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Masato Hirata - One of the best experts on this subject based on the ideXlab platform.

  • inhibition of Ca2 signalling by p130 a Phospholipase C related CatalytiCally inaCtive protein CritiCal role of the p130 pleCkstrin homology domain
    Biochemical Journal, 2000
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Matilda Katan, Hugh Paterson, Masahiro Oike, Victoria Allen, Yushi Ito, Christophe Erneux, Masato Hirata
    Abstract:

    p130 was originally identified as an Ins(1,4,5)P(3)-binding protein similar to Phospholipase C-Delta but laCking any Phospholipase aCtivity. In the present study we have further analysed the interaCtions of p130 with inositol Compounds in vitro. To determine whiCh of the potential ligands interaCts with p130 in Cells, we performed an analysis of the Cellular loCalization of this protein, the isolation of a protein-ligand Complex from Cell lysates and studied the effeCts of p130 on Ins(1,4,5)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfeCted COS-1 Cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P(3) with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) Cells by immunopreCipitation, it was found to be assoCiated with Ins(1,4,5)P(3). LoCalization studies demonstrated the presenCe of the full-length p130 in the Cytoplasm of living Cells, not at the plasma membrane. In Cell-based assays, p130 had an inhibitory effeCt on Ca(2+) signalling. When fura-2-loaded COS-1(p130) Cells were stimulated with bradykinin, epidermal growth faCtor or ATP, it was found that the agonist-induCed inCrease in free Ca(2+) ConCentration, observed in Control Cells, was inhibited in COS-1(p130). This inhibition was not aCCompanied by the deCreased produCtion of Ins(1,4,5)P(3); the intaCt p130 pleCkstrin homology domain, known to be the ligand-binding site in vitro, was required for this effeCt in Cells. These results suggest that Ins(1,4,5)P(3) Could be the main p130 ligand in Cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.

  • 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, 1996
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hassan Bin Yaakob, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Masato Hirata
    Abstract:

    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.

  • 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, 1996
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Hassan Bin Yaakob, Masato Hirata
    Abstract:

    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.

  • A new inositol 1,4,5-trisphosphate binding protein similar to Phospholipase C-Delta 1.
    Biochemical Journal, 1996
    Co-Authors: Takashi Kanematsu, Yoshio Misumi, Yukio Ikehara, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, Sadaaki Iwanaga, Masato Hirata
    Abstract:

    We have reported that two inositol 1,4,5-trisphosphate binding proteins, with moleCular masses of 85 and 130 kDa, were purified from rat brain; the former protein was found to be the Delta 1-isoenzyme of Phospholipase C (PLC-Delta 1) and the latter was an unidentified novel protein [Kanematsu, Takeya, Watanabe, Ozaki, Yoshida, Koga, Iwanaga and Hirata (1992) J. Biol. Chem. 267, 6518-6525]. Here we desCribe the isolation of the full-length CDNA for the 130 kDa Ins(1,4,5)P3 binding protein, whiCh enCodes 1096 amino aCids. The prediCted sequenCe of the 130 kDa protein had 38.2% homology to that of PLC-Delta 1. Three known domains of PLC-Delta 1 (pleCkstrin homology and putative CatalytiC X and Y domains) were loCated at residues 110-222, 377-544 and 585-804 with 35.2%, 48.2% and 45.8% homologies respeCtively. However, the protein showed no PLC aCtivity to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol. The 130 kDa protein expressed by transfeCtion in COS-1 Cells bound Ins(1,4,5)P3 in the same way as the moleCule purified from brain. Thus the 130 kDa protein is a novel Ins(1,4,5)P3 binding protein homologous to PLC-Delta 1, but with no CatalytiC aCtivity. The funCtional signifiCanCe of the 130 kDa protein is disCussed.

  • D-myo-Inositol 1,4,5-Trisphosphate Binding Domain of Phospholipase C-δ1
    Biochemical and biophysical research communications, 1994
    Co-Authors: Masato Hirata, Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, K. Sakuma, Hitoshi Yagisawa
    Abstract:

    D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding domain of Phospholipase C-Delta 1 (PLC-Delta 1) was determined by examining binding aCtivity of the synthetiC peptide Corresponding to residues 30-43 of PLC-Delta 1. The peptide Coupled with Carrier proteins suCh as keyhole limpet hemoCyanin or bovine serum albumin bound Ins(1,4,5)P3, whereas a sCrambled peptide with the same amino aCids did not do so. PolyClonal antibody against the peptide was examined to determine whether it would Cause inhibition of the Ins(1,4,5)P3 binding to PLC-Delta 1. Fab fragment of antibody to the peptide did inhibit binding to PLC-Delta 1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-Delta 1 is responsible for the binding of Ins(1,4,5)P3.

Hitoshi Yagisawa - One of the best experts on this subject based on the ideXlab platform.

  • Phosphorylation of Phospholipase C‐δ1 Regulates its EnzymatiC ACtivity
    Journal of cellular biochemistry, 2009
    Co-Authors: Makoto Fujii, Myung Jong Kim, Sung Ho Ryu, Pann-ghill Suh, Hitoshi Yagisawa
    Abstract:

    Phosphorylation of Phospholipase C-Delta(1) (PLC-Delta(1)) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of baCterially expressed PLC-Delta(1) most potently. It was also demonstrated that PLC-Delta(1) direCtly bound PKC-alpha via its pleCkstrin homology (PH) domain. Using deletion mutants of PLC-Delta(1) and synthetiC peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC-Delta(1). In vitro phosphorylation of PLC-Delta(1) by PKC stimulated [(3)H]PtdIns(4,5)P(2) hydrolyzing aCtivity and [(3)H]Ins(1,4,5)P(3)-binding of the PLC-Delta(1). On the other hand, endogenous PLC-Delta(1) was Constitutively phosphorylated and phosphoamino aCid analysis revealed that major phosphorylation sites were threonine residues in quiesCent Cells. The phosphorylation level and the speCies of phosphoamino aCid were not Changed by various stimuli suCh as PMA, EGF, NGF, and forskolin. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass speCtrometry, we determined that Thr209 of PLC-Delta(1) is one of the Constitutively phosphorylated sites in quiesCent Cells. The PLC aCtivity was potentiated when Constitutively phosphorylated PLC-Delta(1) was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, Coexpression with PKC-alpha reduCed serine phosphorylation of PLC-Delta(1) deteCted by an anti-phosphoserine antibody and PLC-Delta(1)-dependent basal produCtion of inositol phosphates in NIH-3T3 Cells, suggesting PKC-alpha aCtivates phosphatase or inaCtivates another kinase involved in PLC-Delta(1) serine phosphorylation to modulate the PLC-Delta(1) aCtivity in vivo. Taken together, these results suggest that PLC-Delta(1) has multiple phosphorylation sites and phosphorylation status of PLC-Delta(1) regulates its aCtivity positively or negatively depends on the phosphorylation sites.

  • Coordinated intraCellular transloCation of phosphoinositide-speCifiC Phospholipase C-Delta with the Cell CyCle.
    Biochimica et biophysica acta, 2006
    Co-Authors: Hitoshi Yagisawa, Masashi Okada, Yoko Naito, Koh Sasaki, Masaki Yamaga, Makoto Fujii
    Abstract:

    The Delta family phosphoinositide (PI)-speCifiC Phospholipase C (PLC) are most fundamental forms of eukaryotiC PI-PLCs. Despite the presenCe of lipid targeting domains suCh as the PH domain and C2 domain, the isoforms are also found in the Cytoplasm and nuCleus as well as at the plasma membrane. The isoforms have sequenCes or regions that Can serve as a nuClear loCalization signal (NLS) and a nuClear export signal (NES). Their intraCellular loCalization differs from one isoform to another, presumably due to the differenCe in the transport equilibrium balanCed by the strength of the two signals of eaCh isoform. Even for a partiCular isoform, its intraCellular loCalization seems to vary during the Cell CyCle. As an example, PLCDelta(1), whiCh is generally found at the plasma membrane and in the Cytoplasm of quiesCent Cells, loCalizes to disCrete nuClear struCtures in the G(1)/S boundary of the Cell CyCle. This may be at least partly due to an inCrease in intraCellular Ca(2+), sinCe Ca(2+) faCilitates the formation of a nuClear transport Complex Comprised of PLCDelta(1) and importin beta1, a Carrier moleCule for the nuClear import. PLCDelta(1) as well as PLCDelta(4) may play a pivotal role in Controlling the initiation of DNA synthesis in S phase. Spatio-temporal Changes in the levels of PtdIns(4,5)P(2) seem to be another major determinant for the loCalization and regulation of the Delta isoforms. High nuClear PtdIns(4,5)P(2) levels are assoCiated with the G(1)/S phases. After entering M phase, PtdIns(4,5)P(2) synthesis at sites of Cell division oCCurs and PLCs seem to loCalize to the Cleavage furrow during Cytokinesis. Coordinated transloCation of PLCs with the Cell CyCle or with stress responses may result in Changes in intra-nuClear environments and loCal membrane arChiteCtures that modulate proliferation and differentiation. In this review, reCent findings regarding the moleCular maChineries and meChanisms of the nuCleoCytoplasmiC shuttling as well as roles in the Cell CyCle progression of the Delta isoforms of PLC will be disCussed.

  • 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, 1996
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hassan Bin Yaakob, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Masato Hirata
    Abstract:

    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.

  • 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, 1996
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Hassan Bin Yaakob, Masato Hirata
    Abstract:

    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.

  • D-myo-Inositol 1,4,5-Trisphosphate Binding Domain of Phospholipase C-δ1
    Biochemical and biophysical research communications, 1994
    Co-Authors: Masato Hirata, Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, K. Sakuma, Hitoshi Yagisawa
    Abstract:

    D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding domain of Phospholipase C-Delta 1 (PLC-Delta 1) was determined by examining binding aCtivity of the synthetiC peptide Corresponding to residues 30-43 of PLC-Delta 1. The peptide Coupled with Carrier proteins suCh as keyhole limpet hemoCyanin or bovine serum albumin bound Ins(1,4,5)P3, whereas a sCrambled peptide with the same amino aCids did not do so. PolyClonal antibody against the peptide was examined to determine whether it would Cause inhibition of the Ins(1,4,5)P3 binding to PLC-Delta 1. Fab fragment of antibody to the peptide did inhibit binding to PLC-Delta 1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-Delta 1 is responsible for the binding of Ins(1,4,5)P3.

Takashi Kanematsu - One of the best experts on this subject based on the ideXlab platform.

  • inhibition of Ca2 signalling by p130 a Phospholipase C related CatalytiCally inaCtive protein CritiCal role of the p130 pleCkstrin homology domain
    Biochemical Journal, 2000
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Matilda Katan, Hugh Paterson, Masahiro Oike, Victoria Allen, Yushi Ito, Christophe Erneux, Masato Hirata
    Abstract:

    p130 was originally identified as an Ins(1,4,5)P(3)-binding protein similar to Phospholipase C-Delta but laCking any Phospholipase aCtivity. In the present study we have further analysed the interaCtions of p130 with inositol Compounds in vitro. To determine whiCh of the potential ligands interaCts with p130 in Cells, we performed an analysis of the Cellular loCalization of this protein, the isolation of a protein-ligand Complex from Cell lysates and studied the effeCts of p130 on Ins(1,4,5)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfeCted COS-1 Cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P(3) with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) Cells by immunopreCipitation, it was found to be assoCiated with Ins(1,4,5)P(3). LoCalization studies demonstrated the presenCe of the full-length p130 in the Cytoplasm of living Cells, not at the plasma membrane. In Cell-based assays, p130 had an inhibitory effeCt on Ca(2+) signalling. When fura-2-loaded COS-1(p130) Cells were stimulated with bradykinin, epidermal growth faCtor or ATP, it was found that the agonist-induCed inCrease in free Ca(2+) ConCentration, observed in Control Cells, was inhibited in COS-1(p130). This inhibition was not aCCompanied by the deCreased produCtion of Ins(1,4,5)P(3); the intaCt p130 pleCkstrin homology domain, known to be the ligand-binding site in vitro, was required for this effeCt in Cells. These results suggest that Ins(1,4,5)P(3) Could be the main p130 ligand in Cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.

  • 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, 1996
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hassan Bin Yaakob, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Masato Hirata
    Abstract:

    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.

  • 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, 1996
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Hassan Bin Yaakob, Masato Hirata
    Abstract:

    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.

  • A new inositol 1,4,5-trisphosphate binding protein similar to Phospholipase C-Delta 1.
    Biochemical Journal, 1996
    Co-Authors: Takashi Kanematsu, Yoshio Misumi, Yukio Ikehara, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, Sadaaki Iwanaga, Masato Hirata
    Abstract:

    We have reported that two inositol 1,4,5-trisphosphate binding proteins, with moleCular masses of 85 and 130 kDa, were purified from rat brain; the former protein was found to be the Delta 1-isoenzyme of Phospholipase C (PLC-Delta 1) and the latter was an unidentified novel protein [Kanematsu, Takeya, Watanabe, Ozaki, Yoshida, Koga, Iwanaga and Hirata (1992) J. Biol. Chem. 267, 6518-6525]. Here we desCribe the isolation of the full-length CDNA for the 130 kDa Ins(1,4,5)P3 binding protein, whiCh enCodes 1096 amino aCids. The prediCted sequenCe of the 130 kDa protein had 38.2% homology to that of PLC-Delta 1. Three known domains of PLC-Delta 1 (pleCkstrin homology and putative CatalytiC X and Y domains) were loCated at residues 110-222, 377-544 and 585-804 with 35.2%, 48.2% and 45.8% homologies respeCtively. However, the protein showed no PLC aCtivity to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol. The 130 kDa protein expressed by transfeCtion in COS-1 Cells bound Ins(1,4,5)P3 in the same way as the moleCule purified from brain. Thus the 130 kDa protein is a novel Ins(1,4,5)P3 binding protein homologous to PLC-Delta 1, but with no CatalytiC aCtivity. The funCtional signifiCanCe of the 130 kDa protein is disCussed.

  • D-myo-Inositol 1,4,5-Trisphosphate Binding Domain of Phospholipase C-δ1
    Biochemical and biophysical research communications, 1994
    Co-Authors: Masato Hirata, Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, K. Sakuma, Hitoshi Yagisawa
    Abstract:

    D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding domain of Phospholipase C-Delta 1 (PLC-Delta 1) was determined by examining binding aCtivity of the synthetiC peptide Corresponding to residues 30-43 of PLC-Delta 1. The peptide Coupled with Carrier proteins suCh as keyhole limpet hemoCyanin or bovine serum albumin bound Ins(1,4,5)P3, whereas a sCrambled peptide with the same amino aCids did not do so. PolyClonal antibody against the peptide was examined to determine whether it would Cause inhibition of the Ins(1,4,5)P3 binding to PLC-Delta 1. Fab fragment of antibody to the peptide did inhibit binding to PLC-Delta 1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-Delta 1 is responsible for the binding of Ins(1,4,5)P3.

Yutaka Watanabe - One of the best experts on this subject based on the ideXlab platform.

  • 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, 1996
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hassan Bin Yaakob, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Masato Hirata
    Abstract:

    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.

  • 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, 1996
    Co-Authors: Hiroshi Takeuchi, Takashi Kanematsu, Yoshio Misumi, Hitoshi Yagisawa, Yukio Ikehara, Yutaka Watanabe, Zheng Tan, Stephen B Shears, Hassan Bin Yaakob, Masato Hirata
    Abstract:

    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.

  • A new inositol 1,4,5-trisphosphate binding protein similar to Phospholipase C-Delta 1.
    Biochemical Journal, 1996
    Co-Authors: Takashi Kanematsu, Yoshio Misumi, Yukio Ikehara, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, Sadaaki Iwanaga, Masato Hirata
    Abstract:

    We have reported that two inositol 1,4,5-trisphosphate binding proteins, with moleCular masses of 85 and 130 kDa, were purified from rat brain; the former protein was found to be the Delta 1-isoenzyme of Phospholipase C (PLC-Delta 1) and the latter was an unidentified novel protein [Kanematsu, Takeya, Watanabe, Ozaki, Yoshida, Koga, Iwanaga and Hirata (1992) J. Biol. Chem. 267, 6518-6525]. Here we desCribe the isolation of the full-length CDNA for the 130 kDa Ins(1,4,5)P3 binding protein, whiCh enCodes 1096 amino aCids. The prediCted sequenCe of the 130 kDa protein had 38.2% homology to that of PLC-Delta 1. Three known domains of PLC-Delta 1 (pleCkstrin homology and putative CatalytiC X and Y domains) were loCated at residues 110-222, 377-544 and 585-804 with 35.2%, 48.2% and 45.8% homologies respeCtively. However, the protein showed no PLC aCtivity to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol. The 130 kDa protein expressed by transfeCtion in COS-1 Cells bound Ins(1,4,5)P3 in the same way as the moleCule purified from brain. Thus the 130 kDa protein is a novel Ins(1,4,5)P3 binding protein homologous to PLC-Delta 1, but with no CatalytiC aCtivity. The funCtional signifiCanCe of the 130 kDa protein is disCussed.

  • D-myo-Inositol 1,4,5-Trisphosphate Binding Domain of Phospholipase C-δ1
    Biochemical and biophysical research communications, 1994
    Co-Authors: Masato Hirata, Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, K. Sakuma, Hitoshi Yagisawa
    Abstract:

    D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding domain of Phospholipase C-Delta 1 (PLC-Delta 1) was determined by examining binding aCtivity of the synthetiC peptide Corresponding to residues 30-43 of PLC-Delta 1. The peptide Coupled with Carrier proteins suCh as keyhole limpet hemoCyanin or bovine serum albumin bound Ins(1,4,5)P3, whereas a sCrambled peptide with the same amino aCids did not do so. PolyClonal antibody against the peptide was examined to determine whether it would Cause inhibition of the Ins(1,4,5)P3 binding to PLC-Delta 1. Fab fragment of antibody to the peptide did inhibit binding to PLC-Delta 1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-Delta 1 is responsible for the binding of Ins(1,4,5)P3.

  • Putative Inositol 1,4,5-Trisphosphate Binding Proteins in Rat Brain Cytosol*
    The Journal of biological chemistry, 1992
    Co-Authors: Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, Sadaaki Iwanaga, Hiroyuki Takeya, Masako Yoshida, Masato Hirata
    Abstract:

    In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the seCond Carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) BioChem. Biophys. Res. Commun. 168, 379-386). When the Cytosol fraCtion from the rat brain was applied to an Ins(1,4,5)P3 affinity Column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding aCtivity. The aCtive fraCtion was further fraCtionated with gel filtration Chromatography, and two proteins with an apparent moleCular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing aCtivities. Partial amino aCid sequenCes determined after proteolysis and reversed-phase Chromatography revealed that the protein with an apparent moleCular mass of 85 kDa is the Delta-isozyme of Phospholipase C and that of 130 kDa has no sequenCe the same as the Ins(1,4,5)P3-reCognizing proteins hitherto examined. Ins(1,4,5)P3 at ConCentrations greater than 1 miCroM strongly inhibited 85-kDa Phospholipase C Delta aCtivity, without Changing its dependenCe on the ConCentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at muCh the same ConCentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidenCe for the presenCe of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of whiCh is the Delta isozyme of Phospholipase C.

Shoichiro Ozaki - One of the best experts on this subject based on the ideXlab platform.

  • A new inositol 1,4,5-trisphosphate binding protein similar to Phospholipase C-Delta 1.
    Biochemical Journal, 1996
    Co-Authors: Takashi Kanematsu, Yoshio Misumi, Yukio Ikehara, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, Sadaaki Iwanaga, Masato Hirata
    Abstract:

    We have reported that two inositol 1,4,5-trisphosphate binding proteins, with moleCular masses of 85 and 130 kDa, were purified from rat brain; the former protein was found to be the Delta 1-isoenzyme of Phospholipase C (PLC-Delta 1) and the latter was an unidentified novel protein [Kanematsu, Takeya, Watanabe, Ozaki, Yoshida, Koga, Iwanaga and Hirata (1992) J. Biol. Chem. 267, 6518-6525]. Here we desCribe the isolation of the full-length CDNA for the 130 kDa Ins(1,4,5)P3 binding protein, whiCh enCodes 1096 amino aCids. The prediCted sequenCe of the 130 kDa protein had 38.2% homology to that of PLC-Delta 1. Three known domains of PLC-Delta 1 (pleCkstrin homology and putative CatalytiC X and Y domains) were loCated at residues 110-222, 377-544 and 585-804 with 35.2%, 48.2% and 45.8% homologies respeCtively. However, the protein showed no PLC aCtivity to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol. The 130 kDa protein expressed by transfeCtion in COS-1 Cells bound Ins(1,4,5)P3 in the same way as the moleCule purified from brain. Thus the 130 kDa protein is a novel Ins(1,4,5)P3 binding protein homologous to PLC-Delta 1, but with no CatalytiC aCtivity. The funCtional signifiCanCe of the 130 kDa protein is disCussed.

  • D-myo-Inositol 1,4,5-Trisphosphate Binding Domain of Phospholipase C-δ1
    Biochemical and biophysical research communications, 1994
    Co-Authors: Masato Hirata, Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, K. Sakuma, Hitoshi Yagisawa
    Abstract:

    D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding domain of Phospholipase C-Delta 1 (PLC-Delta 1) was determined by examining binding aCtivity of the synthetiC peptide Corresponding to residues 30-43 of PLC-Delta 1. The peptide Coupled with Carrier proteins suCh as keyhole limpet hemoCyanin or bovine serum albumin bound Ins(1,4,5)P3, whereas a sCrambled peptide with the same amino aCids did not do so. PolyClonal antibody against the peptide was examined to determine whether it would Cause inhibition of the Ins(1,4,5)P3 binding to PLC-Delta 1. Fab fragment of antibody to the peptide did inhibit binding to PLC-Delta 1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-Delta 1 is responsible for the binding of Ins(1,4,5)P3.

  • Expression and CharaCterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-speCifiC Phospholipase C-Delta 1.
    The Journal of biological chemistry, 1994
    Co-Authors: Hitoshi Yagisawa, Takashi Kanematsu, Masato Hirata, Shoichiro Ozaki, Yoshimasa Watanabe, K. Sakuma, H Tanaka, N Yabuta, Hideaki Kamata, Hajime Hirata
    Abstract:

    It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the Delta 1 isoform of phosphatidylinositol-speCifiC Phospholipase C (PLC). We expressed rat PLC-Delta 1 in EsCheriChia Coli as a fusion protein with glutathione S-transferase, and found that the baCterial lysate shows a signifiCant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(1,4,5)P3-immobilized Column Chromatography and the eluate with 2 M NaCl solution Containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding. The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system. BaCterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding CharaCteristiCs as the brain-derived enzyme. PLC-Delta 1 Consists of the amino-terminal nonConserved region and two well-Conserved regions among isozymes, designated as X and Y, whiCh are thought to Constitute a CatalytiC Core of the enzyme. Using a Combination of deletion mutants and proteolytiC produCts of the enzyme, we were able to loCate an Ins(1,4,5)P3 binding domain in the moleCule. Deletion of 223 residues from the amino terminus Completely abolished the binding aCtivity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affeCt the binding. A 76-kDa proteolytiC produCt of the expressed PLC-Delta 1 whiCh laCked 60 amino aCids at the amino terminus showed a minimal Ins(1,4,5)P3 binding aCtivity. A peptide Consists of 14 amino aCids Corresponding to residues 30-43 of PLC-Delta 1, whiCh Contains 6 basiC amino aCids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was bloCked by phospholipid vesiCles Containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indiCate that the amino-terminal domain of PLC-Delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.

  • Putative Inositol 1,4,5-Trisphosphate Binding Proteins in Rat Brain Cytosol*
    The Journal of biological chemistry, 1992
    Co-Authors: Takashi Kanematsu, Yutaka Watanabe, Shoichiro Ozaki, Toshitaka Koga, Sadaaki Iwanaga, Hiroyuki Takeya, Masako Yoshida, Masato Hirata
    Abstract:

    In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the seCond Carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) BioChem. Biophys. Res. Commun. 168, 379-386). When the Cytosol fraCtion from the rat brain was applied to an Ins(1,4,5)P3 affinity Column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding aCtivity. The aCtive fraCtion was further fraCtionated with gel filtration Chromatography, and two proteins with an apparent moleCular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing aCtivities. Partial amino aCid sequenCes determined after proteolysis and reversed-phase Chromatography revealed that the protein with an apparent moleCular mass of 85 kDa is the Delta-isozyme of Phospholipase C and that of 130 kDa has no sequenCe the same as the Ins(1,4,5)P3-reCognizing proteins hitherto examined. Ins(1,4,5)P3 at ConCentrations greater than 1 miCroM strongly inhibited 85-kDa Phospholipase C Delta aCtivity, without Changing its dependenCe on the ConCentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at muCh the same ConCentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidenCe for the presenCe of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of whiCh is the Delta isozyme of Phospholipase C.