Molecular analysis of a dual action F box protein in cell cycle regulation

Scientists refer to cells as being 'programmed' to divide, to die or to differentiate. This describes the elaborate, and seemingly automatic, changes that take place in the way that a cell behaves under different circumstances. For example, cells grow when provided with the right nutrients, die if irrevocably damage, or differentiate into distinctly different types of cells when in the appropriate niche environment. Cells are sensitive to the signals that dictate changes to their programmes and thus to their behaviour at a particular time in their growth cycles. Often, to execute one programme means that others are disabled. These time-sensitive switches allow coordinated and absolute decisiveness to cells' fate. These switches are comprised of a family of proteins called SCF ligases, which directly control the cell's growth machinery. Understanding the how cells make their decisions and regulate cell fate is a matter of fundamental interest, and this proposal outlines experiments designed to understand the activity of SCF proteins, and how they achieve this. In particular we will focus on a component of this SCF called Fbxo7. It differentially regulates at least three different proteins that affect cellular proliferation, death, and differentiation. In other words, it is a master regulator. We want to understand how Fbxo7 is capable of varying its activity towards the proteins it binds, and ultimately how it controls cell fate. We will use a variety of methods to understand the protein's function, to solve its structure, and find other proteins that it regulates. By doing experiments to broaden our understanding of the proteins at the intersection of different cell programmes we hope to be able to affect and ultimately direct these programmes in clinically relevant settings.

SCF-type E3 ubiquitin ligases control cell cycle progression by promoting the ubiquitin-mediated degradation of key regulators. These holoenzymes contain F box proteins (FBPs) which direct their interaction with substrates. The experiments outlined in this proposal will investigate how an unconventional F box protein, Fbxo7, has disparate activities towards its interacting proteins, stabilising some but enabling the degradation of others. This dual capacity distinguishes Fbxo7 from other F box proteins, and emphasizes the fact that F box proteins have novel activities that have not been investigated. The global objectives for this project are to understand the basis for the different capacities of Fbxo7, to shed light on how the family of FBPs target their substrates, and ultimately how SCF ligases direct and coordinate cellular processes, like proliferation and apoptosis. This will involve pursuing the following experimental objectives: - Determination of the important residues within Fbxo7 which affect its activity. This investigation will be based on structure/function and bioinformatics analyses, and analyse aspects such as dimerization, phosphorylation, and interactions with other proteins, which may regulate Fbxo7 activity; - Molecular analysis of essential residues in Fbxo7 for its major functions: as part of an SCF, in degradation or as a stabilizer of its substrates, or in determination of cellular responses in proliferation, apoptosis, and transformation; - Analysis of sub-cellular localization and kinetics of Fbxo7 expression and correlate these findings with substrate binding and its function; - Determination of the crystal structure of Fbxo7in complex with cyclin D/cdk6; - Identification of additional Fbxo7 interacting proteins, - Analysis of the mechanisms of Fbxo7-mediated apoptosis induction, including the pathways that regulate its activities and the downstream effectors that transduce its activity.