The Experts below are selected from a list of 69711 Experts worldwide ranked by ideXlab platform
Kozo Kaibuchi - One of the best experts on this subject based on the ideXlab platform.
-
positive role of iqgap1 an effector of RAC1 in actin meshwork formation at sites of cell cell contact
Molecular Biology of the Cell, 2003Co-Authors: Jun Noritake, Masaki Fukata, Masato Nakagawa, Kazumasa Sato, Takashi Watanabe, Nanae Izumi, Shujie Wang, Yuko Fukata, Kozo KaibuchiAbstract:The small guanosine triphosphatase RAC1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of RAC1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of RAC1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of RAC1 reduced them. Knockdown of RAC1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in RAC1/Cdc42 binding. Knockdown of either RAC1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal RAC1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the RAC1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that RAC1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→RAC1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”
-
recruitment and activation of RAC1 by the formation of e cadherin mediated cell cell adhesion sites
Journal of Cell Science, 2001Co-Authors: Masato Nakagawa, Masaki Fukata, Masaki Yamaga, Naohiro Itoh, Kozo KaibuchiAbstract:RAC1, a member of the Rho family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of RAC1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged RAC1, we demonstrate that EGFP-RAC1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca(2+) chelation. Re-establishment of cell-cell adhesion by restoration of Ca(2)(+) caused EGFP-RAC1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-RAC1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced RAC1 activation by measuring the amounts of GTP-bound RAC1 based on its specific binding to the Cdc42/RAC1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced RAC1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of RAC1, and EGFP-RAC1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. RAC1 activation was also confirmed by measuring the amounts of coimmunoprecipitated RAC1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that RAC1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase.
-
recruitment and activation of RAC1 by the formation of e cadherin mediated cell cell adhesion sites
Journal of Cell Science, 2001Co-Authors: Masato Nakagawa, Masaki Fukata, Masaki Yamaga, Naohiro Itoh, Kozo KaibuchiAbstract:RAC1, a member of the Ρ family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of RAC1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged RAC1, we demonstrate that EGFP-RAC1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca(2+) chelation. Re-establishment of cell-cell adhesion by restoration of Ca(2)(+) caused EGFP-RAC1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-RAC1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced RAC1 activation by measuring the amounts of GTP-bound RAC1 based on its specific binding to the Cdc42/RAC1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced RAC1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of RAC1, and EGFP-RAC1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. RAC1 activation was also confirmed by measuring the amounts of coimmunoprecipitated RAC1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that RAC1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase. http://www/biologists.com/JCS/movies/jcs2094.html
-
p140sra 1 specifically RAC1 associated protein is a novel specific target for RAC1 small gtpase
Journal of Biological Chemistry, 1998Co-Authors: Kenta Kobayashi, Nobuko Yoshidakubomura, Takahiro Nagase, Akihiro Iwamatsu, Shinya Kuroda, Tomoko Nakamura, Nobuo Nomura, Masaki Fukata, Yoshiharu Matsuura, Kozo KaibuchiAbstract:Abstract RAC1 small GTPase plays pivotal roles in various cell functions such as cell morphology, cell polarity, and cell proliferation. We have previously identified IQGAP1 from bovine brain cytosol as a target for RAC1 by an affinity purification method. By using the same method, we purified a specifically RAC1-associated protein with a molecular mass of about 140 kDa (p140) from bovine brain cytosol. This protein interacted with guanosine 5′-(3-O-thio)triphosphate (GTPγS)·glutathioneS-transferase (GST)-RAC1 but not with the GDP·GST-RAC1, GTPγS·GST-Cdc42, or GTPγS·GST-RhoA. The amino acid sequences of this protein revealed that p140 is identified as a product of KIAA0068 gene. We denoted this protein as Sra-1 (SpecificallyRAC1-associated protein). Recombinant Sra-1 interacted with GTPγS·GST-RAC1 and weakly with GDP·RAC1 but not with GST-Cdc42 or GST-RhoA. The N-terminal domain of Sra-1 (1–407 amino acids) was responsible for the interaction with RAC1. Myc-tagged Sra-1 and the deletion mutant capable of interacting with RAC1, but not the mutants unable to bind RAC1, were colocalized with dominant active RAC1Val-12 and cortical actin filament at the RAC1Val-12-induced membrane ruffling area in KB cells. Sra-1 was cosedimented with filamentous actin (F-actin), indicating that Sra-1 directly interacts with F-actin. These results suggest that Sra-1 is a novel and specific target for RAC1.
Peter L Hordijk - One of the best experts on this subject based on the ideXlab platform.
-
the f bar domain protein pacsin2 associates with RAC1 and regulates cell spreading and migration
Journal of Cell Science, 2011Co-Authors: Bart Jan De Kreuk, Eloise C Anthony, Paul J. Hensbergen, Andre M Deelder, Markus Plomann, Micha Nethe, Mar Fernandezborja, Peter L HordijkAbstract:The RAC1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading and migration mediated by signaling through effector proteins, such as the PAK kinases and the Scar and WAVE proteins. We previously identified a series of regulatory proteins that associate with RAC1 through its hypervariable C-terminal domain, including the RAC1 activator β-Pix (also known as Rho guanine-nucleotide-exchange factor 7) and the membrane adapter caveolin-1. Here, we show that RAC1 associates, through its C-terminus, with the F-BAR domain protein PACSIN2, an inducer of membrane tubulation and a regulator of endocytosis. We show that RAC1 localizes with PACSIN2 at intracellular tubular structures and on early endosomes. Active RAC1 induces a loss of PACSIN2-positive tubular structures. By contrast, RAC1 inhibition results in an accumulation of PACSIN2-positive tubules. In addition, PACSIN2 appears to regulate RAC1 signaling; siRNA-mediated loss of PACSIN2 increases the levels of RAC1-GTP and promotes cell spreading and migration in a wound healing assay. Moreover, ectopic expression of PACSIN2 reduces RAC1-GTP levels in a fashion that is dependent on the PACSIN2-RAC1 interaction, on the membrane-tubulating capacity of PACSIN2 and on dynamin. These data identify the BAR-domain protein PACSIN2 as a RAC1 interactor that regulates RAC1-mediated cell spreading and migration.
-
RAC1 recruits the adapter protein cms cd2ap to cell cell contacts
Journal of Biological Chemistry, 2010Co-Authors: Trynette J Van Duijn, Eloise C Anthony, Paul J. Hensbergen, Andre M Deelder, Peter L HordijkAbstract:RAC1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in RAC1 mediates specific protein-protein interactions. Most recently, we found that the RAC1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of RAC1 but not with that of other Rho family members. Efficient interaction between RAC1 and CD2AP requires both the proline-rich domain and the poly-basic region in the RAC1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with RAC1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links RAC1 to CAPZ and cortactin. Finally, expression of constitutive active RAC1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel RAC1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.
-
RAC1 recruits the adapter protein CMS/CD2AP to cell-cell contacts
Journal of Biological Chemistry, 2010Co-Authors: Trynette J Van Duijn, Eloise C Anthony, Paul J. Hensbergen, Andre M Deelder, Peter L HordijkAbstract:RAC1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in RAC1 mediates specific protein-protein interactions. Most recently, we found that the RAC1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of RAC1 but not with that of other Rho family members. Efficient interaction between RAC1 and CD2AP requires both the proline-rich domain and the poly-basic region in the RAC1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with RAC1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links RAC1 to CAPZ and cortactin. Finally, expression of constitutive active RAC1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel RAC1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.
-
targeting and activation of RAC1 are mediated by the exchange factor β pix
Journal of Cell Biology, 2006Co-Authors: Jean Paul Ten Klooster, Zahara M. Jaffer, Jonathan Chernoff, Peter L HordijkAbstract:Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase RAC1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator β-Pix (p21-activated kinase [Pak]–interacting exchange factor). The interaction with β-Pix is nucleotide independent and is necessary and sufficient for RAC1 recruitment to membrane ruffles and to focal adhesions. In addition, the RAC1–β-Pix interaction is required for RAC1 activation by β-Pix as well as for RAC1-mediated spreading. Finally, using cells deficient for the β-Pix–binding kinase Pak1, we show that Pak1 regulates the RAC1–β-Pix interaction and controls cell spreading and adhesion-induced RAC1 activation. These data provide a model for the intracellular targeting and localized activation of RAC1 through its exchange factor β-Pix.
Masaki Fukata - One of the best experts on this subject based on the ideXlab platform.
-
positive role of iqgap1 an effector of RAC1 in actin meshwork formation at sites of cell cell contact
Molecular Biology of the Cell, 2003Co-Authors: Jun Noritake, Masaki Fukata, Masato Nakagawa, Kazumasa Sato, Takashi Watanabe, Nanae Izumi, Shujie Wang, Yuko Fukata, Kozo KaibuchiAbstract:The small guanosine triphosphatase RAC1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of RAC1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of RAC1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of RAC1 reduced them. Knockdown of RAC1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in RAC1/Cdc42 binding. Knockdown of either RAC1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal RAC1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the RAC1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that RAC1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→RAC1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”
-
recruitment and activation of RAC1 by the formation of e cadherin mediated cell cell adhesion sites
Journal of Cell Science, 2001Co-Authors: Masato Nakagawa, Masaki Fukata, Masaki Yamaga, Naohiro Itoh, Kozo KaibuchiAbstract:RAC1, a member of the Rho family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of RAC1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged RAC1, we demonstrate that EGFP-RAC1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca(2+) chelation. Re-establishment of cell-cell adhesion by restoration of Ca(2)(+) caused EGFP-RAC1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-RAC1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced RAC1 activation by measuring the amounts of GTP-bound RAC1 based on its specific binding to the Cdc42/RAC1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced RAC1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of RAC1, and EGFP-RAC1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. RAC1 activation was also confirmed by measuring the amounts of coimmunoprecipitated RAC1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that RAC1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase.
-
recruitment and activation of RAC1 by the formation of e cadherin mediated cell cell adhesion sites
Journal of Cell Science, 2001Co-Authors: Masato Nakagawa, Masaki Fukata, Masaki Yamaga, Naohiro Itoh, Kozo KaibuchiAbstract:RAC1, a member of the Ρ family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of RAC1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged RAC1, we demonstrate that EGFP-RAC1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca(2+) chelation. Re-establishment of cell-cell adhesion by restoration of Ca(2)(+) caused EGFP-RAC1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-RAC1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced RAC1 activation by measuring the amounts of GTP-bound RAC1 based on its specific binding to the Cdc42/RAC1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced RAC1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of RAC1, and EGFP-RAC1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. RAC1 activation was also confirmed by measuring the amounts of coimmunoprecipitated RAC1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that RAC1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase. http://www/biologists.com/JCS/movies/jcs2094.html
-
p140sra 1 specifically RAC1 associated protein is a novel specific target for RAC1 small gtpase
Journal of Biological Chemistry, 1998Co-Authors: Kenta Kobayashi, Nobuko Yoshidakubomura, Takahiro Nagase, Akihiro Iwamatsu, Shinya Kuroda, Tomoko Nakamura, Nobuo Nomura, Masaki Fukata, Yoshiharu Matsuura, Kozo KaibuchiAbstract:Abstract RAC1 small GTPase plays pivotal roles in various cell functions such as cell morphology, cell polarity, and cell proliferation. We have previously identified IQGAP1 from bovine brain cytosol as a target for RAC1 by an affinity purification method. By using the same method, we purified a specifically RAC1-associated protein with a molecular mass of about 140 kDa (p140) from bovine brain cytosol. This protein interacted with guanosine 5′-(3-O-thio)triphosphate (GTPγS)·glutathioneS-transferase (GST)-RAC1 but not with the GDP·GST-RAC1, GTPγS·GST-Cdc42, or GTPγS·GST-RhoA. The amino acid sequences of this protein revealed that p140 is identified as a product of KIAA0068 gene. We denoted this protein as Sra-1 (SpecificallyRAC1-associated protein). Recombinant Sra-1 interacted with GTPγS·GST-RAC1 and weakly with GDP·RAC1 but not with GST-Cdc42 or GST-RhoA. The N-terminal domain of Sra-1 (1–407 amino acids) was responsible for the interaction with RAC1. Myc-tagged Sra-1 and the deletion mutant capable of interacting with RAC1, but not the mutants unable to bind RAC1, were colocalized with dominant active RAC1Val-12 and cortical actin filament at the RAC1Val-12-induced membrane ruffling area in KB cells. Sra-1 was cosedimented with filamentous actin (F-actin), indicating that Sra-1 directly interacts with F-actin. These results suggest that Sra-1 is a novel and specific target for RAC1.
Masato Nakagawa - One of the best experts on this subject based on the ideXlab platform.
-
positive role of iqgap1 an effector of RAC1 in actin meshwork formation at sites of cell cell contact
Molecular Biology of the Cell, 2003Co-Authors: Jun Noritake, Masaki Fukata, Masato Nakagawa, Kazumasa Sato, Takashi Watanabe, Nanae Izumi, Shujie Wang, Yuko Fukata, Kozo KaibuchiAbstract:The small guanosine triphosphatase RAC1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of RAC1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of RAC1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of RAC1 reduced them. Knockdown of RAC1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in RAC1/Cdc42 binding. Knockdown of either RAC1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal RAC1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the RAC1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that RAC1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→RAC1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”
-
recruitment and activation of RAC1 by the formation of e cadherin mediated cell cell adhesion sites
Journal of Cell Science, 2001Co-Authors: Masato Nakagawa, Masaki Fukata, Masaki Yamaga, Naohiro Itoh, Kozo KaibuchiAbstract:RAC1, a member of the Ρ family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of RAC1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged RAC1, we demonstrate that EGFP-RAC1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca(2+) chelation. Re-establishment of cell-cell adhesion by restoration of Ca(2)(+) caused EGFP-RAC1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-RAC1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced RAC1 activation by measuring the amounts of GTP-bound RAC1 based on its specific binding to the Cdc42/RAC1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced RAC1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of RAC1, and EGFP-RAC1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. RAC1 activation was also confirmed by measuring the amounts of coimmunoprecipitated RAC1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that RAC1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase. http://www/biologists.com/JCS/movies/jcs2094.html
-
recruitment and activation of RAC1 by the formation of e cadherin mediated cell cell adhesion sites
Journal of Cell Science, 2001Co-Authors: Masato Nakagawa, Masaki Fukata, Masaki Yamaga, Naohiro Itoh, Kozo KaibuchiAbstract:RAC1, a member of the Rho family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of RAC1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged RAC1, we demonstrate that EGFP-RAC1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca(2+) chelation. Re-establishment of cell-cell adhesion by restoration of Ca(2)(+) caused EGFP-RAC1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-RAC1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced RAC1 activation by measuring the amounts of GTP-bound RAC1 based on its specific binding to the Cdc42/RAC1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced RAC1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of RAC1, and EGFP-RAC1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. RAC1 activation was also confirmed by measuring the amounts of coimmunoprecipitated RAC1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that RAC1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase.
Lykke Sylow - One of the best experts on this subject based on the ideXlab platform.
-
RAC1 muscle knockout exacerbates the detrimental effect of high fat diet on insulin stimulated muscle glucose uptake independently of akt
The Journal of Physiology, 2018Co-Authors: Steffen H Raun, Thomas E Jensen, Erik A Richter, Mona Ali, Rasmus Kjobsted, Lisbeth L V Moller, Morten A Federspiel, Lykke SylowAbstract:Key points The actin cytoskeleton regulating GTPase, RAC1, is a novel player in insulin-stimulated glucose uptake in muscle in vivo. High-fat diet (HFD) exacerbates muscle insulin resistance in RAC1 muscle knockout (mKO) mice. Muscle RAC1 KO protects against HFD-induced insulin resistance in fat tissue indicating tissue cross-talk. A fatty diet markedly reduces insulin clearance in mice. Abstract Insulin resistance and perturbations in glucose metabolism underpin common lifestyle diseases such as type 2 diabetes and obesity. Insulin resistance in muscle is characterized by compromised activity of the GTPase, Ras-related C3 Botulinum toxin substrate 1 (RAC1), yet the role of RAC1 in insulin-stimulated glucose uptake in vivo and diet-induced insulin resistance is unknown. Inducible muscle-specific RAC1 knockout (RAC1 mKO) and wild type (WT) littermate mice were either fed a chow or a 60% high-fat diet (HFD). Insulin-stimulated 2-deoxy-glucose uptake, intracellular signalling, protein expression, substrate utilization, and glucose and insulin tolerance were assessed. In chow-fed mice, in vivo insulin-stimulated glucose uptake was reduced in triceps, soleus and gastrocnemius muscles from RAC1 mKO mice. HFD-induced whole body insulin resistance was exacerbated by the lack of muscle RAC1 and glucose uptake was reduced in all muscles, except for soleus. Muscle Akt (also known as protein kinase B) signalling was unaffected by diet or genotype. In adipose tissue, RAC1 mKO mice were protected from HFD-induced insulin resistance (with respect to both glucose uptake and phosphorylated-Akt), rendering their whole body glucose tolerance comparable to WT mice on HFD. Our findings show that lack of RAC1 exacerbates HFD-induced insulin resistance in skeletal muscle. Whole body glucose tolerance, however, was largely unaffected in RAC1 mKO mice, likely due to improved insulin-stimulated glucose uptake in adipose tissue. We conclude that lack of RAC1 in the context of obesity is detrimental to insulin-stimulated muscle glucose uptake in muscle independently of Akt signalling.
-
RAC1 signaling is required for insulin stimulated glucose uptake and is dysregulated in insulin resistant murine and human skeletal muscle
Diabetes, 2013Co-Authors: Lykke Sylow, Thomas E Jensen, Maximilian Kleinert, Kurt Hojlund, Bente Kiens, Jorgen F P Wojtaszewski, Clara Prats, Peter Schjerling, Erik A RichterAbstract:The actin cytoskeleton–regulating GTPase RAC1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of RAC1 and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been investigated. We hypothesized that RAC1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle and are dysregulated in insulin-resistant states. Muscle-specific inducible RAC1 knockout (KO) mice and pharmacological inhibition of RAC1 were used to determine whether RAC1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, RAC1 and PAK1 expression and signaling were investigated in muscle of insulin-resistant mice and humans. Inhibition and KO of RAC1 decreased insulin-stimulated glucose transport in mouse soleus and extensor digitorum longus muscles ex vivo. RAC1 KO mice showed decreased insulin and glucose tolerance and trended toward higher plasma insulin concentrations after intraperitoneal glucose injection. RAC1 protein expression and insulin-stimulated PAKThr423 phosphorylation were decreased in muscles of high fat–fed mice. In humans, insulin-stimulated PAK activation was decreased in both acute insulin-resistant (intralipid infusion) and chronic insulin-resistant states (obesity and diabetes). These findings show that RAC1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.
