Systematic identification of cellular signals reactivating Kaposi sarcoma-associated herpesvirus.

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication.

Yeast screens for inhibitors of Ras-Raf interaction and characterization of MCP inhibitors of Ras-Raf interaction.

Because of the central role of Ras in cancer cell signaling, there has been considerable interest in developing small molecule inhibitors of the Ras signaling pathways as potential chemotherapeutic agents. This chapter describes the use of a two-hybrid approach to identify the MCP compounds, small molecules that disrupt the interaction between Ras and its effector Raf. We first outline the reagent development and selection/counter selection methods required to successfully apply a two-hybrid approach to isolation of MCP compounds.

Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast.

Rheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine-17, serine-21, lysine-120 or asparagine-153.

Farnesyltransferase inhibitors reverse altered growth and distribution of actin filaments in Tsc-deficient cells via inhibition of both rapamycin-sensitive and -insensitive pathways.

Farnesyltransferase inhibitors (FTI) have been developed as anticancer drugs and are currently being evaluated in clinical trials. In this study, we have examined the effects of FTIs on Tsc-null cells to gain insight into their effects on farnesylated Rheb GTPase. This protein is involved in the activation of mTOR/S6K signaling and is down-regulated by the Tsc1/Tsc2 complex.

Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells.

The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (MCP1 and its derivatives, 53 and 110) reduced serum-induced transcriptional activation of serum response element as well as Ras-induced transcription by way of the AP-1 site.