Yoshimasa Uehara

Irreversible inhibition of v-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds.

Herbimycin A, an antibiotic which reverses Rous sarcoma virus transformation, inhibited irreversibly the auto- and trans-phosphorylation activities of p60v-src in in vitro immune complex kinase assays. The addition of a sulfhydryl compound such as dithiothreitol, 2-mercaptoethanol, glutathione (reduced form) or cysteine abolished the ability of herbimycin A to inactivate p60v-src kinase as well as the ability to reverse transformed cell morphology, whereas the addition of oxidized glutathione, cystine or methionine showed no effect. The sulfhydryl alkylating reagent N-ethylmaleimide also, although less effectively, inactivated p60v-src kinase activity in vitro. These results suggest the likelihood that sulfhydryl groups of p60v-src are involved in the inactivation of v-src tyrosine kinase activity by herbimycin A.

Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway

While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenic transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When rat normal fibroblasts were cultured in suspension without substrate attachment, the cell cycle arrested in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulated. Conditional expression of oncogenic Ras induced the G1-S transition of the cell cycle and significantly shortened the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevented the oncogenic Ras-induced degradation of p27Kip1. These results suggest that the loss of substrate attachment induces the cell cycle arrest through the up-regulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the MAP kinase signaling pathway. Furthermore, we have found that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2.

Use and selectivity of herbimycin A as inhibitor of protein-tyrosine kinases.

Publisher Summary This chapter discusses the use and selectivity of herbimycin A as inhibitor of protein-tyrosine kinases. Protein-tyrosine phosphorylation is one of the basic mechanisms of signal transduction for cell growth and differentiation. Specific inhibitors of protein tyrosine kinases, therefore, may provide useful means for examining the role of tyrosine phosphorylation in a variety of cellular events. Herbimycin A induces inactivation of v-src tyrosine kinase and reduces cellular phosphotyrosine content in RSV-transformed cells. Because of restricted water solubility of herbimycin A, the preparation of concentrated stock solutions can best be done in an organic solvent such as dimethyl sulfoxide (DMSO) or methanol. The concentration required to reverse morphology varies among cells. The beginning of morphological changes can usually be observed within several hours, and is preceded by a reduction in tyrosine kinase activity.

Inhibition of transforming activity of tyrosine kinase oncogenes by herbimycin A

We studied the effectiveness of herbimycin A, an inhibitor of the function of the temperature-sensitive src oncogene, to reverse the morphologies of chicken and mammalian cells transformed by various oncogenes. It was found that the antibiotic was effective against the cells transformed by tyrosine kinase oncogenes src, yes, fps, ros, abl, erbB, but did not reverse the transformed morphologies induced by oncogenes raf, ras, and myc. Moreover, decreases in phosphotyrosine content of the total cellular proteins and in 36K protein phosphorylation by herbimycin treatment supported the selective inhibition of the antibiotic on the transforming activity of tyrosine kinase oncogenes.

Labeling of v-Src and BCR-ABL tyrosine kinases with [14C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism

The ansamycin antibiotic, herbimycin A, selectively inactivates cytoplasmic tyrosine kinases, most likely by binding irreversibly to the reactive SH group(s) of kinases. To further investigate the mechanism of herbimycin A action, we attempted to label tyrosine kinases with [14C]herbimycin A. p60 v‐src and p2 10 BCR‐ABL in immune complexes were labeled with [14C]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [14C]herbimycin A‐labeled p60 v‐src with Staphylococcus taureus V8 protease revealed that the herbimycin A binding site is within the C‐terminal 26‐kDa fragment of p60 v‐src , which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60 v‐src by [14]C]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A‐modified p60 v‐src cannot interact with ATP. The results suggest that herbimycin A inactivates tyrosine kinases by binding directly to the kinase domain, thereby inhibiting access to ATP.

Induction of Hsp 72/73 by herbimycin A, an inhibitor of transformation by tyrosine kinase oncogenes

Herbimycin A, which has been known to inactivate and degrade p60v-src tyrosine kinase, induced an elevated synthesis of a protein with a molecular size of 70 kDa in A431 human epidermoid carcinoma cells. This protein showed the same migration distance on SDS-polyacrylamide gel electrophoresis as that of the protein induced in the cells by heat shock treatment, and this 70-kDa protein was identified as a member of the heat shock protein 70 family (hsp70) through immunoprecipitation with anti-hsp72/73 antibody and partial digestion with V8 protease. The induced level of the 70-kDa protein was dependent on the length of period and the concentration of herbimycin A treatment. Cellular fractionation and indirect immunofluorescence analyses revealed that the 70-kDa protein induced by herbimycin A was localized in the cytoplasm, in contrast to the nuclear distribution of hsp70 induced by heat treatment. Induction of hsp70 by herbimycin A was also observed in several other cells, including HeLa S3 cells, chicken embryo fibroblasts, NIH3T3 cells, and Rous sarcoma virus-transformed NIH3T3 cells.

Small molecules inhibiting the nuclear localization of YAP/TAZ for chemotherapeutics and chemosensitizers against breast cancers

YAP and TAZ oncoproteins confer malignancy and drug resistance to various cancer types. We screened for small molecules that inhibit the nuclear localization of YAP/TAZ. Dasatinib, statins and pazopanib inhibited the nuclear localization and target gene expression of YAP and TAZ. All three drugs induced phosphorylation of YAP and TAZ, and pazopanib induced proteasomal degradation of YAP/TAZ. The sensitivities to these drugs are correlated with dependence on YAP/TAZ in breast cancer cell lines. Combinations of these compounds with each other or with other anti‐cancer drugs efficiently reduced cell proliferation of YAP/TAZ‐dependent breast cancer cells. These results suggest that these drugs can be therapeutics and chemosensitizers for YAP/TAZ‐dependent breast cancers.

Effects of herbimycin A and various SH-reagents on p60v-src kinase activity invitro

Herbimycin A is an antiobiotic which reverses transformation caused by src family oncogenes. It inactivates p60v-src in vitro, possibly by binding to reactive SH-group(s) of the kinase. We examined effects of various SH-reagents on p60v-src and observed that N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) or N-(9-acridinyl)maleimide (NAM) were potent inactivators of the kinase, whereas N-ethylmaleimide (NEM) required high concentrations, and iodoacetamide was totally ineffective in reducing the kinase activity. Pretreatment of p60v-src immune-complex with NEM and iodoacetamide, however, protected the kinase from inactivation by herbimycin A, BIPM, and NAM. The results suggest that SH-group(s) to which herbimycin A binds is not essential for the kinase activity, but is positioned in the vicinity of the active center.

Novel Approach to Designing Primers for Identification and Distinction of the Human Pathogenic Fungi Coccidioides immitis and Coccidioides posadasii by PCR Amplification

ABSTRACT We developed a pair of primers that specifically identifies Coccidioides species, etiologic agents of the human fungal disease coccidioidomycosis. These primers could be used for distinguishing Coccidioides immitis and Coccidioides posadasii by simply comparing the amplicon sizes on an agarose gel.

Deletion of the CaBIG1 Gene Reduces β-1,6-Glucan Synthesis, Filamentation, Adhesion, and Virulence in Candida albicans

ABSTRACT The human fungal pathogen Candida albicans is able to change its shape in response to various environmental signals. We analyzed the C. albicans BIG1 homolog, which might be involved in β-1,6-glucan biosynthesis in Saccharomyces cerevisiae. C. albicans BIG1 is a functional homolog of an S. cerevisiae BIG1 gene, because the slow growth of an S. cerevisiae big1 mutant was restored by introduction of C. albicans BIG1. CaBig1p was expressed constitutively in both the yeast and hyphal forms. A specific localization of CaBig1p at the endoplasmic reticulum or plasma membrane similar to the subcellular localization of S. cerevisiae Big1p was observed in yeast form. The content of β-1,6-glucan in the cell wall was decreased in the Cabig1Δ strain in comparison with the wild-type or reconstituted strain. The C. albicans BIG1 disruptant showed reduced filamentation on a solid agar medium and in a liquid medium. The Cabig1Δ mutant showed markedly attenuated virulence in a mouse model of systemic candidiasis. Adherence to human epithelial HeLa cells and fungal burden in kidneys of infected mice were reduced in the Cabig1Δ mutant. Deletion of CaBIG1 abolished hyphal growth and invasiveness in the kidneys of infected mice. Our results indicate that adhesion failure and morphological abnormality contribute to the attenuated virulence of the Cabig1Δ mutant.