Yuko Murakami

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.

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.

Nek11, a new member of the NIMA family of kinases, involved in DNA replication and genotoxic stress responses

DNA replication and genotoxic stresses activate various checkpoint-associated protein kinases, and checkpoint dysfunction often leads to cell lethality. Here, we have identified new members of the mammalian NIMA family of kinases, termed Nek11L and Nek11S (NIMA-related kinase11 Long and Short isoform) as novel DNA replication/damage stresses-responsive kinases. Molecular cloning and biochemical studies showed that the catalytic domain of Nek11 is most similar to Nek4 and Nek3, and substrate specificity of Nek11L is distinguishable from those of NIMA and Nek2. The expression ofnek11L mRNA increased through S to G2/M phase, and subcellular localization of Nek11 protein altered between interphase and prometaphase, suggesting multiple roles of Nek11. We found an activation of Nek11 kinase activity when cells were treated with various DNA-damaging agents and replication inhibitors, and this activation of Nek11 was suppressed by caffeine in HeLaS3 cells. The transient expression of wild-type Nek11L enhanced the aphidicolin-induced S-phase arrest, whereas the aphidicolin-induced S-phase arrest was reduced in the U2OS cell lines expressing kinase-negative Nek11L (K61R), and these cells were more sensitive to aphidicolin-induced cell lethality. Collectively, these results suggest that Nek11 has a role in the S-phase checkpoint downstream of the caffeine-sensitive pathway.

Ets-1-dependent expression of vascular endothelial growth factor receptors is activated by latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus through interaction with Daxx

Vascular endothelial growth factor (VEGF) and its receptors are highly expressed in Kaposis sarcoma (KS) lesion and play a key role in angiogenesis. Latency-associated nuclear antigen (LANA) of Kaposis sarcoma-associated herpesvirus (KSHV/HHV8) has multiple functions related to viral latency and KSHV-induced oncogenesis. In this report, we have identified Daxx as a LANA-binding protein by co-immunoprecipitation analysis of HeLa cells stably expressing LANA. LANA associated with Daxx in a PEL cell line infected with KSHV. LANA and Daxx also bound in vitro, suggesting direct interaction. From the results of binding assays, a region containing the Glu/Asp-rich domain within LANA, and a central region including the second paired amphipathic helix within Daxx contributed to the interaction. To address the physiological significance of this interaction, we focused on a Daxx-mediated VEGF receptor gene regulation. We found that Daxx repressed Ets-1-dependent Flt-1/VEGF receptor-1 gene expression, and that LANA inhibited the repression by Daxx in a reporter assay. Analyses of flow cytometry and real-time PCR revealed that expression of VEGF receptor-1 and -2 in LANA-expressing human umbilical vein endothelial cells (HUVECs) significantly increased. Co-immunoprecipitation and immunoblotting experiments suggested that LANA-bound Daxx to inhibit the interaction between Daxx and Ets-1. Chromatin immunoprecipitation assays showed that Daxx associated with VEGF receptor-1 promoter in HUVECs, and that LANA expression reduced this association. These results suggested that LANA contributes to a high expression of VEGF receptors in KS lesion by interfering with the interaction between Daxx and Ets-1.

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.

Anti-Candida-biofilm activity of micafungin is attenuated by voriconazole but restored by pharmacological inhibition of Hsp90-related stress responses

We have conducted an in vitro evaluation of the efficacy of a voriconazole-micafungin combination against Candida albicans. When used alone, both micafungin and voriconazole decreased the metabolic activity of planktonic cells, but only micafungin displayed potent anti-biofilm activity. Their combination appeared to have an additive effect against planktonic cells. However, voriconazole significantly antagonized the fungicidal effect of micafungin against Candida biofilms. Time-lag experiments showed that pre-treatment with voriconazole induced resistance to micafungin in Candida biofilms. The micafungin-antagonizing effect of voriconazole persisted even when the biofilm was no longer exposed to voriconazole. In contrast, voriconazole addition after 24 h of micafungin treatment did not alter micafungin sensitivity. To investigate the mechanism of antagonism, we used inhibitors of Hsp90 and its effectors because Hsp90 seems to be implicated in the resistance to micafungin. These molecules reversed the voriconazole-induced resistance to micafungin which suggests that Hsp90-related stress responses are involved in the antagonism. Our results may provide clues as to the mechanism of increased drug resistance in Candida biofilms and raises concerns about the use of the voriconazole-micafungin combination in clinical settings.

A mammalian two-hybrid screening system for inhibitors of interaction between HIV Nef and the cellular tyrosine kinase Hck.

In the scope of the search for new anti-HIV agents interacting with a new target, we developed a high-throughput screening system to detect the interactions between Nef and Hck. Nef is an accessory protein of HIV, which is involved in the pathogenicity of the acquired immunodeficiency syndrome (AIDS). Nef is also a signaling molecule because it binds to many host molecules. It has especially high affinity to Hck, a member of src family tyrosine kinase. Using a mammalian two-hybrid system, the interaction between Nef and the SH3 domain of Hck induced luciferase activity with high sensitivity and a Nef-PXXP peptide inhibited this interaction; and so did the anticancer drug adriamycin. We also developed another assay system by coexpression of full-length Hck and Nef, and found that Hck kinase was activated depending on the dose of Nef plasmid. Using the second system, we found that adriamycin interfered with the Nef-Hck interaction by reducing the amount of the Hck protein. The mammalian two-hybrid system may show utility in screening inhibitors of Nef-Hck interaction.

γ-Herpesviruses and cellular signaling in AIDS-associated malignancies

γ‐Herpesviruses, Epstein–Barr virus (EBV/HHV‐4) and Kaposis sarcoma‐associated herpesvirus (KSHV/HHV‐8), are involved in human carcinogenesis, particularly in immunocompromised patients. Virus‐associated malignancies are becoming of significant concern for the mortality of long‐lived immunocompromised patients, and therefore, research of advanced strategies for AIDS‐related malignancies is an important field in cancer chemotherapy. Detailed understanding of the EBV and KSHV lifecycle and related cancers at the molecular level is required for novel strategies of molecular‐targeted cancer chemotherapy. The present review gives a simple outline of the functional interactions between KSHV‐ and EBV‐viral gene products and host cell deregulated signaling pathways as possible targets of chemotherapy against AIDS‐related malignancies. (Cancer Sci 2007; 98: 1288–1296)