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James A Decaprio - One of the best experts on this subject based on the ideXlab platform.
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Cyclin D–CDK4 relieves cooperative repression of proliferation and cell cycle gene expression by DREAM and RB
Oncogene, 2019Co-Authors: Amy E Schade, Matthew Oser, Hilary E Nicholson, James A DecaprioAbstract:The retinoblastoma Protein (RB) restricts cell cycle gene expression and entry into the cell cycle. The RB-related Protein P130 forms the DREAM (DP, RB-like, E2F, and MuvB) complex and contributes to repression of cell cycle-dependent genes during quiescence. Although both RB and DREAM bind and repress an overlapping set of E2F-dependent gene promoters, it remains unclear whether they cooperate to restrict cell cycle entry. To test the specific contributions of RB and DREAM, we generated RB and P130 knockout cells in primary human fibroblasts. Knockout of both P130 and RB yielded higher levels of cell cycle gene expression in G0 and G1 cells compared to cells with knockout of RB alone, indicating a role for DREAM and RB in repression of cell cycle genes. We observed that RB had a dominant role in E2F-dependent gene repression during mid to late G1 while DREAM activity was more prominent during G0 and early G1. Cyclin D–Cyclin-Dependent Kinase 4 (CDK4)-dependent phosphorylation of P130 occurred during early G1, and led to the release of P130 and MuvB from E2F4 and decreased P130 and MuvB binding to cell cycle promoters. Specific inhibition of CDK4 activity by palbociclib blocked DREAM complex disassembly during cell cycle entry. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as in palbociclib-sensitive cancer cell lines. Although RB knockout cells were partially resistant to CDK4 inhibition, RB and P130 double knockout cells were significantly more resistant to palbociclib treatment. These results indicate that DREAM cooperates with RB in repressing E2F-dependent gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.
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cyclin d cdk4 relieves cooperative repression of proliferation and cell cycle gene expression by dream and rb
Oncogene, 2019Co-Authors: Amy E Schade, James A Decaprio, Matthew Oser, Hilary E NicholsonAbstract:The retinoblastoma Protein (RB) restricts cell cycle gene expression and entry into the cell cycle. The RB-related Protein P130 forms the DREAM (DP, RB-like, E2F, and MuvB) complex and contributes to repression of cell cycle-dependent genes during quiescence. Although both RB and DREAM bind and repress an overlapping set of E2F-dependent gene promoters, it remains unclear whether they cooperate to restrict cell cycle entry. To test the specific contributions of RB and DREAM, we generated RB and P130 knockout cells in primary human fibroblasts. Knockout of both P130 and RB yielded higher levels of cell cycle gene expression in G0 and G1 cells compared to cells with knockout of RB alone, indicating a role for DREAM and RB in repression of cell cycle genes. We observed that RB had a dominant role in E2F-dependent gene repression during mid to late G1 while DREAM activity was more prominent during G0 and early G1. Cyclin D-Cyclin-Dependent Kinase 4 (CDK4)-dependent phosphorylation of P130 occurred during early G1, and led to the release of P130 and MuvB from E2F4 and decreased P130 and MuvB binding to cell cycle promoters. Specific inhibition of CDK4 activity by palbociclib blocked DREAM complex disassembly during cell cycle entry. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as in palbociclib-sensitive cancer cell lines. Although RB knockout cells were partially resistant to CDK4 inhibition, RB and P130 double knockout cells were significantly more resistant to palbociclib treatment. These results indicate that DREAM cooperates with RB in repressing E2F-dependent gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.
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The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression
Genes & Development, 2012Co-Authors: Subhashini Sadasivam, Shenghua Duan, James A DecaprioAbstract:Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex—comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4—serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like Protein P130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from P130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis.
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Phosphorylation of the retinoblastoma-related Protein P130 in growth-arrested cells
Oncogene, 2000Co-Authors: Alfredo J Canhoto, Larisa Litovchick, Anton Chestukhin, James A DecaprioAbstract:The retinoblastoma family of Proteins including pRB, p107 and P130 undergoes cell cycle dependent phosphorylation during the mid-G1 to S phase transition. This phosphorylation is dependent upon the activity of cyclin D/cdk4. In contrast to pRB and p107, P130 is phosphorylated during G0 and the early G1 phase of the cell cycle. We observed that P130 is specifically phosphorylated on serine and threonine residues in T98G cells arrested in G0 by serum deprivation or density arrest. Identification of the phospho-serine and phospho-threonine residues revealed that most were clustered within a short co-linear region unique to P130, defined as the Loop. Deletion of the Loop region resulted in a change in the phosphorylation status of P130 under growth arrest conditions. Notably, deletion of the Loop did not affect the ability of P130 to bind to E2F-4 or SV40 Large T antigen, to induce growth arrest in Saos-2 cells, and to become hyperphosphorylated during the proliferative phase of the cell cycle. P130 undergoes specific G0 phosphorylation in a manner that distinguishes it from pRB and p107.
Takashi Kanematsu - One of the best experts on this subject based on the ideXlab platform.
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Role of the PLC‐related, catalytically inactive Protein P130 in GABAA receptor function
The EMBO journal, 2002Co-Authors: Takashi Kanematsu, Il Sung Jang, Taku Yamaguchi, Hiroyasu Nagahama, Kenji Yoshimura, Kiyoshi Hidaka, Miho Matsuda, Hiroshi Takeuchi, Yoshio Misumi, Keiko NakayamaAbstract:The Protein P130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding Protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. We show that P130 plays an important role in signaling by the type A receptor for γ-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABAA receptor-associated Protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a Protein that interacts with P130. Furthermore, P130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl– current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of P130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that P130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit.
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role of the plc related catalytically inactive Protein P130 in gabaa receptor function
The EMBO Journal, 2002Co-Authors: Takashi Kanematsu, Il Sung Jang, Taku Yamaguchi, Hiroyasu Nagahama, Kenji Yoshimura, Kiyoshi Hidaka, Miho Matsuda, Hiroshi Takeuchi, Yoshio Misumi, Keiko NakayamaAbstract:The Protein P130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding Protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. We show that P130 plays an important role in signaling by the type A receptor for γ-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABAA receptor-associated Protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a Protein that interacts with P130. Furthermore, P130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl– current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of P130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that P130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit.
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Interaction of P130 with, and Consequent Inhibition of, the Catalytic Subunit of Protein Phosphatase 1α
The Journal of biological chemistry, 2001Co-Authors: Kenji Yoshimura, Kiyoshi Hidaka, Hiroshi Takeuchi, Naoko Doira, Yushi Ito, Osamu Sato, Miho Terunuma, Kae Harada, Yasuo Ogawa, Takashi KanematsuAbstract:Abstract The Protein P130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding Protein with a domain organization similar to that of phospholipase C-δ1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode Proteins that interact with P130 has now led to the identification of the catalytic subunit of Protein phosphatase 1α (PP1cα) as a P130-binding Protein. The association between P130 and PP1cα was also confirmedin vitro by an overlay assay, a “pull-down” assay, and surface plasmon resonance analysis. The interaction of P130 with PP1cα resulted in inhibition of the catalytic activity of the latter in a P130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express P130 and of mouse brain extract with antibodies to P130 and to PP1cα also detected the presence of a complex of P130 and PP1cα. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1cα, was higher in COS-1 cells that stably express P130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca2+ signaling, P130 might also contribute to regulation of Protein dephosphorylation through its interaction with PP1cα.
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Hyperinsulinemia in PRIP-1 Gene Deleted Mice
Biomedical Research, 2001Co-Authors: Naoko Doira, Takashi Kanematsu, Miho Matsuda, Hiroshi Takeuchi, Keiko Nakayama, H. Nakano, Yushi Ito, Keiichi I. Nakayama, Masato HirataAbstract:The Protein P130, named from its molecular size, was originally identified as an inositol 1, 4,5-trisphosphate binding Protein similar to phospholipase C (PLC)-δ1, but lacking any PLC activity. It was recently renamed PLC-related catalytically inactive Protein-1 (PRIP-1). In the present study, PRIP-1 gene-targeted mice were analyzed for glycogen metabolism based on the previous finding that PRIP-1 interacts with Protein phosphatase-1 (26). Compared to the control mice, mutant mice exhibited lower phosphorylase activity and higher levels of glycogen in the liver, which appeared to be consistent with the inhibition of Protein phosphatase-1 by PRIP-1. These observation could also be attributed to the increased levels of plasma insulin. Hyperinsulinemia, observed even in the young mice, was progressive with aging, and was accompanied by the accumulation of fat tissues, increased body weight and decreased sensitivity to insulin in the mutant mice at the age of 12 months. These results suggest that PRIP-1 is a molecule involved in the control of plasma insulin.
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domain organization of P130 plc related catalytically inactive Protein and structural basis for the lack of enzyme activity
FEBS Journal, 2000Co-Authors: Takashi Kanematsu, Kenji Yoshimura, Kiyoshi Hidaka, Hiroshi Takeuchi, Matilda Katan, Masato HirataAbstract:The 130-kDa Protein (P130) was isolated as a novel inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]-binding Protein similar to phospholipase C-delta1 (PLC-delta1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of P130 and structural basis for lack of PLC activity, we subjected P130 to limited proteolysis and also constructed a number of chimeras with PLC-delta1. Trypsin treatment of P130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta1 and demonstrating that all domains of P130, including the unique region at the C-terminus, are stable, tightly folded structures. P130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that P130 has intrinsically no PLC activity. A number of chimeric molecules between P130 and PLC-delta1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two Proteins, as only some domains of P130 could replace the corresponding structures in PLC-delta1 to form a functional enzyme. These results suggest that P130 and the related Proteins could represent a new Protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.
Masato Hirata - One of the best experts on this subject based on the ideXlab platform.
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Hyperinsulinemia in PRIP-1 Gene Deleted Mice
Biomedical Research, 2001Co-Authors: Naoko Doira, Takashi Kanematsu, Miho Matsuda, Hiroshi Takeuchi, Keiko Nakayama, H. Nakano, Yushi Ito, Keiichi I. Nakayama, Masato HirataAbstract:The Protein P130, named from its molecular size, was originally identified as an inositol 1, 4,5-trisphosphate binding Protein similar to phospholipase C (PLC)-δ1, but lacking any PLC activity. It was recently renamed PLC-related catalytically inactive Protein-1 (PRIP-1). In the present study, PRIP-1 gene-targeted mice were analyzed for glycogen metabolism based on the previous finding that PRIP-1 interacts with Protein phosphatase-1 (26). Compared to the control mice, mutant mice exhibited lower phosphorylase activity and higher levels of glycogen in the liver, which appeared to be consistent with the inhibition of Protein phosphatase-1 by PRIP-1. These observation could also be attributed to the increased levels of plasma insulin. Hyperinsulinemia, observed even in the young mice, was progressive with aging, and was accompanied by the accumulation of fat tissues, increased body weight and decreased sensitivity to insulin in the mutant mice at the age of 12 months. These results suggest that PRIP-1 is a molecule involved in the control of plasma insulin.
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domain organization of P130 plc related catalytically inactive Protein and structural basis for the lack of enzyme activity
FEBS Journal, 2000Co-Authors: Takashi Kanematsu, Kenji Yoshimura, Kiyoshi Hidaka, Hiroshi Takeuchi, Matilda Katan, Masato HirataAbstract:The 130-kDa Protein (P130) was isolated as a novel inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]-binding Protein similar to phospholipase C-delta1 (PLC-delta1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of P130 and structural basis for lack of PLC activity, we subjected P130 to limited proteolysis and also constructed a number of chimeras with PLC-delta1. Trypsin treatment of P130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta1 and demonstrating that all domains of P130, including the unique region at the C-terminus, are stable, tightly folded structures. P130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that P130 has intrinsically no PLC activity. A number of chimeric molecules between P130 and PLC-delta1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two Proteins, as only some domains of P130 could replace the corresponding structures in PLC-delta1 to form a functional enzyme. These results suggest that P130 and the related Proteins could represent a new Protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.
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Membrane association of a new inositol 1,4,5-trisphosphate binding Protein, P130 is not dependent on the pleckstrin homology domain
Chemistry and physics of lipids, 1999Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Masato HirataAbstract:Abstract The 130-kDa Protein was isolated as a novel inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) binding Protein from rat brain and was molecularly cloned to be found similar to phospholipase C-δ 1 (Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M., 1992. Putative inositol 1,4,5-trisphosphate binding Proteins in rat brain cytosol, J. Biol. Chem. 267, 6518–6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M., 1996. A new inositol 1,4,5-trisphosphate binding Protein similar to phospholipase C-δ 1 , Biochem. J. 313, 319–325). The 130-kDa Protein and its deleted Protein expressed in COS-1 cells were seen in both the membrane and the cytosol fractions. Truncation of 232 residues from the N-terminus, the Protein molecule lacking the pleckstrin homology (PH) domain was also localized in the membrane fraction as much as seen with a full-length Protein and other deleted Proteins, thereby indicating that the PH domain is not primarily involved in the membrane localization. The addition of Mg 2+ to homogenates of COS-1 cells caused the translocation of expressed Proteins from the cytosol to the membrane fraction, yet further addition of AlF 4 − which induced the activation of GTP binding Proteins did not cause a further translocation. The Protein translocated to the membrane by the addition of Mg 2+ was hardly extracted with Triton X-100. The inclusion of Ins(1,4,5)P 3 or phosphatidylinositol 4,5-bisphosphate in cell homogenates caused the very small reduction in the amounts of membrane-associated Proteins expressed by some constructs. These results indicate that (i) the PH domain is not primarily involved in the membrane localization of the 130-kDa Protein, (ii) the activation of GTP binding Protein does not appear to cause the translocation of the 130-kDa Protein, and (iii) intrinsic phosphatidylinositol 4,5-bisphosphate present in the membrane appears to be involved in the membrane association of the 130-kDa Protein to a very small extent, probably through the binding site in the PH domain.
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Localization of a novel inositol 1,4,5-trisphosphate binding Protein, P130 in rat brain
Neuroscience letters, 1998Co-Authors: Miho Matsuda, Takashi Kanematsu, Hiroshi Takeuchi, Toshio Kukita, Masato HirataAbstract:We have isolated a novel inositol 1,4,5-trisphosphate binding Protein with molecular mass of 130 kDa (P130), homologous to phospholipase C-delta1 in amino acid sequence but with no catalytic activity. Here we report the expression and localization of P130 at the mRNA level in rat brain. Northern blotting showed that gene expression encoding P130 was most abundant in brain. Brain localization of P130-mRNA using an in situ hybridization technique revealed that in the cerebellum, the mRNA was detected in the granular cell and Purkinje cell layers, and cerebellar nuclei. In the cerebrum, the mRNA was localized in hippocampal pyramidal cells, dentate granule cells and pyramidal and/or granule cells of the cerebral cortex. The brain localization of P130-mRNA was similar to that of the beta-subtype of phospholipase C, indicating that P130 may be mainly involved in phospholipase Cbeta-mediated signaling.
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localization of a high affinity inositol 1 4 5 trisphosphate inositol 1 4 5 6 tetrakisphosphate binding domain to the pleckstrin homology module of a new 130 kda Protein characterization of the determinants of structural specificity
Biochemical Journal, 1996Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hassan Bin Yaakob, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Masato HirataAbstract:We have previously identified a novel 130 kDa Protein (P130) which binds Ins(1,4,5)P3 and shares 38% sequence identity with phospholipase C-delta 1 [Kanematsu, Misumi, Watanabe, Ozaki, Koga, Iwanaga, Ikehara and Hirata (1996) Biochem. J. 313, 319-325]. We have now transfected COS-1 cells with genes encoding the entire length of the molecule or one of several truncated mutants, in order to locate the region for binding of Ins(1,4,5)P3. Deletion of N-terminal residues 116-232, the region which corresponds to the pleckstrin homology (PH) domain of the molecule, completely abolished binding activity. This result was confirmed when the PH domain itself (residues 95-232), isolated from a bacterial expression system, was found to bind [3H]Ins(1,4,5)P3. We also found that Ins(1,4,5,6)P4 was as efficacious as Ins(1,4,5)P3 in displacing [3H]Ins(1,4,5)P3, suggesting that these two polyphosphates bind to P130 with similar affinity. This conclusion was confirmed by direct binding studies using [3H]Ins(1,4,5,6)P4 with high specific radioactivity which we prepared ourselves. Binding specificity was also examined with a variety of inositol phosphate derivatives. As is the case with other PH domains characterized to date, we found that the 4,5-vicinal phosphate pair was an essential determinant of ligand specificity. However, the PH domain of P130 exhibited some novel features. For example, the 3- and/or 6-phosphates could also contribute to overall binding; this contrasts with some other PH domains where these phosphate groups decrease ligand affinity by imposing a steric constraint. Secondly, a free monoester 1-phosphate substantially increased binding affinity, which is a situation so far unique to the PH domain of P130.
Hisamaru Hirai - One of the best experts on this subject based on the ideXlab platform.
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a novel signaling molecule P130 forms stable complexes in vivo with v crk and v src in a tyrosine phosphorylation dependent manner
The EMBO Journal, 1994Co-Authors: Ryuichi Sakai, Nobuhiro Hirano, Tomoyuki Tanaka, Seishi Ogawa, Akihiro Iwamatsu, H. Mano, Yoshio Yazaki, Hisamaru HiraiAbstract:p47v-crk (v-Crk), a transforming gene product containing Src homology (SH)-2 and -3 domains, induces an elevated level of tyrosine phosphorylation of several cellular Proteins. Among these Proteins, a 125-135 kDa Protein (P130) shows marked phosphorylation at tyrosines and tight association with v-Crk, suggesting a direct signal mediator of v-Crk. Here we report the molecular cloning of rat P130 by immunoaffinity purification. The P130 is a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs of v-Crk. Immunochemical analyses revealed that P130 is highly phosphorylated at tyrosines during transformation by p60v-src (v-Src), as well as by v-Crk, forming stable complexes with these oncoProteins. The P130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v-Src-associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic P130 could move to the membrane upon tyrosine phosphorylation. The P130 (designated Cas for Crk-associated substrate) is a common cellular target of phosphorylation signal via v-Crk and v-Src oncoProteins, and its unique structure indicates the possible role of P130Cas in assembling signals from multiple SH2-containing molecules.
Hiroshi Takeuchi - One of the best experts on this subject based on the ideXlab platform.
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Role of the PLC‐related, catalytically inactive Protein P130 in GABAA receptor function
The EMBO journal, 2002Co-Authors: Takashi Kanematsu, Il Sung Jang, Taku Yamaguchi, Hiroyasu Nagahama, Kenji Yoshimura, Kiyoshi Hidaka, Miho Matsuda, Hiroshi Takeuchi, Yoshio Misumi, Keiko NakayamaAbstract:The Protein P130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding Protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. We show that P130 plays an important role in signaling by the type A receptor for γ-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABAA receptor-associated Protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a Protein that interacts with P130. Furthermore, P130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl– current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of P130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that P130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit.
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role of the plc related catalytically inactive Protein P130 in gabaa receptor function
The EMBO Journal, 2002Co-Authors: Takashi Kanematsu, Il Sung Jang, Taku Yamaguchi, Hiroyasu Nagahama, Kenji Yoshimura, Kiyoshi Hidaka, Miho Matsuda, Hiroshi Takeuchi, Yoshio Misumi, Keiko NakayamaAbstract:The Protein P130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding Protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. We show that P130 plays an important role in signaling by the type A receptor for γ-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABAA receptor-associated Protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a Protein that interacts with P130. Furthermore, P130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl– current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of P130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that P130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit.
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Interaction of P130 with, and Consequent Inhibition of, the Catalytic Subunit of Protein Phosphatase 1α
The Journal of biological chemistry, 2001Co-Authors: Kenji Yoshimura, Kiyoshi Hidaka, Hiroshi Takeuchi, Naoko Doira, Yushi Ito, Osamu Sato, Miho Terunuma, Kae Harada, Yasuo Ogawa, Takashi KanematsuAbstract:Abstract The Protein P130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding Protein with a domain organization similar to that of phospholipase C-δ1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode Proteins that interact with P130 has now led to the identification of the catalytic subunit of Protein phosphatase 1α (PP1cα) as a P130-binding Protein. The association between P130 and PP1cα was also confirmedin vitro by an overlay assay, a “pull-down” assay, and surface plasmon resonance analysis. The interaction of P130 with PP1cα resulted in inhibition of the catalytic activity of the latter in a P130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express P130 and of mouse brain extract with antibodies to P130 and to PP1cα also detected the presence of a complex of P130 and PP1cα. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1cα, was higher in COS-1 cells that stably express P130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca2+ signaling, P130 might also contribute to regulation of Protein dephosphorylation through its interaction with PP1cα.
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Hyperinsulinemia in PRIP-1 Gene Deleted Mice
Biomedical Research, 2001Co-Authors: Naoko Doira, Takashi Kanematsu, Miho Matsuda, Hiroshi Takeuchi, Keiko Nakayama, H. Nakano, Yushi Ito, Keiichi I. Nakayama, Masato HirataAbstract:The Protein P130, named from its molecular size, was originally identified as an inositol 1, 4,5-trisphosphate binding Protein similar to phospholipase C (PLC)-δ1, but lacking any PLC activity. It was recently renamed PLC-related catalytically inactive Protein-1 (PRIP-1). In the present study, PRIP-1 gene-targeted mice were analyzed for glycogen metabolism based on the previous finding that PRIP-1 interacts with Protein phosphatase-1 (26). Compared to the control mice, mutant mice exhibited lower phosphorylase activity and higher levels of glycogen in the liver, which appeared to be consistent with the inhibition of Protein phosphatase-1 by PRIP-1. These observation could also be attributed to the increased levels of plasma insulin. Hyperinsulinemia, observed even in the young mice, was progressive with aging, and was accompanied by the accumulation of fat tissues, increased body weight and decreased sensitivity to insulin in the mutant mice at the age of 12 months. These results suggest that PRIP-1 is a molecule involved in the control of plasma insulin.
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domain organization of P130 plc related catalytically inactive Protein and structural basis for the lack of enzyme activity
FEBS Journal, 2000Co-Authors: Takashi Kanematsu, Kenji Yoshimura, Kiyoshi Hidaka, Hiroshi Takeuchi, Matilda Katan, Masato HirataAbstract:The 130-kDa Protein (P130) was isolated as a novel inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]-binding Protein similar to phospholipase C-delta1 (PLC-delta1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of P130 and structural basis for lack of PLC activity, we subjected P130 to limited proteolysis and also constructed a number of chimeras with PLC-delta1. Trypsin treatment of P130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta1 and demonstrating that all domains of P130, including the unique region at the C-terminus, are stable, tightly folded structures. P130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that P130 has intrinsically no PLC activity. A number of chimeric molecules between P130 and PLC-delta1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two Proteins, as only some domains of P130 could replace the corresponding structures in PLC-delta1 to form a functional enzyme. These results suggest that P130 and the related Proteins could represent a new Protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.
