The Experts below are selected from a list of 93 Experts worldwide ranked by ideXlab platform
Ushio Kikkawa - One of the best experts on this subject based on the ideXlab platform.
-
DISTINCT SPECIFICITY IN THE BINDING OF INOSITOL PHOSPHATES BY PLECKSTRIN HOMOLOGY DOMAINS OF PLECKSTRIN, Rac-Protein KINASE, DIACYLGLYCEROL KINASE AND A NEW 130 KDA Protein
Biochimica et biophysica acta, 1997Co-Authors: Hiroshi Takeuchi, Hiroaki Konishi, Ushio Kikkawa, Takashi Kanematsu, Yoshio Misumi, Fumio Sakane, Yutaka Watanabe, Matilda Katan, Masato HirataAbstract:The pleckstrin homology domains (PH domains) derived from four different Proteins, the N-terminal part of pleckstrin, Rac-Protein kinase, diacylglycerol kinase and the 130 kDa Protein originally cloned as an inositol 1,4,5-trisphosphate binding Protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from Rac-Protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa Protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.
-
activation of Protein kinase b akt Rac Protein kinase by cellular stress and its association with heat shock Protein hsp27
FEBS Letters, 1997Co-Authors: Hiroaki Konishi, Motonari Tanaka, Hidenori Matsuzaki, Shunichi Kuroda, Yukitoshi Takemura, Ushio KikkawaAbstract:Protein kinase B (PKB, also named as Akt or Rac-Protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl2 and NaAsO2 by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoProtein was co-immunoprecipitated with PKB from the cells metabolic labeled with [32P]orthophosphate. The phosphoProtein was identified as Hsp27, a small heat shock Protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock Protein was not co-immunoprecipitated with other Protein kinases such as Protein kinase C and PKN. When the cells were treated with H2O2, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock Protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.
-
expression of a constitutively active phosphatidylinositol 3 kinase induces process formation in rat pc12 cells use of cre loxp recombination system
Journal of Biological Chemistry, 1997Co-Authors: Michimoto Kobayashi, Hiroaki Konishi, Satoshi Nagata, Yoshihiro Kita, Noriyuki Nakatsu, Sayoko Ihara, Kozo Kaibuchi, Shinya Kuroda, Hideo Iba, Ushio KikkawaAbstract:It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated Protein kinase or Protein kinase B/Rac Protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.
-
activation of Rac Protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3 kinase
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Hiroaki Konishi, Motonari Tanaka, Hidenori Matsuzaki, Yoshitaka Ono, Chiharu Tokunaga, Shunichi Kuroda, Ushio KikkawaAbstract:AbstRact Rac Protein kinase (Rac-PK), a serine/threonine Protein kinase containing a pleckstrin homology (PH) domain, was activated by cellular stress such as heat shock and hyperosmolarity. Wortmannin, which is known as a potent inhibitor of phosphatidylinositol 3-kinase and normally inhibits growth factor-induced activation of Rac-PK, did not suppress heat-shock induced activation of Rac-PK, indicating that this stress-induced activation of the kinase is not mediated by phosphatidylinositol 3-kinase. The PH domain was indispensable for stress-induced activation of Rac PK. In heat-treated cells, PKC delta, a member of the Protein kinase C family, was found to associate with the PH domain of Rac-PK. This PKC subspecies was phosphorylated in vitro by Rac-PK. The results suggest that Rac-PK may play a role in the cellular response to stress through its PH domain.
-
molecular cloning and chaRacterization of a new member of the Rac Protein kinase family association of the pleckstrin homology domain of 3 types of Rac Protein kinase with Protein kinase c subspecies and βγ subunits of g Proteins
Biochemical and Biophysical Research Communications, 1995Co-Authors: Hiroaki Konishi, Shoji Kuroda, Motonari Tanaka, Hidenori Matsuzaki, Yoshitaka Ono, K Kameyama, Tatsuya Haga, Ushio KikkawaAbstract:cDNA clones encoding the third member of the Rac Protein kinase family, termed Rac-PK γ, were isolated from a rat brain cDNA library. The deduced amino acid sequence of Rac-PK γ was highly related to those of previously identified family members, Rac-PK α and β, that have a pleckstrin homology domain and a Protein-serine/threonine kinase catalytic domain at the amino- and carboxyl-terminal regions, respectively. Northern blot analysis indicated that RAG-PK γ was expressed abundantly in brain and testis. Specific activities of Rac-PK α, β, and γ purified from transfected COS-7 cells were similar when measured by using myelin basic Protein as a phosphate acceptor. Analysis using fusion Proteins of glutathione S-transferase revealed that the pleckstrin homology domain of the three subtypes of Rac-PK associate with both Protein kinase C subspecies and βγ subunits of G Proteins. These results suggest that the pleckstrin homology domains of Rac Protein kinase family could associate more than one Protein to regulate the activity and/or intRacellular distribution of this enzyme family by different ways.
Hiroaki Konishi - One of the best experts on this subject based on the ideXlab platform.
-
DISTINCT SPECIFICITY IN THE BINDING OF INOSITOL PHOSPHATES BY PLECKSTRIN HOMOLOGY DOMAINS OF PLECKSTRIN, Rac-Protein KINASE, DIACYLGLYCEROL KINASE AND A NEW 130 KDA Protein
Biochimica et biophysica acta, 1997Co-Authors: Hiroshi Takeuchi, Hiroaki Konishi, Ushio Kikkawa, Takashi Kanematsu, Yoshio Misumi, Fumio Sakane, Yutaka Watanabe, Matilda Katan, Masato HirataAbstract:The pleckstrin homology domains (PH domains) derived from four different Proteins, the N-terminal part of pleckstrin, Rac-Protein kinase, diacylglycerol kinase and the 130 kDa Protein originally cloned as an inositol 1,4,5-trisphosphate binding Protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from Rac-Protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa Protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.
-
activation of Protein kinase b akt Rac Protein kinase by cellular stress and its association with heat shock Protein hsp27
FEBS Letters, 1997Co-Authors: Hiroaki Konishi, Motonari Tanaka, Hidenori Matsuzaki, Shunichi Kuroda, Yukitoshi Takemura, Ushio KikkawaAbstract:Protein kinase B (PKB, also named as Akt or Rac-Protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl2 and NaAsO2 by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoProtein was co-immunoprecipitated with PKB from the cells metabolic labeled with [32P]orthophosphate. The phosphoProtein was identified as Hsp27, a small heat shock Protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock Protein was not co-immunoprecipitated with other Protein kinases such as Protein kinase C and PKN. When the cells were treated with H2O2, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock Protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.
-
expression of a constitutively active phosphatidylinositol 3 kinase induces process formation in rat pc12 cells use of cre loxp recombination system
Journal of Biological Chemistry, 1997Co-Authors: Michimoto Kobayashi, Hiroaki Konishi, Satoshi Nagata, Yoshihiro Kita, Noriyuki Nakatsu, Sayoko Ihara, Kozo Kaibuchi, Shinya Kuroda, Hideo Iba, Ushio KikkawaAbstract:It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated Protein kinase or Protein kinase B/Rac Protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.
-
activation of Rac Protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3 kinase
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Hiroaki Konishi, Motonari Tanaka, Hidenori Matsuzaki, Yoshitaka Ono, Chiharu Tokunaga, Shunichi Kuroda, Ushio KikkawaAbstract:AbstRact Rac Protein kinase (Rac-PK), a serine/threonine Protein kinase containing a pleckstrin homology (PH) domain, was activated by cellular stress such as heat shock and hyperosmolarity. Wortmannin, which is known as a potent inhibitor of phosphatidylinositol 3-kinase and normally inhibits growth factor-induced activation of Rac-PK, did not suppress heat-shock induced activation of Rac-PK, indicating that this stress-induced activation of the kinase is not mediated by phosphatidylinositol 3-kinase. The PH domain was indispensable for stress-induced activation of Rac PK. In heat-treated cells, PKC delta, a member of the Protein kinase C family, was found to associate with the PH domain of Rac-PK. This PKC subspecies was phosphorylated in vitro by Rac-PK. The results suggest that Rac-PK may play a role in the cellular response to stress through its PH domain.
-
molecular cloning and chaRacterization of a new member of the Rac Protein kinase family association of the pleckstrin homology domain of 3 types of Rac Protein kinase with Protein kinase c subspecies and βγ subunits of g Proteins
Biochemical and Biophysical Research Communications, 1995Co-Authors: Hiroaki Konishi, Shoji Kuroda, Motonari Tanaka, Hidenori Matsuzaki, Yoshitaka Ono, K Kameyama, Tatsuya Haga, Ushio KikkawaAbstract:cDNA clones encoding the third member of the Rac Protein kinase family, termed Rac-PK γ, were isolated from a rat brain cDNA library. The deduced amino acid sequence of Rac-PK γ was highly related to those of previously identified family members, Rac-PK α and β, that have a pleckstrin homology domain and a Protein-serine/threonine kinase catalytic domain at the amino- and carboxyl-terminal regions, respectively. Northern blot analysis indicated that RAG-PK γ was expressed abundantly in brain and testis. Specific activities of Rac-PK α, β, and γ purified from transfected COS-7 cells were similar when measured by using myelin basic Protein as a phosphate acceptor. Analysis using fusion Proteins of glutathione S-transferase revealed that the pleckstrin homology domain of the three subtypes of Rac-PK associate with both Protein kinase C subspecies and βγ subunits of G Proteins. These results suggest that the pleckstrin homology domains of Rac Protein kinase family could associate more than one Protein to regulate the activity and/or intRacellular distribution of this enzyme family by different ways.
Brian A Hemmings - One of the best experts on this subject based on the ideXlab platform.
-
high affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of Rac Protein kinase b and their influence on kinase activity
Journal of Biological Chemistry, 1997Co-Authors: Matthias Frech, Mirjana Andjelkovic, Evan Ingley, Kishta K Reddy, John R Falck, Brian A HemmingsAbstract:The influence of inositol phosphates and phosphoinositides on the alpha isoform of the Rac-Protein kinase B (Rac/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 microM and 0.5 microM, respectively. Similar values were obtained using Rac/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of Rac/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the Rac/PKB kinase activity with half-maximal inhibition at 2.5 microM. In contrast, phosphatidylinositol 3, 4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 microM). A mutant Rac/PKB Protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of Rac/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of Rac/PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and Rac/PKB regulation.
-
activation and phosphorylation of a pleckstrin homology domain containing Protein kinase Rac pk pkb promoted by serum and Protein phosphatase inhibitors
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: M Andjelkovic, T Jakubowicz, Peter Cron, Xiufen Ming, Brian A HemmingsAbstract:AbstRact Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2- to 11-fold activation of the serine/ threonine Rac Protein kinase (Rac-PK). Kinase activation was accompanied by decreased mobility of Rac-PK on SDS/PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the Rac-PK phosphorylation level 3-to 4-fold. Unstimulated Rac-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of Rac-PK in vitro with Protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect Rac-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/threonine rich C-terminal region impaired both Rac-PKalpha basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in Rac-PKalpha activation. Stimulation of Rac-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that Rac-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for Rac-PK activation by phosphorylation.
-
developmental regulation of expression and activity of multiple forms of the drosophila Rac Protein kinase
Journal of Biological Chemistry, 1995Co-Authors: Mirjana Andjelkovic, Peter Cron, Pamela F Jones, Ueli Grossniklaus, Alexander F Schier, Mathias S Dick, Graeme Bilbe, Brian A HemmingsAbstract:AbstRact We have chaRacterized the Drosophila homologue of the proto-oncogenic Rac Protein kinase (DRac-PK). The DRac-PK gene gives rise to two transcripts with the same coding potential, generated by the use of two different polyadenylation signals. Each transcript encodes two polypeptides because of the presence of a weaker initiator ACG codon, upstream from the major AUG, such that the larger Protein contains an N-terminal extension. Like the human isoforms, DRac-PKs possess a novel signaling region, the pleckstrin homology domain. DRac-PK Proteins have a similar expression pattern, being regulated both maternally and zygotically, and are expressed throughout Drosophila development. Antisera specific for recombinant DRac-PK and for its C terminus detected two polypeptides of 66 and 85 kDa in Drosophila extRacts. The antirecombinant antisera also recognized a polypeptide of 120 kDa from Drosophila, which apparently shared an epitope related to DRac-PK sequences. The role of p120 appears to be restricted compared with that of DRac-PK, since it was not detected in larvae or adult flies. There was no spatial restriction of DRac-PK expression during embryogenesis, suggesting that localized activation might be a regulatory mechanism for its function. DRac-PK possesses an intrinsic kinase activity that is 8-fold higher in adult flies than in 0-3-h embryos undergoing rapid mitotic cycles.
-
molecular cloning of a second form of Rac Protein kinase
Molecular Biology of the Cell, 1991Co-Authors: P F Jones, T Jakubowicz, Brian A HemmingsAbstract:AbstRact A novel serine/threonine Protein kinase (termed Rac-PK) has recently been identified and cloned from cDNA libraries derived from the human cell lines MCF-7 and WI38. A second form of this Protein kinase, termed Rac Protein kinase beta, has been identified from cDNAs derived from the same cell lines. These two closely related forms show 90% homology, although the beta form with a predicted Mr 60,200 has a carboxyl terminal extension of 40 amino acids in comparison to the alpha form. This extension has a high serine content with 11 serine residues in the last 30 amino acids. The beta form of the Protein has been shown by both in vitro translation and bacterial expression to be approximately 5000 Da larger than the alpha form. Rac Protein kinase beta is encoded by a 3.4-kb transcript and the alpha form is encoded by a 3.2-kb mRNA. Using gene-specific probes both transcripts were detected in all cell types analyzed, although levels of expression were different for the two forms. The catalytic domain of Rac Protein kinase beta shows a high degree of homology to both the Protein kinase C and cyclic AMP-dependent Protein kinase families, and hence Rac Protein kinases appear to represent a new subfamily of the second messenger serine/threonine Protein kinases.
-
molecular cloning and identification of a serine threonine Protein kinase of the second messenger subfamily
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Pamela F Jones, Teresa Jakubowicz, Fernando J Pitossi, Fransisca Maurer, Brian A HemmingsAbstract:A partial cDNA was isolated that encoded a Protein kinase, termed Rac (related to the A and C kinases). This cDNA was subsequently used to screen libraries derived from the human cell lines MCF-7 and WI38 and led to the isolation of full-length cDNA clones. DNA sequence analysis identified an open reading frame of 1440 base pairs encoding a Protein of 480 amino acids (Mr, 55,716). This result was supported by the synthesis of a Mr 58,000 Protein in an in vitro translation system that used RNA transcribed from cloned cDNAs with SP6 RNA polymerase. The predicted Protein contains consensus sequences chaRacteristic of a Protein kinase catalytic domain and shows 73% and 68% similarity to Protein kinase C and the cAMP-dependent Protein kinase, respectively. Northern (RNA) analysis revealed a single mRNA transcript of 3.2 kilobases that varied up to 300-fold between different cell lines. Specific antisera directed towards the carboxyl terminal of the Rac Protein kinase were prepared and used to identify that phosphorylated several substrates in immunoprecipitates prepared with the Rac-specific antisera.
Hidenori Matsuzaki - One of the best experts on this subject based on the ideXlab platform.
-
activation of Protein kinase b akt Rac Protein kinase by cellular stress and its association with heat shock Protein hsp27
FEBS Letters, 1997Co-Authors: Hiroaki Konishi, Motonari Tanaka, Hidenori Matsuzaki, Shunichi Kuroda, Yukitoshi Takemura, Ushio KikkawaAbstract:Protein kinase B (PKB, also named as Akt or Rac-Protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl2 and NaAsO2 by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoProtein was co-immunoprecipitated with PKB from the cells metabolic labeled with [32P]orthophosphate. The phosphoProtein was identified as Hsp27, a small heat shock Protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock Protein was not co-immunoprecipitated with other Protein kinases such as Protein kinase C and PKN. When the cells were treated with H2O2, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock Protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.
-
activation of Rac Protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3 kinase
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Hiroaki Konishi, Motonari Tanaka, Hidenori Matsuzaki, Yoshitaka Ono, Chiharu Tokunaga, Shunichi Kuroda, Ushio KikkawaAbstract:AbstRact Rac Protein kinase (Rac-PK), a serine/threonine Protein kinase containing a pleckstrin homology (PH) domain, was activated by cellular stress such as heat shock and hyperosmolarity. Wortmannin, which is known as a potent inhibitor of phosphatidylinositol 3-kinase and normally inhibits growth factor-induced activation of Rac-PK, did not suppress heat-shock induced activation of Rac-PK, indicating that this stress-induced activation of the kinase is not mediated by phosphatidylinositol 3-kinase. The PH domain was indispensable for stress-induced activation of Rac PK. In heat-treated cells, PKC delta, a member of the Protein kinase C family, was found to associate with the PH domain of Rac-PK. This PKC subspecies was phosphorylated in vitro by Rac-PK. The results suggest that Rac-PK may play a role in the cellular response to stress through its PH domain.
-
molecular cloning and chaRacterization of a new member of the Rac Protein kinase family association of the pleckstrin homology domain of 3 types of Rac Protein kinase with Protein kinase c subspecies and βγ subunits of g Proteins
Biochemical and Biophysical Research Communications, 1995Co-Authors: Hiroaki Konishi, Shoji Kuroda, Motonari Tanaka, Hidenori Matsuzaki, Yoshitaka Ono, K Kameyama, Tatsuya Haga, Ushio KikkawaAbstract:cDNA clones encoding the third member of the Rac Protein kinase family, termed Rac-PK γ, were isolated from a rat brain cDNA library. The deduced amino acid sequence of Rac-PK γ was highly related to those of previously identified family members, Rac-PK α and β, that have a pleckstrin homology domain and a Protein-serine/threonine kinase catalytic domain at the amino- and carboxyl-terminal regions, respectively. Northern blot analysis indicated that RAG-PK γ was expressed abundantly in brain and testis. Specific activities of Rac-PK α, β, and γ purified from transfected COS-7 cells were similar when measured by using myelin basic Protein as a phosphate acceptor. Analysis using fusion Proteins of glutathione S-transferase revealed that the pleckstrin homology domain of the three subtypes of Rac-PK associate with both Protein kinase C subspecies and βγ subunits of G Proteins. These results suggest that the pleckstrin homology domains of Rac Protein kinase family could associate more than one Protein to regulate the activity and/or intRacellular distribution of this enzyme family by different ways.
Motonari Tanaka - One of the best experts on this subject based on the ideXlab platform.
-
activation of Protein kinase b akt Rac Protein kinase by cellular stress and its association with heat shock Protein hsp27
FEBS Letters, 1997Co-Authors: Hiroaki Konishi, Motonari Tanaka, Hidenori Matsuzaki, Shunichi Kuroda, Yukitoshi Takemura, Ushio KikkawaAbstract:Protein kinase B (PKB, also named as Akt or Rac-Protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl2 and NaAsO2 by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoProtein was co-immunoprecipitated with PKB from the cells metabolic labeled with [32P]orthophosphate. The phosphoProtein was identified as Hsp27, a small heat shock Protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock Protein was not co-immunoprecipitated with other Protein kinases such as Protein kinase C and PKN. When the cells were treated with H2O2, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock Protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.
-
activation of Rac Protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3 kinase
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Hiroaki Konishi, Motonari Tanaka, Hidenori Matsuzaki, Yoshitaka Ono, Chiharu Tokunaga, Shunichi Kuroda, Ushio KikkawaAbstract:AbstRact Rac Protein kinase (Rac-PK), a serine/threonine Protein kinase containing a pleckstrin homology (PH) domain, was activated by cellular stress such as heat shock and hyperosmolarity. Wortmannin, which is known as a potent inhibitor of phosphatidylinositol 3-kinase and normally inhibits growth factor-induced activation of Rac-PK, did not suppress heat-shock induced activation of Rac-PK, indicating that this stress-induced activation of the kinase is not mediated by phosphatidylinositol 3-kinase. The PH domain was indispensable for stress-induced activation of Rac PK. In heat-treated cells, PKC delta, a member of the Protein kinase C family, was found to associate with the PH domain of Rac-PK. This PKC subspecies was phosphorylated in vitro by Rac-PK. The results suggest that Rac-PK may play a role in the cellular response to stress through its PH domain.
-
molecular cloning and chaRacterization of a new member of the Rac Protein kinase family association of the pleckstrin homology domain of 3 types of Rac Protein kinase with Protein kinase c subspecies and βγ subunits of g Proteins
Biochemical and Biophysical Research Communications, 1995Co-Authors: Hiroaki Konishi, Shoji Kuroda, Motonari Tanaka, Hidenori Matsuzaki, Yoshitaka Ono, K Kameyama, Tatsuya Haga, Ushio KikkawaAbstract:cDNA clones encoding the third member of the Rac Protein kinase family, termed Rac-PK γ, were isolated from a rat brain cDNA library. The deduced amino acid sequence of Rac-PK γ was highly related to those of previously identified family members, Rac-PK α and β, that have a pleckstrin homology domain and a Protein-serine/threonine kinase catalytic domain at the amino- and carboxyl-terminal regions, respectively. Northern blot analysis indicated that RAG-PK γ was expressed abundantly in brain and testis. Specific activities of Rac-PK α, β, and γ purified from transfected COS-7 cells were similar when measured by using myelin basic Protein as a phosphate acceptor. Analysis using fusion Proteins of glutathione S-transferase revealed that the pleckstrin homology domain of the three subtypes of Rac-PK associate with both Protein kinase C subspecies and βγ subunits of G Proteins. These results suggest that the pleckstrin homology domains of Rac Protein kinase family could associate more than one Protein to regulate the activity and/or intRacellular distribution of this enzyme family by different ways.
