Characterization of the Wsc proteins and their interactions with proteins that function in the PKC1-MPK1 signaling pathway

Cells are constantly faced with the challenge of adapting to environmental stresses, such as heat shock, and nutrient deprivation. In order to survive these adverse conditions, cells must elicit a molecular response to protect them from damage. It has been well established that stress-activated protein kinase cascades are involved in transducing signals intracellularly to the nucleus, in response to environmental stresses. In the budding yeast Saccharomyces cerevisiae, the protein kinase C (called the PKC1-MPK1) cascade is involved in the stress response and in maintaining integrity of the cell wall. WSC1, WSC2, and WSC3 (for cell w&barbelow;all integrity and s&barbelow;tress response c&barbelow;omponent) are a novel family of genes that function upstream of the PKC1-MPK1 pathway and are candidate sensors of environmental stresses. The Wsc family of proteins have similar sequence characteristics: at the N-terminus they have a cysteine motif (referred to as the WSC domain) and a serine/threonine rich domain predicted to be extracellular, a hydrophobic domain suggested to be transmembranous, and a variable, highly charged C-terminus that may be involved in intracellular signaling. It is thought that the Wsc proteins may regulate the PKC1-MPK1 signaling pathway through the small GTPase Rho1. To elucidate how the Wsc proteins may regulate the PKC1-MPK1 pathway, we investigated the biochemical properties of the Wsc proteins and their interactions with putative intracellular targets that are known to function in the PKC1-MPK1 pathway. Biochemical experiments demonstrated that Wsc1 is an integral membrane protein that is phosphorylated. Using the yeast two-hybrid system, we found the intracellular C-terminus of Wsc2, but not Wsc1, interacts with Bem4, a novel protein known to interact with members of the Rho GTPases. The C-terminal domain of Wsc1 and Wsc2 was also shown to interact with Rom2, a guanine nucleotide exchange factor for Rho1. Further two-hybrid experiments showed that Rom2 and Bem4 interact, suggesting that Wsc2 forms a complex with Bem4 and Rom2. Altogether, these results suggest that Wsc2 acts as a sensor of stress by regulating the PKC1-MPK1 pathway via Bem4 and Rom2.