B. Structural, biochemical and functional studies of ubiquitination signaling.
Signaling via covalent attachment of ubiquitin plays an essential role in an increasing number of cellular processes. Ubiquitination involves a series of steps involving E1, E2 and E3 ubiquitin ligases that catalyze the activation and subsequent covalent attachment of ubiquitin to substrate proteins. The E3 class of ubiquitin ligases typically involves the coordinated activity of a group of proteins that form a multi-protein complex of substrate, E2 ligase recognition elements, scaffolds, and molecular bridges.
1. The Function of Prp19 in RNA splicing (Botuyan 1999, Ohi 2003)
In collaboration with yeast cell biologist Kathy Gould (Dept. of Cell Biology, HHMI) we are investigating the first evidence for ubiquitin ligase activity in RNA splicing, which we showed is associated with the protein Prp19. Sequence analysis of Prp19 revealed the presence of a U-box domain. Structure determination revealed the U-box has the same architecture as RING domains found in a common class of E3 ubiquitin ligases and subsequent studies revealed that Prp19 does indeed have ubiquitin ligase activity. Proteomic strategies have been used to elucidate some of the Prp19 binding partners. Current efforts are focused on biophysical and structural studies to understand the functional organization of the multi-domain Prp19 (U-box/coiled-coil/WD-40) and discerning which of the yeast E2 ligases is the native partner of the Prp19 U-box for subsequent study of the molecular basis for E2-E3 recognition.
2. Intracellular- S100A6 modulation of SIP function. (Pubs. Nowotny 2000, Lee 2004)
Siah-1 interacting protein (SIP) is an essential component of an E3 ligase complex that ubiquitinates β-catenin as part of the regulation of cell proliferation during embryogenesis. SIP function appears to be regulated by S100A6 through physical interaction. The connection to intracellular signaling via protein ubiquitination represents a fundamentally new area of investigation for S100 proteins. In addition to SIP, we are also working with a highly homologous target protein, Sgt1, which also binds S100A6 and is a member of both the core kinetchore and certain SCF E3 ubiquitin ligase complexes. The overall objective of our research is to understand protein-protein interactions mediating formation of the complex. The objectives in this project are to determine the domain structures and organization of SIP and Sgt1, define the structural basis for their interaction with S100A6, and examine how S100 protein binding affects the structure and dynamics of these targets and their interactions with other known binding partners (e.g. Siah-1, Skp1, HSP90) from their respective E3 complexes.
last updated April 26, 2004