Randall K. Johnson

Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with γ-radiation or with the topoisomerase-I inhibitor topotecan.

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Uptake and retention of adriamycin and daunorubicin by sensitive and anthracycline-resistant sublines of P388 leukemia

Abstract Sublines of P388 leukemia completely resistant to adriamycin (P388/ADR) or daunorubicin (P388/DAU) in vivo were studied in vitro . These sublines were more resistant to the cytotoxic effects of adriamycin (800-fold relative to sensitive parental cell line, P388/S) than to daunorubicin (18-fold for P388/ADR and 56-fold for P388/DAU). When the effects of the drugs on thymidine incorporation were compared in vitro in sensitive and resistant cells, it was observed that slightly higher levels of the drugs were required to inhibit nucleic acid synthesis in the resistant cells. The shift in inhibitory concentration was much less than the shift in cytotoxic concentration, particularly for adriamycin. The uptake and efflux of [G- 3 H]daunorubicin and [14- 14 C]adriamycin were studied. At low concentrations uptake of both drugs was impaired in the resistant sublines, whereas, at high concentrations a difference in uptake between sensitive and resistant cells was not evident. Resistance did not appear to be related to the difference in the rate of uptake. A markedly enhanced efflux of the drugs from the resistant cells was observed which correlated well with the difference in sensitivity of the sublines to adriamycin and daunorubicin. Enhancing the uptake of adriamycin by increasing the pH of the incubation medium and thereby increasing the proportion of non-ionized drug available for diffusion into the cells or by modifiying the cell membrane by the addition of Tween 80 failed to reverse resistance. The binding of daunorubicin to isolated nuclei from P388/S and P388/ADR cells was essentially similar. It is concluded that these anthracycline-resistant cell lines are resistant by virtue of decreased retention of the drugs.

Frondosins, five new sesquiterpene hydroquinone derivatives with novel skeletons from the sponge Dysidea frondosa: Inhibitors of interleukin-8 receptors

Abstract Bioassay-guided fractionation of the EtOAc extract of the sponge Dysidea frondosa collected in Pohnpei yielded five sesquiterpenes, frondosins A - E ( 1–5 ). The structures and relative stereochemistry of the frondosins were established by interpretation of spectral data. Frondosins A - E ( 1–5 ), which possess novel carbon skeletons, were found to be inhibitors of interleukin-8 receptors and protein kinase C in the low micromolar range.

The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface

BACKGROUND Rapamycin is an immunosuppressant natural product, which blocks T-cell mitogenesis and yeast proliferation. In the cytoplasm, rapamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP. The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain). We previously showed that the allylic methoxy group at C-7 of rapamycin could be replaced by a variety of different substituents. We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity. RESULTS Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays. The X-ray crystal structures of four rapamycin analogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 group remained exposed to solvent. We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent. This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP. CONCLUSIONS We conclude that the C-7 methoxy group of rapamycin is part of the effector domain. In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12.

Fasicularin, a novel tricyclic alkaloid from the ascidian Nephteis fasicularis with selective activity against a DNA repair-deficient organism

Abstract A novel tricyclic alkaloid, fasicularin (1), was isolated from the Micronesian ascidian Nephteis fasicularis. The structure of 1 was elucidated primarily by interpretation of spectral data. Fasicularin was found to be active in a DNA damaging assay.

The plakortones, novel bicyclic lactones from the sponge Plakortis halichondrioides: Activators of cardiac SR-Ca2+-pumping ATPase

Abstract Bioassay-guided fractionation of the EtOAc extract of sponge Plakortis halichondrioides yielded four novel bicyclic lactones, plakortones A, B, C, D and a novel acid, plakortide E ( 3, 4, 5, 6 and 7 ). The structures, including stereochemistry, of these compounds were established by interpretation of spectral data. Plakortones A-D ( 3–6 ) comprise a novel class of activators of cardiac SR-Ca 2+ -pumping ATPase which were found to be active at micromolar concentrations As part of an SAR study, the α and β diols 9 and 10 of plakortone D were prepared using Sharpless AD procedure.

PULCHERRIMINS A - D, NOVEL DITERPENE DIBENZOATES FROM CAESALPINIA PULCHERRIMA WITH SELECTIVE ACTIVITY AGAINST DNA REPAIR-DEFICIENT YEAST MUTANTS

Abstract Bioassay-guided fractionation of the MeOH/CH2Cl2 extract of the roots of Caesalpinia pulcherrima yielded four novel dibenzoate diterpenes, pulcherrimins A, B, C and D (1–4). The structures of these compounds including their absolute configuration were established by interpretation of spectral data and CD measurements. Pulcherrimins A and B (1 and 2) were found to be active in DNA repair-deficient yeast mutant. Bioassay-guided fractionation of the MeOH/CH2Cl2 extract of the roots of Caesalpinia pulcherrima yielded four novel dibenzoate tetracyclic diterpenes, pulcherrimins A, B, C and D (1–4_. The structures of these compounds including their absolute configuration were established by interpretation of spectral data and CD measurements. Pulcherrimins A and B (1 and 2)_ were found to be active in DNA repair-deficient yeast mutant.

Yeast TOR (DRR) proteins: amino-acid sequence alignment and identification of structural motifs

The yeast TOR1 (DRR1) and TOR2 (DRR2) proteins are putative targets of the immunosuppressive drug rapamycin (Rm), defined by dominant drug-resistance mutations. They share a large C-terminal domain that exhibits sequence similarity to the 110-kDa subunit of phosphatidylinositol (PI) 3-kinases. In this report, we present an amino acid (aa) sequence alignment of TOR1 (DRR1) and TOR2 (DRR2) and identify conserved and nonconserved motifs within the N-terminal domain that are indicative of possible nuclear localization. We also show that the mutations responsible for Rm resistance in four independent drr2dom alleles alter the identical aa (Ser1975-->Arg) previously identified in drr1dom mutants (Ser1972-->Arg or Asn). Models for TOR (DRR) protein function are discussed.