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Potent and specific inhibitors of protein kinase C have been found in streptomyces and fungi: Staurosporine, an alkaloid from Streptomyces sp., is the most potent inhibitor of protein kinases with an IC50 in the nanomolar range. UCN-01 (7-hydroxy staurosporine), isolated from Streptomyces sp., is a selective inhibitor of protein kinase C with antitumor activity. Calphostin, isolated from the fungus Cladosporium cladosporioides, specifically inhibits protein kinase C (IC50 = 0.05 microM) without inhibiting other protein kinases. Microbial metabolites appear to be a promising source of inhibitors that target signal transduction pathways of eukaryotes.

A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the Sa. cerevisiae RAS2 protein. The NH2-terminal 657 amino acids were found to be dispensable for the activaition. However, almost all 2-amino acid insertions in the middle 600 residues comprising leucine-rich repeats and deletions in the COOHterminal 66 residues completely abolished activation by the RAS2 protein, whereas insertion mutations in the other regions generally had no effect. Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the Sa. cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein. We found that the fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the Sa. cerevisiae cyclase rendered the catalytic domain of the Sc. pombe cyclase, which otherwise did not respond to RAS proteins, activatable by the RAS2 protein. Thus the leucine-rich repeats and the COOH terminus of the Sa. cerevisiae adenylate cyclase appear to be required for interaction with RAS proteins.The ras oncogenes were identified as the transforming genes of retroviruses and the physiological function of ras proteins still remains to be elucidated (for review, see ref.

These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

A Schizosaccharomyces pombe gene encoding adenylate cyclase has been cloned by cross-hybridization with the Saccharomyces cerevisiae adenylate cyclase gene. (3)(4)(5). Although the precise mode of interaction between RAS proteins and yeast adenylate cyclase is unknown, regulatory proteins of adenylate cyclase appear to have somehow switched during the course of evolution. Specifically, it was shown that the cAMP concentration of the fission yeast Schizosaccharomyces pombe cells was not affected by the disruption or mutational activation of its sole ras gene, suggesting that its adenylate cyclase is not regulated by ras proteins (6). Sa. cerevisiae adenylate cyclase comprises 2026 amino acids in which the COOH-terminal 417 residues, called the catalytic domain, retains a Mn2+_ dependent cyclase activity (7). The remaining portion of the NH2 terminus was proposed to be essential for the RAS protein-and GTP-dependent activation in the presence of Mg2+ (7)(8)(9). In this report, we describe the primary structure of Sc. pombe adenylate cyclase.* Sequence comparison with Sa. cerevisiae adenylate cyclase reveals four segments displaying varying extents of homology. The functional significance of these segments is discussed. MATERIALS AND METHODSCell Strains, Growth Media, and Transformation. A Sa. cerevisiae strain, T50-3A (MATa, leu2, his3, trpl, ura3, cyrl-2) was described (7). A Sc. pombe strain 972h-s was obtained from D. Beach, Cold Spring Harbor Laboratory. Culture media for yeast cells and method of yeast transformation were as described (2-4, 10).DNA and RNA Preparation and Hybridization Analyses.