Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.

Kssl protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to a-factor, Kssl was rapidly phosphorylated on both Thrl83 and Tyrl85, and both sites were required for Kssl function in vivo. De novo protein synthesis was required for sustained pheromoneinduced phosphorylation of Kssl. Catalytically inactive Kssl mutants displayed a-factor-induced phosphorylation on both residues, even in ksslA cells; hence, autophosphorylation is not obligatory for these modifications. In

The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broadspectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a ␤-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and ␤-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

These results form a rational basis for the design of clinical trials to evaluate the expression of these enzymes as predictors for treatment outcome.

This study examined the pathological complete response (pCR) rate and safety of sequential gemcitabine-based combinations in breast cancer. We also examined gene expression profiles from tumour biopsies to identify biomarkers predictive of response. Indian women with large or locally advanced breast cancer received 4 cycles of gemcitabine 1200 mg m À2 plus doxorubicin 60 mg m À2(Gem þ Dox), then 4 cycles of gemcitabine 1000 mg m À2 plus cisplatin 70 mg m À2 (Gem þ Cis), and surgery. Three alternate dosing sequences were used during cycle 1 to examine dynamic changes in molecular profiles. Of 65 women treated, 13 (24.5% of 53 patients with surgery) had a pCR and 22 (33.8%) had a complete clinical response. Patients administered Gem d1, 8 and Dox d2 in cycle 1 (20 of 65) reported more toxicities, with G3/4 neutropenic infection/febrile neutropenia (7 of 20) as the most common cycle-1 event. Four drug-related deaths occurred. In 46 of 65 patients, 10-fold cross validated supervised analyses identified gene expression patterns that predicted with X73% accuracy (1) clinical complete response after eight cycles, (2) overall clinical complete response, and (3) pCR. This regimen shows strong activity. Patients receiving Gem d1, 8 and Dox d2 experienced unacceptable toxicity, whereas patients on other sequences had manageable safety profiles. Gene expression patterns may predict benefit from gemcitabine-containing neoadjuvant therapy.