Investigation of Cdc34 Molecular Recognition by its Enzyme Partners, Uba1 and SCF Ligases.

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PROJECT SUMMARY Cdc34 is a key regulator in cell cycle progression at the G1 to S phase checkpoint1-3. It acts as an E2 ubiquitin conjugating enzyme that is first charged with ubiquitin by E1 enzyme, Uba1, and then functions with the SCF family of E3 enzymes to modify other cell cycle proteins with ubiquitin for targeted degradation by the proteasome4,5. In years since its discovery, Cdc34 dysregulation has been implicated in several types of cancer6-8. Many downstream targets of Cdc34 are cyclin-dependent kinase inhibitors such as p27 and p40 that are critically important for cancer progression9-14. Together, these findings have led to investigations of Cdc34 as a target for cancer therapeutics but a more directed approach, such as specifically targeting Cdc34 interactions with its enzyme partners is needed. However, a detailed structural understanding for molecular recognition of Cdc34 by its E1 partner, Uba1, and E3 ligase partners is not available. Further, regulation of Cdc34 is not completely understood. Previous studies have shown positive regulation of Cdc34 by phosphorylation; however, we have exciting preliminary data that implicates phosphorylation of residue serine 10 as a negative regulator of Cdc34 activity and this is supported in the literature by a phosphoproteome mass spectrometry screen that identified this residue in vivo15-18. Further, based on structural predictions we believe Ser10 residues on the surface of Cdc34 that interacts with both E1 and E3 enzymes. Thus, we aim to identify key residues in the molecular recognition of Cdc34 by its enzyme partners and hypothesize that phosphorylation of Ser10 will negatively regulate this molecular recognition through steric clash and electrostatic repulsions. Our hypothesis will be addressed through the following Specific Aims. Aim 1 will identify key residues for Cdc34 interaction with E1 and E3 enzyme partners. We will use x-ray crystallography in concert with biochemical and genetic techniques to accomplish this aim. Aim 2 will elucidate the regulatory effects of Cdc34 phosphorylation at residue Ser10. We will confirm the phosphorylation of Ser10 in vivo and examine the effects of S10D phosphomimetic mutation on molecular recognition of Cdc34 by its enzyme partners in vitro with biochemical assays and in vivo using yeast genetics.