Hidesuke Fukazawa

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.

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.

Bisebromoamide, a Potent Cytotoxic Peptide from the Marine Cyanobacterium Lyngbya sp.: Isolation, Stereostructure, and Biological Activity

A novel cytotoxic peptide, termed bisebromoamide (1), has been isolated from the marine cyanobacterium Lyngbya sp. Its planar structure was determined by 1D and 2D NMR spectroscopy. The absolute stereostructure of 1 was determined by chemical degradation followed by chiral HPLC analysis. Bisebromoamide (1) exhibited potent protein kinase inhibition: the phosphorylation of ERK in NRK cells by PDGF-stimulation was selectively inhibited by treatment with 10-0.1 microM of 1.

Highly Effective Recombinant Format of a Humanized IgG-like Bispecific Antibody for Cancer Immunotherapy with Retargeting of Lymphocytes to Tumor Cells

We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e. humanized anti-EGFR scFv and anti-CD3 scFv, that contains the same four variable domains as hEx3, on the platform of human IgG1 (hEx3-scFv-Fc). hEx3-scFv-Fc prepared from mammalian cells showed specific binding to both EGFR and CD3 target antigens. At one-thousandth (0.1–100 fmol/ml) of the dose of normal hEx3, hEx3-scFv-Fc showed intense cytotoxicity to an EGFR-positive cell line in a growth-inhibition assay using lymphokine-activated killer cells with the T-cell phenotype (T-LAK cells). The enhanced antitumor effect was more clearly observed when peripheral blood mononuclear cells (PBMCs) were used as effector cells, indicating the utility of IgG-like fabrication. These results suggested that the intense antitumor activity is attributable to the multivalency and the presence of the fused human Fc, a hypothesis that was supported by the results of flow cytometry, PBMC proliferation assay, and protein kinase inhibition assay. Furthermore, the growth inhibition effects of hEx3-scFv-Fc were considerably superior to those of the approved therapeutic antibody, cetuximab, which recognizes the same EGFR antigen even when using PBMCs as effector cells. The high potency of hEx3-scFv-Fc may translate into improved antitumor therapy and lower costs of production because of the smaller doses needed.

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.

Novel compounds, '1,3-selenazine derivatives' as specific inhibitors of eukaryotic elongation factor-2 kinase.

The inhibitory activities of 5,6-dihydro-4H-1,3-selenazine derivatives on protein kinases were investigated. In a multiple protein kinase assay using a postnuclear fraction of v-src-transformed NIH3T3 cells, 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-6-isopropyl-4-methyl-2-p-tolyl-5,6-dihydro-4H-1, 3-selenazine (TS-4) exhibited selective inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K) over protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). In further experiments using purified kinases, TS-2 (IC(50)=0.36 microM) and TS-4 (IC(50)=0.31 microM) inhibited eEF-2K about 25-fold more effectively than calmodulin-dependent protein kinase-I (CaMK-I), and about 6-fold (TS-2) or 33-fold (TS-4) more effectively than calmodulin-dependent protein kinase-II (CaMK-II), respectively. TS-2 and TS-4 showed much weaker inhibitory activity toward PKA and PKC, while TS-4, but not TS-2, moderately inhibited immunoprecipitated v-src kinase. TS-2 (10.7-fold) and TS-4 (12.5-fold) demonstrated more potent and more specific eEF-2K inhibitory activity than rottlerin, a previously identified eEF-2K inhibitor. TS-2 inhibited ATP or eEF-2 binding to eEF-2K in a competitive or non-competitive manner, respectively. In cultured v-src-transformed NIH3T3 cells, TS-2 also decreased phospho-eEF-2 protein level (IC(50)=4.7 microM) without changing the total eEF-2 protein level. Taken together, these results suggest that TS-2 and TS-4 are the first identified selective eEF-2K inhibitors and should be useful tools for studying the function of eEF-2K.

Regulation of SV40 large T-antigen stability by reversible acetylation

Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as α-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells.Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as α-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells.