*4 Year WT PhD Programme* The Regulation of PHDs During the Cell Cycle

Prolyl Hydroxylase enzymes (PHDs) respond to oxygen levels and modify critical proteins involved in the cells response to changes in oxygen [1]. PHDs are known for targeting HIF-alpha for degradation [2]. In recent work we have shown PHD1 controls the stability of the centrosomal protein Cep192 [3]. Hydroxylation of Cep192 on Pro1717 by PHD1 creates a binding site for Skp2, which targets Cep192 for degradation. This system controls the levels of Cep192 and thus determines the assembly of the primary cilium through interphase and the centrosome and the mitotic spindle. Our recent experiments also show the kinetochore component CENP-N is prolyl hydroxylated and that this modification is required for proper mitotic kinetochore assembly. Thus we have evidence that assembly of both the centrosome and kinetochore are controlled by PHDs.

This project will measure PHD activity and function throughout the mammalian cell cycle to establish if PHD activity is sensitive to modulation by either Cyclin Dependent Kinases, and/or other cell cycle regulators. Using mass spectrometry we have detected two sites on PHD1 [4] and one site on PHD2 that are phosphorylated; all three of these sites match the consensus for CDK phosphorylation. The project will also address whether other factors that control mitotic spindle assembly and function are modified by the PHDs. Additional mitotic factors controlled by PHDs will also be investigated using state of the art mass spectrometry approaches and quantitative imaging. In this way, we will establish a direct synergistic link between our mechanistic experimental programme and metabolic disease, where oxygen sensing is impaired.