Identification of Radicicol as an Inhibitor of In Vivo Ras/Raf Interaction with the Yeast Two-hybrid Screening System.

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

doi:10.7164/antibiotics.51.936

Cited by 16 publications

(10 citation statements)

References 29 publications

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“…1, C and D). Although these results confirmed our previously published observations (20,21), we now for the first time demonstrate that blocking hyperactive H-Ras with radicicol (56,57) or inhibiting downstream proliferative signaling with Mek/Erk inhibitor, PD98059 (58), as well as inhibition of cdk4 with PD0332991 (59) (Fig. 1E) all resulted in a significant up-regulation in the synthesis of tropoelastin (Fig.…”

Section: Resultssupporting

confidence: 78%

Retinoblastoma Protein Modulates the Inverse Relationship between Cellular Proliferation and Elastogenesis

Sen

Bunda

Shi

et al. 2011

Journal of Biological Chemistry

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The mechanism that leads to the inverse relationship between heightened cellular proliferation and the cessation of elastic fibers production, observed during formation of the arterial occlusions and dermal scars, is not fully understood. Because the retinoblastoma protein (Rb), responsible for cell cycle initiation, has also been implicated in insulin-like growth factor-Imediated signaling stimulating elastin gene activation, we explored whether differential phosphorylation of Rb by various cyclin⅐cyclin-dependent kinase complexes would be responsible for promoting either elastogenic or pro-proliferative signals. We first tested cultures of dermal fibroblasts derived from Costello syndrome patients, in which heightened proliferation driven by mutated oncogenic H-Ras coincides with inhibition of elastogenesis. We found that Costello syndrome fibroblasts display elevated level of Rb phosphorylation on serine 780 (Ser(P)-780-Rb) and that pharmacological inhibition of Ras with radicicol, Mek/Erk with PD98059, or cyclin-dependent kinase 4 with PD0332991 not only leads to down-regulation of Ser(P)-780-Rb levels but also enhances Rb phosphorylation on threonine-821 (Thr(P)-821-Rb), which coincides with the recovery of elastin production. Then we demonstrated that treatment of normal skin fibroblasts with the pro-proliferative PDGF BB also up-regulates Ser(P)-780-Rb levels, but treatment with the pro-elastogenic insulin-like growth factor-I activates cyclinE-cdk2 complex to phosphorylate Rb on Thr-821. Importantly, we have established that elevation of Thr(P)-821-Rb promotes Rb binding to the Sp1 transcription factor and that successive binding of the Rb-Sp1 complex to the retinoblastoma control element within the elastin gene promoter stimulates tropoelastin transcription. In summary, we provide novel insight into the role of Rb in mediating the inverse relationship between elastogenesis and cellular proliferation.Elastic fibers are composed of a microfibrillar scaffold made up of several glycoproteins and a polymeric elastin core. They are particularly numerous in the extracellular matrix of blood vessels lungs, heart, skin, ligaments, periosteum, and auricular cartilage (1-3) Mature elastic fibers, deposited almost exclusively during the late gestation and the early childhood, constitute the most durable element of the extracellular matrix that in undisturbed tissues may last over the entire human lifespan (4, 5). However, after local inflammation or mechanical injuries, the elastic fibers can be degraded (6 -9). Interestingly, the healing of wounded connective tissue frameworks and the remodeling of injured blood vessels or heart mostly engages the intense proliferation of local fibroblasts or smooth muscle cells and seldom concludes with the recovery of normal elastic fibers that are otherwise replaced with the abundant collagen fibers (6 -15). On the other hand, patients with genetic diseases caused by primary haploinsufficiency of the elastin gene (supravalvular aortic stenosis or Williams-Beuren syndrome) devel...

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Section: Resultssupporting

confidence: 78%

Retinoblastoma Protein Modulates the Inverse Relationship between Cellular Proliferation and Elastogenesis

Sen

Bunda

Shi

et al. 2011

Journal of Biological Chemistry

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“…The 1 H NMR data (Table 1) All isolates were evaluated for their inhibitory activities of Ras-Raf-1 interaction in the yeast two-hybrid assay (Table 2) according to a literature method. 3 In brief, we looked for a compound giving a clear zone of inhibition (ZOI) that inhibited growth of LZ cells in the SD His -plates and not in the SD His + plates, together with inhibition of b-galactosidase activity. This would indicate the compound is an inhibitor of Ras-Raf-1 interaction.…”

Section: Resultsmentioning

confidence: 99%

“…18 Cycloheximide (6) was isolated from the cultures of Streptomyces griseus and has been demonstrated to inhibit protein synthesis in mammalian systems. 3,19 AH-135Y (5) has been reported from Streptomyces albovinaceus is reported to have antiherpetic, cytotoxic and antifungal activities. 9 Many terrestrial yeast, lichens, and fungi are known to produce diketopiperazines by condensation of two amino acids.…”

Section: Resultsmentioning

confidence: 99%

“…3 To identify such inhibitors, we employed the yeast two-hybrid system in our screening. 3 The actinomycete family continues to be a rich reservoir of microorganisms that produce a diverse array of biologically active metabolites effective against a broad spectrum of health problems. For example, staurosporine from Streptomyces staurosporeus exhibited potent inhibition Three New Amides from Streptomyces sp.…”

Section: Introductionmentioning

confidence: 99%

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Three new amides from Streptomyces sp. H7372

Cheenpracha¹,

Borris²,

Tran³

et al. 2011

J. Braz. Chem. Soc.

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As amidas fenatato de metila (1), actifenamida (2) e 1-b-D-glucopiranosídeo-actinofenol (3), juntamente com outros 13 compostos de estruturas conhecidas, foram isolados do extrato de uma cultura de Streptomyces sp H7372. As estruturas destes compostos foram determinadas por análises de RMN uni e bidimensionais e por espectrometria de massas. Cicloheximida (6) Three new amides, methyl phenatate A (1), actiphenamide (2) and actiphenol 1-b-Dglucopyranoside (3), along with thirteen known compounds, were isolated from the organic extract of a fermentation culture of Streptomyces sp. H7372. The structures were elucidated by spectroscopic methods including 1D-and 2D-NMR techniques, and MS analyses. Cycloheximide (6) and cyclo(DAla-L-Val) (8) gave a clear zone of inhibition of Ras-Raf-1 interaction in the yeast twohybrid assay which showed high potency with 10 and 25 mm clear ZOIs on SD His -and inactive on SD His + at 2.5 mg per disk, respectively.

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“…Increased expression of gelsolin, an actin regulatory protein, has also been observed during the induction of morphological changes in various transformed cells (11). Recent studies showed that radicicol disrupted the Ras-activated signaling pathway by selectively depleting Raf kinase or reducing Ras/Raf molecular interaction (12,13). The target molecule of radicicol was proposed to be Hsp90, because it strongly binds Hsp90 in a manner competitive with ATP and geldanamycin, a known Hsp90 inhibitor (14,15).…”

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confidence: 99%

Radicicol Binds and Inhibits Mammalian ATP Citrate Lyase

Ki¹,

Ishigami²,

Kitahara³

et al. 2000

Journal of Biological Chemistry

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Six different biotinylated radicicol derivatives were synthesized as affinity probes for identification of cellular radicicol-binding proteins. Derivatives biotinylated at the C-17 (BR-1) and C-11 (BR-6) positions retained the activity of morphological reversion in v-src-transformed 3Y1 fibroblasts. Two radicicol-binding proteins, 120 and 90-kDa in size, were detected in HeLa cell extracts by employing BR-1 and BR-6, respectively. The 90-kDa protein bound to BR-6 was identified to be Hsp90 by immunoblotting. The 120-kDa protein bound to BR-1 was purified from rabbit reticulocyte lysate, and its internal amino acid sequence was identical to that of human and rat ATP citrate lyase. The identity of the 120-kDa protein as ATP citrate lyase was confirmed by immunoblotting. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. These results suggest that radicicol binds the two proteins through different molecular portions of its structure. BR-1-bound ATP citrate lyase isolated from rabbit reticulocyte lysate showed no enzymatic activity. The activity of rat liver ATP citrate lyase was inhibited by radicicol and BR-1 but not by BR-6. Kinetic analysis demonstrated that radicicol was a noncompetitive inhibitor of ATP citrate lyase with K i values for citrate and ATP of 13 and 7 M, respectively.Radicicol (also known as monorden), a 14-membered macrolide originally isolated from Monosporium bonorden as an antifungal antibiotic in 1953 (1), is a compound showing a variety of biological activities. It was reported again as a potent tranquilizer with low toxicity in 1964 (2). We rediscovered radicicol as a potent inducer of reversal of the transformed phenotype in v-src-transformed fibroblasts to the normal one (3, 4). We also showed that radicicol caused cell cycle arrest in G 1 and G 2 phases, and Oikawa et al. (5) demonstrated that it inhibited in vivo angiogenesis. Furthermore, radicicol was reported to induce morphological reversion of not only src but also ras, mos, raf, fos, and SV40-transformed cell lines and inhibited the expression of mitogen-inducible cyclooxygenase in macrophages (6 -8). Some of leukemia cell lines were differentiated in response to radicicol (4, 9). Recently, KF25706, a novel oxime derivative of radicicol, was reported to show potent antitumor activity and is currently under consideration as an anticancer drug (10). The in vivo inhibition of tyrosine kinases and MAP kinases has been suggested to be involved in these characteristic phenotypes elicited by radicicol (6, 7). Increased expression of gelsolin, an actin regulatory protein, has also been observed during the induction of morphological changes in various transformed cells (11). Recent studies showed that radicicol disrupted the Ras-activated signaling pathway by selectively depleting Raf kinase or reducing Ras/Raf molecular interaction (12, 13). The target molecule of radicicol was proposed to be Hsp90, because it strongly binds Hsp90 in a manner competitive with ATP and...

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Identification of Radicicol as an Inhibitor of In Vivo Ras/Raf Interaction with the Yeast Two-hybrid Screening System.

Cited by 16 publications

References 29 publications

Retinoblastoma Protein Modulates the Inverse Relationship between Cellular Proliferation and Elastogenesis

Retinoblastoma Protein Modulates the Inverse Relationship between Cellular Proliferation and Elastogenesis

Three new amides from Streptomyces sp. H7372

Radicicol Binds and Inhibits Mammalian ATP Citrate Lyase

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