Studies of Ste5

Structural and functional study of the transition of Ste5 from a signaling scaffold to an E3 ubiquitin ligase

MAP-kinase signaling networks are conserved from yeast to humans and transduce many internal and external signals. For example, a classical three-tier MAP-kinase cascade coordinates the response of yeast cells to mating pheromones, and serves as a paradigm to study the function and regulation of these pathways. The protein Ste5 functions as a scaffold and couples pheromone receptor stimulation to the activation of the MAPK cascade. Upon activation of the pathway, Ste5 is recruited to the plasma membrane by binding to the activated heterotrimeric G-protein, where it facilitates recruitment and activation of the MAPK kinase components. Importantly, the laboratory of Prof. Peter recently found that Ste5 also functions as a regulated E3 ubiquitin ligase that triggers the covalent attachment of ubiquitin to the MAPK kinase kinase Ste11. This ubiqutination targets Ste11 for proteasomal degradation, thereby limiting MAPK activation. For ubiquitination three enzymes are required: E1 (the ubiquitin-activator), E2 (the ubiquitin-conjugator) and E3 (the ubiquitin ligase). Phosphorylation of a single residue (Ser 185) in the RING-H2 domain of Ste5 promotes the interaction with the E2-enzyme Ubc4, and abolishes binding to the receptor-linked G-protein. Because Ser 185 is phosphorylated in a MAPK-dependent manner, this mechanism provides an activity-dependent negative feedback loop that restricts MAPK signaling output by controlling membrane turnover of Ste5 and the stability of activated Ste11.

Importantly, bioinformatic investigations and preliminary experiments suggest that a subset of RING domains may similarly be regulated by phosphorylation of specific Ser/Thr residues, thus providing a novel mechanism to activate RING E3 ubiquitin ligase activity. The aim of the project is thus to investigate the structural basis for this mechanism. We will use NMR titrations and biophysical methods to quantify the interaction of Ubc4 and mutant RING-H2 domains that mimic the phosphorylated (S185E) and non-phosphorylated (S185A) forms. As crystallization trials have so far failed, we will use NMR to determine the structure of the phosphorylated Ste5-RING domain in complex with Ubc4. Based on the structural data, we plan in vitro and in vivo assays for functional analysis of the complex between Ubc4 and the Ste5 RING-H2 domain. We expect that this study provides a major advance in understanding the regulation of RING-finger E3 ligases and may provide an exciting new link between MAP-kinase signaling networks and the ubiquitin-proteasome system.

Supported by

ETH Zurich

In Collaboration With

M. Walczak, Prof. M. Peter