Publications by category
Journal articles
Takesono A, Kudoh T, Tyler CR (2022). Application of Transgenic Zebrafish Models for Studying the Effects of Estrogenic Endocrine Disrupting Chemicals on Embryonic Brain Development. Frontiers in Pharmacology, 13
Takesono A, Heasman SJ, Wojciak-Stothard B, Garg R, Ridley AJ (2010). Microtubules regulate migratory polarity through Rho/ROCK signaling in T cells.
PLoS One,
5(1).
Abstract:
Microtubules regulate migratory polarity through Rho/ROCK signaling in T cells.
Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs. CONCLUSIONS/SIGNIFICANCE: Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.
Abstract.
David R, Ma L, Ivetic A, Takesono A, Ridley AJ, Chai J-G, Tybulewicz VL, Marelli-Berg FM (2009). T-cell receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue.
Blood,
113(16), 3696-3705.
Abstract:
T-cell receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue.
Localization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)- and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into nonlymphoid antigen-rich tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signaling pathways that regulate T-cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4(+) T cells to antigenic sites. Severe migratory defects displayed by Vav1(-/-) T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1(-/-) T cells in vivo. In contrast, Vav1(-/-) T-cell retention into antigen-rich tissue was severely impaired, reflecting T cells' inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, and TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T cell-mediated tissue damage.
Abstract.
Author URL.
Jarmin SJ, David R, Ma L, Chai J-G, Dewchand H, Takesono A, Ridley AJ, Okkenhaug K, Marelli-Berg FM (2008). T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice.
J Clin Invest,
118(3), 1154-1164.
Abstract:
T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice.
The establishment of T cell-mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110delta contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110delta displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110delta activity. These observations suggest that pharmacological targeting of p110delta activity is a viable strategy for the therapy of T cell-mediated pathology.
Abstract.
Author URL.
Dombroski D, Houghtling RA, Labno CM, Precht P, Takesono A, Caplen NJ, Billadeau DD, Wange RL, Burkhardt JK, Schwartzberg PL, et al (2005). Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.
J Immunol,
174(3), 1385-1392.
Abstract:
Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.
The Tec family kinase Itk is an important regulator of Ca(2+) mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.
Abstract.
Author URL.
Dutra, A. Takesono, A. Garrett-Beal, L. (2004). Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media. Genesis, 39(2):100-4
tak101, Dombroski D, Horai R, Mandai M (2004). Requirement for Tec kihases in chemokine-induced migration and activation of Cdc42 and Rac. Current Biology, 14(10), 917-922.
tak101, Burkhardt JK, Finkelstein LD, Labno CM (2003). Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP. Current Biology, 13(18), 1619-1624.
tak101, Cismowski M, Duzic E, Lanier SM (2002). Activator of G-protein signaling 1 blocks GIRK channel activation by a G-protein-coupled receptor - Apparent disruptor of receptor signaling complexes. Journal of Biological Chemistry, 277(16), 13827-13830.
Takesono A, Finkelstein LD, Schwartzberg PL (2002). Beyond calcium: new signaling pathways for Tec family kinases.
J Cell Sci,
115(Pt 15), 3039-3048.
Abstract:
Beyond calcium: new signaling pathways for Tec family kinases.
The Tec kinases represent the second largest family of mammalian non-receptor tyrosine kinases and are distinguished by the presence of distinct proline-rich regions and pleckstrin homology domains that are required for proper regulation and activation. Best studied in lymphocyte and mast cells, these kinases are critical for the full activation of phospholipase-C gamma (PLC-gamma) and Ca(2+) mobilization downstream of antigen receptors. However, it has become increasingly clear that these kinases are activated downstream of many cell-surface receptors, including receptor tyrosine kinases, cytokine receptors, integrins and G-protein-coupled receptors. Evidence suggests that the Tec kinases influence a wide range of signaling pathways controlling activation of MAP kinases, actin reorganization, transcriptional regulation, cell survival and cellular transformation. Their impact on cellular physiology suggests that the Tec kinases help regulate multiple cellular processes beyond Ca(2+) mobilization.
Abstract.
Author URL.
Cismowski MJ, Takesono A, Ma C, Lanier SM, Duzic E (2002). Identification of modulators of mammalian G-protein signaling by functional screens in the yeast Saccharomyces cerevisiae.
Methods Enzymol,
344, 153-168.
Author URL.
Takesono, A. Sato, M. Hildebrandt, J.D. (2002). Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator. Journal of Biological Chemistry, 27;277(52):50223-5
Pizzinat N, Takesono A, Lanier SM (2001). Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains.
J Biol Chem,
276(20), 16601-16610.
Abstract:
Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains.
AGS3, a 650-amino acid protein encoded by an approximately 4-kilobase (kb) mRNA enriched in rat brain, is a Galpha(i)/Galpha(t)-binding protein that competes with Gbetagamma for interaction with Galpha(GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins. An approximately 2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, and determined its ability to interact with and regulate the guanine nucleotide-binding properties of G-proteins. Screening of a rat heart cDNA library, 5'-rapid amplification of cDNA ends, and RNase protection assays identified two populations of cDNAs (1979 and 2134 nucleotides plus the polyadenylation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the middle of the protein coding region. Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the expression of a major immunoreactive AGS3 polypeptide (M(r) approximately 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M(r) approximately 23,000 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectively interacted with the GDP-bound versus guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound conformation of Galpha(i2) and inhibited GTPgammaS binding to Galpha(i2). Protein interaction assays with glutathione S-transferase-AGS3-SHORT and heart lysates indicated interaction of AGS3-SHORT with Galpha(i1/2) and Galpha(i3), but not Galpha(s) or Galpha(q). Immunofluorescent imaging and subcellular fractionation following transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese hamster ovary cells indicated distinct subcellular distributions of the two forms of AGS3. Thus, AGS3 exists as a short and long form, both of which apparently stabilize the GDP-bound conformation of Galpha(i), but which differ in their tissue distribution and trafficking within the cell.
Abstract.
Author URL.
Cismowski MJ, Takesono A, Ma C, Lizano JS, Xie X, Fuernkranz H, Lanier SM, Duzic E (1999). Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.
Nat Biotechnol,
17(9), 878-883.
Abstract:
Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.
We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.
Abstract.
Author URL.
Takesono A, Zahner J, Blumer KJ, Nagao T, Kurose H (1999). Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway.
Biochem J,
343 Pt 1(Pt 1), 77-85.
Abstract:
Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway.
Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic receptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-mediated inhibition was not observed when alpha(2A)AR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to G(i)-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates G(i) signalling in a protein-kinase-C-dependent manner.
Abstract.
Author URL.
Takesono A, Cismowski MJ, Ribas C, Bernard M, Chung P, Hazard S, Duzic E, Lanier SM (1999). Receptor-independent activators of heterotrimeric G-protein signaling pathways.
J Biol Chem,
274(47), 33202-33205.
Abstract:
Receptor-independent activators of heterotrimeric G-protein signaling pathways.
Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.
Abstract.
Author URL.
Publications by year
2022
Takesono A, Kudoh T, Tyler CR (2022). Application of Transgenic Zebrafish Models for Studying the Effects of Estrogenic Endocrine Disrupting Chemicals on Embryonic Brain Development. Frontiers in Pharmacology, 13
2010
Takesono A, Heasman SJ, Wojciak-Stothard B, Garg R, Ridley AJ (2010). Microtubules regulate migratory polarity through Rho/ROCK signaling in T cells.
PLoS One,
5(1).
Abstract:
Microtubules regulate migratory polarity through Rho/ROCK signaling in T cells.
Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs. CONCLUSIONS/SIGNIFICANCE: Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.
Abstract.
2009
David R, Ma L, Ivetic A, Takesono A, Ridley AJ, Chai J-G, Tybulewicz VL, Marelli-Berg FM (2009). T-cell receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue.
Blood,
113(16), 3696-3705.
Abstract:
T-cell receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue.
Localization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)- and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into nonlymphoid antigen-rich tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signaling pathways that regulate T-cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4(+) T cells to antigenic sites. Severe migratory defects displayed by Vav1(-/-) T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1(-/-) T cells in vivo. In contrast, Vav1(-/-) T-cell retention into antigen-rich tissue was severely impaired, reflecting T cells' inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, and TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T cell-mediated tissue damage.
Abstract.
Author URL.
2008
Jarmin SJ, David R, Ma L, Chai J-G, Dewchand H, Takesono A, Ridley AJ, Okkenhaug K, Marelli-Berg FM (2008). T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice.
J Clin Invest,
118(3), 1154-1164.
Abstract:
T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice.
The establishment of T cell-mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110delta contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110delta displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110delta activity. These observations suggest that pharmacological targeting of p110delta activity is a viable strategy for the therapy of T cell-mediated pathology.
Abstract.
Author URL.
2005
Dombroski D, Houghtling RA, Labno CM, Precht P, Takesono A, Caplen NJ, Billadeau DD, Wange RL, Burkhardt JK, Schwartzberg PL, et al (2005). Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.
J Immunol,
174(3), 1385-1392.
Abstract:
Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.
The Tec family kinase Itk is an important regulator of Ca(2+) mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.
Abstract.
Author URL.
2004
Dutra, A. Takesono, A. Garrett-Beal, L. (2004). Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media. Genesis, 39(2):100-4
tak101, Dombroski D, Horai R, Mandai M (2004). Requirement for Tec kihases in chemokine-induced migration and activation of Cdc42 and Rac. Current Biology, 14(10), 917-922.
2003
tak101, Burkhardt JK, Finkelstein LD, Labno CM (2003). Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP. Current Biology, 13(18), 1619-1624.
2002
tak101, Cismowski M, Duzic E, Lanier SM (2002). Activator of G-protein signaling 1 blocks GIRK channel activation by a G-protein-coupled receptor - Apparent disruptor of receptor signaling complexes. Journal of Biological Chemistry, 277(16), 13827-13830.
Takesono A, Finkelstein LD, Schwartzberg PL (2002). Beyond calcium: new signaling pathways for Tec family kinases.
J Cell Sci,
115(Pt 15), 3039-3048.
Abstract:
Beyond calcium: new signaling pathways for Tec family kinases.
The Tec kinases represent the second largest family of mammalian non-receptor tyrosine kinases and are distinguished by the presence of distinct proline-rich regions and pleckstrin homology domains that are required for proper regulation and activation. Best studied in lymphocyte and mast cells, these kinases are critical for the full activation of phospholipase-C gamma (PLC-gamma) and Ca(2+) mobilization downstream of antigen receptors. However, it has become increasingly clear that these kinases are activated downstream of many cell-surface receptors, including receptor tyrosine kinases, cytokine receptors, integrins and G-protein-coupled receptors. Evidence suggests that the Tec kinases influence a wide range of signaling pathways controlling activation of MAP kinases, actin reorganization, transcriptional regulation, cell survival and cellular transformation. Their impact on cellular physiology suggests that the Tec kinases help regulate multiple cellular processes beyond Ca(2+) mobilization.
Abstract.
Author URL.
Cismowski MJ, Takesono A, Ma C, Lanier SM, Duzic E (2002). Identification of modulators of mammalian G-protein signaling by functional screens in the yeast Saccharomyces cerevisiae.
Methods Enzymol,
344, 153-168.
Author URL.
Takesono, A. Sato, M. Hildebrandt, J.D. (2002). Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator. Journal of Biological Chemistry, 27;277(52):50223-5
2001
Pizzinat N, Takesono A, Lanier SM (2001). Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains.
J Biol Chem,
276(20), 16601-16610.
Abstract:
Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains.
AGS3, a 650-amino acid protein encoded by an approximately 4-kilobase (kb) mRNA enriched in rat brain, is a Galpha(i)/Galpha(t)-binding protein that competes with Gbetagamma for interaction with Galpha(GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins. An approximately 2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, and determined its ability to interact with and regulate the guanine nucleotide-binding properties of G-proteins. Screening of a rat heart cDNA library, 5'-rapid amplification of cDNA ends, and RNase protection assays identified two populations of cDNAs (1979 and 2134 nucleotides plus the polyadenylation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the middle of the protein coding region. Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the expression of a major immunoreactive AGS3 polypeptide (M(r) approximately 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M(r) approximately 23,000 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectively interacted with the GDP-bound versus guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound conformation of Galpha(i2) and inhibited GTPgammaS binding to Galpha(i2). Protein interaction assays with glutathione S-transferase-AGS3-SHORT and heart lysates indicated interaction of AGS3-SHORT with Galpha(i1/2) and Galpha(i3), but not Galpha(s) or Galpha(q). Immunofluorescent imaging and subcellular fractionation following transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese hamster ovary cells indicated distinct subcellular distributions of the two forms of AGS3. Thus, AGS3 exists as a short and long form, both of which apparently stabilize the GDP-bound conformation of Galpha(i), but which differ in their tissue distribution and trafficking within the cell.
Abstract.
Author URL.
1999
Cismowski MJ, Takesono A, Ma C, Lizano JS, Xie X, Fuernkranz H, Lanier SM, Duzic E (1999). Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.
Nat Biotechnol,
17(9), 878-883.
Abstract:
Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.
We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.
Abstract.
Author URL.
Takesono A, Zahner J, Blumer KJ, Nagao T, Kurose H (1999). Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway.
Biochem J,
343 Pt 1(Pt 1), 77-85.
Abstract:
Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway.
Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic receptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-mediated inhibition was not observed when alpha(2A)AR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to G(i)-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates G(i) signalling in a protein-kinase-C-dependent manner.
Abstract.
Author URL.
Takesono A, Cismowski MJ, Ribas C, Bernard M, Chung P, Hazard S, Duzic E, Lanier SM (1999). Receptor-independent activators of heterotrimeric G-protein signaling pathways.
J Biol Chem,
274(47), 33202-33205.
Abstract:
Receptor-independent activators of heterotrimeric G-protein signaling pathways.
Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.
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