMK1 in brain-derived neurotrophic factor (BDNF)-induced neuritogenesis in primary-cultured rat cortical neurons. Knockdown of LIMK1 or expression of a Kinase-dead LIMK1 mutant suppressed BDNF-induced enhancement of primary neurite formation. By contrast, expression of an active form of LIMK1 promoted primary neuritogenesis in the absence of BDNF. BDNF-induced neuritogenesis was inhibited by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein Kinases (CaMKs), but not by STO-609, an inhibitor of CaMK-Kinase (CaMKK). CaMKK activity is required for the activation of CaMKI and CaMKIV, but not CaMKII, which suggests that CaMKII is principally involved in BDNF-induced enhancement of neuritogenesis. Knockdown of CaMKIIβ, but not CaMKIIα, suppressed BDNF-induced neuritogenesis. Active CaMKIIβ promoted neuritogenesis, and this promotion was inhibited by knockdown of LIMK1, indicating that CaMKIIβ is involved in BDNF-induced neuritogenesis via activation of LIMK1. Furthermore, in vitro Kinase assays revealed that CaMKIIβ phosphorylates LIMK1 at Thr-508 in the Kinase domain and activates the cofilin-phosphorylating activity of LIMK1. In summary, these results suggest that CaMKIIβ-mediated activation of LIMK1 plays a crucial role in BDNF-induced enhancement of primary neurite formation.
-
LIM Kinase has a dual role in regulating lamellipodium extension by decelerating the rate of actin retrograde flow and the rate of actin polymerization
2011Co-Authors: Kazumasa Ohashi, Hiroshi Kondo, Tai Kiuchi, Masaaki Sato, Sachiko Fujiwara, Takuya Watanabe, Kensaku MizunoAbstract:Abstract Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM-Kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. We examined the role of LIMK1 in lamellipodium extension by measuring the rates of actin polymerization, actin retrograde flow and lamellipodium extension, using time-lapse imaging of fluorescence recovery after photobleaching. In the non-extending lamellipodia of active Rac-expressing N1E-115 cells, LIMK1 expression decelerated and LIMK1 knockdown accelerated actin retrograde flow. In the extending lamellipodia of neuregulin- stimulated MCF-7 cells, LIMK1 knockdown accelerated both the rate of actin polymerization and the rate of actin retrograde flow, but the accelerating effect on retrograde flow was greater than the effect on polymerization, thus resulting in a decreased rate of lamellipodium extension. These results indicate that LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization and that in MCF-7 cells, endogenous LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow more effectively than decelerating actin polymerization.
-
LIM-Kinase is critical for the mesenchymal-to-amoeboid cell morphological transition in 3D matrices
2010Co-Authors: Toshiaki Mishima, Kazumasa Ohashi, Moyu Naotsuka, Yuji Horita, Masaaki Sato, Kensaku MizunoAbstract:Tumor cells can migrate in 3D matrices in either a mesenchymal-like or amoeboid mode. HT1080 fibrosarcoma cells cultured in 3D collagen gels change their morphology from mesenchymal-like (elongated) to amoeboid (round) following protease inhibitor (PI) treatment or active Rho or ROCK expression. In this study, we examined the role of LIM-Kinase 1 (LIMK1) in the PI- or Rho/ROCK-induced cell morphological change. We showed that LIMK1 was activated after PI treatment of HT1080 cells in 3D collagen gels and this activation was blocked by a ROCK inhibitor. While overexpression of LIMK1 induced cell rounding, knockdown of LIMK1 or the expression of Kinase-inactive LIMK1 suppressed PI- or Rho/ROCK-induced cell rounding. These results suggest that LIMK1 plays an essential role in the PI- or Rho/ROCK-induced mesenchymal-to-amoeboid cell morphological transition of HT1080 cells cultured in 3D collagen gels. Furthermore, LIMK1 knockdown suppressed the invasive activity of HT1080 cells in collagen gels with or without PIs, indicating that LIMK1 mediates both the mesenchymal and amoeboid modes of invasion of HT1080 cells.
-
ca2 calmodulin dependent protein Kinase iv mediated LIM Kinase activation is critical for calcium signal induced neurite outgrowth
2009Co-Authors: Miyohiko Takemura, Kazumasa Ohashi, Toshiaki Mishima, Yan Wang, Jiro Kasahara, Kohji Fukunaga, Kensaku MizunoAbstract:Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM Kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a Kinase-dead mutant of LIMK1 suppressed ionomycin-induced cofilin phosphorylation and neurite outgrowth in Neuro-2a cells. Ionomycin-induced cofilin phosphorylation and neurite outgrowth were also blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein Kinases (CaMKs), and STO-609, an inhibitor of CaMK Kinase. An active form of CaMKIV but not CaMKI enhanced Thr-508 phosphorylation of LIMK1 and increased the Kinase activity of LIMK1. Moreover, the active form of CaMKIV induced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV suppressed ionomycin-induced neurite outgrowth. Taken together, our results suggest that LIMK1-mediated cofilin phosphorylation is critical for ionomycin-induced neurite outgrowth and that CaMKIV mediates ionomycin-induced LIMK1 activation.
Ora Bernard - One of the best experts on this subject based on the ideXlab platform.
-
the phosphorylation of p25 tppp by LIM Kinase 1 inhibits its ability to assemble microtubules
2007Co-Authors: Karla Acevedo, Priscilla Soo, Randy Suryadinata, Boris Sarcevic, Valentina A Valova, Mark E Graham, Phillip J Robinson, Ora BernardAbstract:LIM Kinase 1 (LIMK1) is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin-depolymerizing factor. LIMK1 activity is also required for microtubule disassembly in endothelial cells. A search for LIMK1-interacting proteins identified p25alpha, a phosphoprotein that promotes tubulin polymerization. We found that p25 is phosphorylated by LIMK1 on serine residues in vitro and in cells. Immunoblotting analysis revealed that p25 is not a brain specific protein as previously reported, but is expressed in all mouse tissues. Immunofluorescence analysis demonstrated that endogenous p25 is co-localized with microtubules and is also found in the nucleus. Down-regulation of p25 by siRNA decreased microtubule levels while its overexpression in stable NIH-3T3 cell lines increased cell size and levels of stable tubulin. Bacterially expressed unphosphorylated p25 promotes microtubule assembly in vitro; however, when phosphorylated in cells, p25 lost its ability to assemble microtubule. Our results represent a surprising connection between the tubulin and the actin cytoskeleton mediated by LIMK1. We propose that the LIMK1 phosphorylation of p25 blocks p25 activity, thus promoting microtubule disassembly.
-
hsp90 increases LIM Kinase activity by promoting its homo dimerization
2006Co-Authors: Juliana Soosairajah, John T. Price, Craig J. Morton, Yosef Yarden, Daniel Harari, Ami Citri, Michael W Parker, Ora BernardAbstract:LIM Kinase 1 (LIMK1) is a serine protein Kinase that regulates the actin cytoskeleton by phosphorylation and inactivation of actin depolymerizing factor cofilin. LIMK1 activity is regulated by the Rho-GTPases via their serine/threonine Kinase effectors Rho-Kinase and p21-activated Kinases 1 and 4 that phosphorylate LIMK1 on threonine 508 in its activation loop. The purpose of this study was to elucidate the pathway leading to the stability of LIMK1, a protein with a half-life of approximately 20 h. Because the half-life of Kinase-dead LIMK1 is only 4 h, it is suggestive that trans- or auto-phosphorylation is responsible for the stabilization of LIMK1. Using known Hsp90 inhibitors, we have shown that the half-life of LIMK1 in cells depends on the presence of active Hsp90. Furthermore, endogenous LIMK1 coimmunoprecipitated with endogenous Hsp90 and this interaction promoted LIMK1 homodimer formation as seen by cross-linking experiments. Hsp90 binds LIMK1 via a recognition sequence within the LIMK1 Kinase domain, homologous to that of ErbB-2. Mutation of a proline residue within this sequence to glutamic acid reduces its interaction with Hsp90, inhibits homodimer formation, and reduces its half-life to 4 h. These findings implicate Hsp90 in the stabilization of LIMK1 by promoting homodimer formation and transphosphorylation.
-
LIM Kinase 2 is widely expressed in all tissues.
2006Co-Authors: Karla Acevedo, Nathalie Moussi, Priscilla Soo, Ora BernardAbstract:The LIM Kinase family includes two proteins: LIMK1 and LIMK2. These proteins have identical genomic structure and overall amino acid identity of 50%. Both proteins regulate actin polymerization via phosphorylation and inactivation of the actin depolymerizing factors ADF/cofilin. Although the function of endogenous LIMK1 is well established, little is known about the function of the endogenous LIMK2 protein. To understand the specific role of endogenous LIMK2 protein, we examined its expression in embryonic and adult mice using a rat monoclonal antibody, which recognizes specifically the PDZ domain of LIMK2 but not that of LIMK1. Immunoblotting and immunoprecipitation analyses of mouse tissues and human and mouse cell lines revealed widespread expression of the 75-kDa LIMK2 protein. Immunofluorescence analysis demonstrated that the cellular localization of LIMK2 is different from that of LIMK1. LIMK2 protein is found in the cytoplasm localized to punctae and is not enriched within focal adhesions like LIMK1. Immunohistochemical studies revealed that LIMK2 is widely expressed in embryonic and adult mouse tissues and that its expression pattern is similar to that of LIMK1 except in the testes. We have also demonstrated that endogenous LIMK1 and LIMK2 form heterodimers, and that LIMK2 does not always interact with the same proteins as LIMK1.
-
LIM Kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells
2005Co-Authors: Matvey Gorovoy, Ora Bernard, Jiaxin Niu, Jasmina Profirovic, Richard D Minshall, Radu Neamu, Tatyana A VoynoyasenetskayaAbstract:Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM Kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the Kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho Kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.
-
interplay between components of a novel LIM Kinase slingshot phosphatase complex regulates cofilin
2005Co-Authors: Juliana Soosairajah, Nathalie Moussi, James R. Bamburg, Boris Sarcevic, Patrick D Sarmiere, Sankar Maiti, Oneil Wiggan, Rashmi Sampath, Ora BernardAbstract:Slingshot (SSH) phosphatases and LIM Kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3ζ, is involved, along with the Kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3ζ binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.
Toshikazu Nakamura - One of the best experts on this subject based on the ideXlab platform.
-
regulation of adf cofilin phosphorylation and synaptic function by LIM Kinase
2004Co-Authors: Yanghong Meng, Jinsong Meng, Hisaaki Takahashi, Yu Zhang, Suhail Asrar, Toshikazu Nakamura, Zhengping JiaAbstract:To investigate the role of the LIM-Kinase (LIMK) family in the regulation of ADF/cofilin phosphorylation and synaptic function in the mammalian central nervous system (CNS), we conducted biochemical and electrophysiological analysis using mice that were genetically altered in the expression of LIMK-1 and LIMK-2. We showed here that while LIMK-2 knockout mice exhibited minimal abnormalities, the LIMK-1/2 double knockout mice were more severely impaired in both ADF/cofilin phosphorylation and excitatory synaptic function in the CA1 region of the hippocampus. These results indicate a critical role for the LIMK family in the regulation of ADF/cofilin and synaptic function in the brain.
-
Mitosis-Dependent Phosphorylation and Activation of LIM-Kinase 1
2002Co-Authors: Tomoyuki Sumi, Kunio Matsumoto, Toshikazu NakamuraAbstract:LIM-Kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylation of cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of endogenous LIMK1. When HeLa cells were synchronized at prometaphase by nocodazole-treatment, LIMK1 was hyperphosphorylated, and its activity toward cofilin phosphorylation was markedly increased. During cell cycle progression, LIMK1 activity was low in interphase but reached a maximal level during mitosis. Activation of LIMK1 during mitosis was abrogated by roscovitine, a specific inhibitor of cyclin-dependent Kinases (CDKs), suggesting that activation of CDKs directly or indirectly participates in LIMK1 activation. These results strongly suggest that LIMK1 may play an important role in the cell cycle progression through regulation of actin cytoskeletal rearrangements.
-
impaired spermatogenic ability of testicular germ cells in mice deficient in the LIM Kinase 2 gene
2002Co-Authors: Hisaaki Takahashi, Uichi Koshimizu, Junichi Miyazaki, Toshikazu NakamuraAbstract:LIM-Kinase (LIMK), including LIMK1 and LIMK2, is the only known catalytic protein among LIM-family molecules. It is well known that LIMK phosphorylates and inactivates cofilin, an actin-depolymerizing factor regulating actin reorganization, while in vivo functions have remained to be elucidated. In the present study, we generated LIMk2 gene-deficient mice in which three LIMK2 isoforms were disrupted in a Cre-mediated fashion. Impaired cofilin phosphorylation was clearly observed in LIMk2-/- fibroblasts stimulated with bradykinin or lysophosphatidic acid, thereby suggesting that Cdc42 or Rho-dependent LIMK activation did not occur. However, LIMk2-/- mice did not exhibit embryonic lethality or any phenotypic abnormalities in postnatal growth and development, except for spermatogenesis in the testis. The testes of LIMk2-/- mice were smaller in size and partial degeneration of spermatogenic cells in the seminiferous tubules was apparent in association with increased apoptosis. In addition, the viability of LIMk2-/- spermatogenic cells, when cultured under stressed conditions, was diminished. Furthermore, the potential for germ cells to differentiate in a regenerative state was severely impaired in LIMk2-/- testis. Experimental hyperthermia induced impairment of ADF/cofilin phosphorylation and the formation of intranuclear cofilin inclusions in mutant germ cells. Based on these findings, we propose that LIMK2, especially the testis-specific isoform tLIMK2, plays an important role in proper progression of spermatogenesis by regulation of cofilin activity and/or localization in germ cells.
-
Specific activation of LIM Kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein Kinase.
2001Co-Authors: Tomoyuki Sumi, Kunio Matsumoto, Toshikazu NakamuraAbstract:Abstract LIM-Kinase 1 (LIMK1) and LIM-Kinase 2 (LIMK2) regulate actin cytoskeletal reorganization via cofilin phosphorylation downstream of distinct Rho family GTPases. We report our findings that ROCK, a downstream protein Kinase of Rho, specifically activates LIMK2 but not LIMK1 downstream of RhoA. LIMK1 and LIMK2 activities toward cofilin phosphorylation were stimulated by co-expression with the active form of ROCK (ROCK-Δ3), whereas full-length ROCK selectively activates LIMK2 but not LIMK1. Activation of LIMK2 by RhoA was inhibited by Y-27632, a specific inhibitor of ROCK, but Rac1-mediated activation of LIMK1 was not. ROCK directly phosphorylated the threonine 505 residue within the activation segment of LIMK2 and markedly stimulated LIMK2 activity. A LIMK2 mutant with replacement of threonine 505 by valine abolished LIMK2 activities for cofilin phosphorylation and actin cytoskeletal changes, whereas replacement by glutamate enhanced the protein Kinase activity and stress fiber formation by LIMK2. These results indicate that ROCK directly phosphorylates threonine 505 and activates LIMK2 downstream of RhoA and that this phosphorylation is essential for LIMK2 to induce actin cytoskeletal reorganization. Together with the finding that LIMK1 is regulated by Pak1, LIMK1 and LIMK2 are regulated by different protein Kinases downstream of distinct Rho family GTPases.
-
cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho and cdc42 activated LIM Kinase 2
1999Co-Authors: Tomoyuki Sumi, Kunio Matsumoto, Yoshimi Takai, Toshikazu NakamuraAbstract:The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and Kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.
Richard Treisman - One of the best experts on this subject based on the ideXlab platform.
-
LIM Kinase and diaphanous cooperate to regulate serum response factor and actin dynamics
2002Co-Authors: O Geneste, John W Copeland, Richard TreismanAbstract:The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho Kinase (ROCK)–LIM Kinase (LIMK)–cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.
-
signal regulated activation of serum response factor is mediated by changes in actin dynamics
1999Co-Authors: Athanassia Sotiropoulos, Dziugas Gineitis, John Copeland, Richard TreismanAbstract:Abstract Serum response factor (SRF) regulates transcription of many serum-inducible and muscle-specific genes. Using a functional screen, we identified LIM Kinase-1 as a potent activator of SRF. We show that SRF activation by LIM Kinase-1 is dependent on its ability to regulate actin treadmilling. LIM Kinase activity is not essential for SRF activation by serum, but signals depend on alterations in actin dynamics. Studies with actin-binding drugs, the actin-specific C2 toxin, and actin overexpression demonstrate that G-actin level controls SRF. Regulation of actin dynamics is necessary for serum induction of a subset of SRF target genes, including vinculin, cytoskeletal actin, and srf itself, and also suffices for their activation. Actin treadmilling provides a convergence point for both serum- and LIM Kinase-1-induced signaling to SRF.