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Synthesis and characterization of the regulatory domains of protein kinase C

Research Project

Project/Area Number 11660109
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Bioproduction chemistry/Bioorganic chemistry
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

IRIE Kazuhiro  Kyoto University, Gradute School of Agriculture, Division of Applied Life Sciences, Associate Professor, 農学研究科, 助教授 (00168535)

Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥2,900,000 (Direct Cost: ¥2,900,000)
Keywordscysteine-rich domain / phorbol / protein kinase C / tumor promoter / zinc finger / エレクトロスプレー質量分析法 / HATU
Research Abstract

Protein kinase C (PKC) isozymes are major receptors of tumor-promoting phorbol esters. Conventional and novel PKC isozymes (α, β, γ, δ, ε, η, θ) contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12,13-dibutyrate (PDBu) binding sites. To determine the phorbol ester binding sites of these isozymes, the C1 domains consisting of about 50 amino acids of all PKC isozymes have been synthesized by a solid phase Fmoc strategy. All C1B peptides except for α-C1B were successfully folded by zinc treatment as monitored by CD and ESI-MS spectroscopy, and showed potent PDBu binding affinities with the dissociation constants (K_d) of nanomolar range (0.45-1.5 nM), comparable to those of the native PKC isozymes. However, the K_d values of PDBu for many of the C1A peptides could not be determined. We found that some of the C1A peptides experience the temperature dependent inactivation and that elongation of the 50-mer C1 peptides at both N-and C-termini increases their folding efficiency. These findings enabled us to determine the K_d's of PDBu for all PKC C1 peptides except for θ-C1A.The major PDBu binding sites of novel PKC isozymes (δ, ε, η, θ) were C1B domains with K_d values of 0.45-0.81 nM.In contrast, all C1A peptides of conventional PKC isozymes (α, β, γ) exhibited nanomolar K_d values (0.97-1.3 nM). It is noteworthy that both C1 peptides of PKCβ and PKCγ showed strong PDBu binding affinity of nanomolar range. The above results provide a structural blueprint for the rational design of PKCγ-selective modulators for the treatment of neuropathic pain. To extend this approach, the 116-mer peptide containing the double cysteine-rich motifs of PKCγ (γ-C1A-C1B) has been synthesized for the first time.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Kazuhiro Irie: "Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein kinase D"Bioorg.Med.Chem.Lett.. 9. 2487-2490 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kazuhiro Irie: "Synthesis and phorbol ester bindings of the zinc-finger like sequences of all protein kinase C isozymes"Peptide Science 1998. 65-68 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hiroyuki Fukuda: "Solid-phase synthesis, mass spectrometric analysis of the zinc-folding, and phorbol ester-binding studies of the 116-mer peptide containing the tandem cysteine-rich C1 domains of PKC gamma"Bioorg.Med.Chem.. 7. 1213-1221 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Paul A.Wender: "Selective binding of bryostatin analogues to the cysteine-rich domains of protein kinase C isozymes"Bioorg.Med.Chem.Lett.. 9. 1687-1690 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kazuhiro Irie: "Synthesis and tumor-promoting activities of 12-epi-phorbol-12,13-dibutyrate"Biosci.Biotechnol.Biochem.. 64. 2429-2436 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Minoru Tanaka: "The C4 hydroxyl group of phorbol esters is not necessary for protein kinase C binding"Bioorg.Med.Chem.Lett.. 11. 719-722 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kazuhiro Irie et al.: "Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein kinase D"Bioorg. Med. Chem. Lett.. 9(17). 2487-2490 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kazuhiro Irie et al.: "Synthesis and phorbol ester binding of the zinc-finger like sequences of all protein kinase C isozymes"Peptide Science 1998. 65-68 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hiroyuki Fukuda, Kazuhiro Irie et al.: "Solid-phase synthesis, mass spectrometric analysis of the zinc folding, and phorbol ester-binding studies of the 116-mer peptide containing the tandem cysteine-rich C1 domains of protein kinase C gamma"Bioorg. Med. Chem.. 7(6). 1213-1221 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Paul A.Wender, Kazuhiro Irie et al.: "Selective binding of bryostatin analogues to the cysteine rich domains of protein kinase C isozymes"Bioorg. Med. Chem. Lett.. 9(12). 1687-1690 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kazuhiro Irie et al.: "Synthesis and tumor-promoting activities of 12-epi-phorbol-12, 13-dibutyrate"Biosci. Biotechnol. Biochem.. 64(11). 2429-2436 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Minoru Tanaka, Kazuhiro Irie et al.: "The C4 hydroxyl group of phorbol esters is not necessary for protein kinase C binding"Bioorg. Med. Chem. Lett.. 11. 719-722 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kazuhiro Irie: "Synthesis and tumor-promoting activities of 12-epi-phorbol-12,13-dibutyrate"Biosci. Biotechnol. Biochem.. 64・11. 2429-2436 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Minoru Tanaka: "The C4 hydroxyl group of phorbol esters is not necessary for protein kinase C binding"Bioorg. Med. Chem. Lett.. 11・5. 719-722 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kazuhiro lrie: "Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein kinase D"Bioorg.Med.Chem.Lett.. 9・17. 2487-2490 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kazuhiro lrie: "Synthesis and phorbol ester binding of the zinc-finger like sequences of all protein kinase C isozymes"Peptide Science 1998. 65-68 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Hiroyuki Fukuda: "Solid-phase synthesis,mass spectrometric analysis of the zinc-folding,and phorbol ester-binding studies of the 116-mer peptide containing the tandem cysteine-rich C1 domains of protein kinase C gamma"Bioorg.Med.Chem.. 7・6. 1213-1221 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Paul A.Wender: "Selective binding of bryostatin analogues to the cysteine rich domains of protein kinase C isozymes"Bioorg.Med.Chem.Lett.. 9・12. 1687-1690 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Paul A.Wender: "Mapping phorbol ester binding domains of protein kinase C (PKC) : the design,synthesis and biological activity of novel phorbol ester dimers"Synthesis. SI. 1401-1406 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Paul A.Wender: "A new class of simplified phorbol ester analogues: synthesis and binding to PKC and ηPKC-C1B (ηPKC-CRD2)"Org.Lett.. 1・7. 1009-1012 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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