Identification of radicicol as an inhibitor of in vivo Ras/Raf interaction with the yeast two-hybrid screening system

Se Won Ki, Koji Kasahara, Ho Jeong Kwon, Jun Eishima, Kazutoh Takesako, Jonathan A. Cooper, Minoru Yoshida, Sueharu Horinouchi

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Activation of cytoplasmic serine/threonine kinase Raf-1, an important effector of Ras, requires direct binding to Ras. The yeast two-hybrid screening system used for identification of inhibitors of Ras/Raf-1 interaction showed radicicol to be an inhibitor. Radicicol has been shown to induce morphological reversion of transformed cells. Immunoprecipitation with an anti-Ras antibody revealed that the in vivo Ras/Raf-1 binding in v-Ha-ras-transformed cells was also blocked by low concentrations of radicicol (0.1 ~ 1 μg/ml), while degradation of Raf-1 was induced at concentrations higher than 2 μg/ml. However, in vitro binding of glutathion S-transferase-fused Ras to a maltose binding protein-fused RIP3 containing the Ras-binding domain (RBD) of Raf-1 was not inhibited by radicicol. Similar two-hybrid assays with several truncated forms of Raf-1 showed that both the conserved serine/threonine-rich domain (CR2) and the C-terminal protein kinase domain (CR3) were required for the full inhibition by radicicol. These results suggest that radicicol interacts directly or indirectly with the region except with RBD of Raf-1, thereby inhibiting a conformational change of Raf-1 prerequisite for binding to Ras.

Original languageEnglish
Pages (from-to)936-944
Number of pages9
JournalJournal of Antibiotics
Volume51
Issue number10
DOIs
Publication statusPublished - 1998 Jan 1

Fingerprint

Two-Hybrid System Techniques
Proto-Oncogene Proteins c-raf
Maltose-Binding Proteins
Protein-Serine-Threonine Kinases
Threonine
Glutathione Transferase
Immunoprecipitation
Serine
monorden
Anti-Idiotypic Antibodies

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Drug Discovery

Cite this

Ki, Se Won ; Kasahara, Koji ; Kwon, Ho Jeong ; Eishima, Jun ; Takesako, Kazutoh ; Cooper, Jonathan A. ; Yoshida, Minoru ; Horinouchi, Sueharu. / Identification of radicicol as an inhibitor of in vivo Ras/Raf interaction with the yeast two-hybrid screening system. In: Journal of Antibiotics. 1998 ; Vol. 51, No. 10. pp. 936-944.
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Identification of radicicol as an inhibitor of in vivo Ras/Raf interaction with the yeast two-hybrid screening system. / Ki, Se Won; Kasahara, Koji; Kwon, Ho Jeong; Eishima, Jun; Takesako, Kazutoh; Cooper, Jonathan A.; Yoshida, Minoru; Horinouchi, Sueharu.

In: Journal of Antibiotics, Vol. 51, No. 10, 01.01.1998, p. 936-944.

Research output: Contribution to journalArticle

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