The long-term objective of this proposal is to determine if phosphorylation is involved in normal or pathological prion protein (PrP) biology. Endogenously expressed normal cellular prion protein converts into a proteinase K resistant (PKRES) and fibrillar form when neurons in brain are exposed to prion transmissible material or express a familial mutant form of prion protein. The reason for this conversion is unclear. We show here new preliminary evidence that purified recombinant human, mouse and hamster prion protein can be phosphorylated by the neuronal specific and proline-directed cyclin dependent kinase-5 (cdk5) in vitro. A fragment of the phosphorylated prion protein becomes resistant to proteinase K as it often does in prion diseases. Furthermore, electron microscopy shows the formation of fibrillar and globular aggregate structures in phosphorylated prion protein. Therefore, phosphorylation could be a logical mechanism for the conversion of prion protein observed in disease states and could even be involved in the transmissible nature of the prion protein. While in vitro phosphorylation is easily tested, it is much more difficult to determine if phosphorylation of a specific protein occurs in vivo. Therefore, the goal of this exploratory application is to determine if phosphorylation exists in vivo. We will assess phosphorylation with a three-prong approach: using phospho-columns and western blot analyses, generating phospho-PrP specific antibodies to assess phospho-PrP by western blotting or immunohistochemistry, and immunoprecipitating prion protein from gamma-32P- orthophosphate-labeled cells. We will perform these techniques in systems known to be sensitive to transmissible prions and capable of generating protease-resistant prion protein: normal or mutant prion protein- transfected mouse neuroblastoma N2A cell lines, and human and mouse brains affected by prion diseases. Our preliminary evidence indicates cdk5-independent phosphorylation of PrP in N2A cells and in scrapie- infected mouse brains. Therefore, another goal of this application is to determine which other kinases are involved in prion protein phosphorylation, whether phosphorylation of prion protein at different sites generates identical or different protease resistant fragments, and whether phosphorylated prion protein is transmissible or induced by scrapie exposure. If positive, the result of the present application would not only add novel and potentially important mechanisms to the current state of knowledge of prion protein conversion in vivo, but could have a very high impact on how we view and treat prion diseases.
Prion diseases are transmitted through the conversion of the normally inoffensive prion protein of a host (human, bovine, Elk) into a disease-causing protein. The mechanism for the conversion of this protein is unknown. The present application examines one possible mechanism that would explain the conversion of prion protein and offer new avenues for the prevention or treatment of prion diseases.