Xerostomia is the medical term for dry mouth due to a lack of saliva because of salivary glad hypofunction. Xerostomia is a side effect of irradiation treatment administered to head and neck cancers and greatly diminishes the effectiveness of anti-cancer therapies as well as the life quality of patients. The main cause of Xerostomia is salivary epithelial cells apoptosis. Our lab has identified PKC5 as a critical regulator of apoptosis in the salivary gland both in vitro and in vivo. We have also shown that nuclear accumulation of PKC5 is both required and sufficient for apoptosis, suggesting that tight regulation of the nuclear of this ubiquitous kinase is essential to avoid in-appropriate induction of apoptosis. Here we are exploring a regulatory interaction between the regulatory of PKC6, where we have shown that phosphryaltion of Y64 and Y155 is essential for nuclear localization of PKC5 and c-terminal Nuclear localization signal (NLS) that while required for nuclear accumulation of PKC5 is not sufficient. Interestingly, we have identified a PxxP motif overlapping the NLS that resembles a an SH3 binding site such that found in SH3 containing tyrosine kinases. Here, we are exploring the hypothesis that binding of tyrosine Kinase to the PxxP site, regulates phosphorylation of Y64 and Y155 which induces a conformational change allowing access of nuclear import machinery to the NLS.
In aim 1 I will use site directed mutagenesis to determine the role of the PxxP site in regulating nuclear import of PKC5 and phosphoryaltion at Y64 and Y155, in addition I will use pull down assays to determine if nuclear import in ultimately regulated by binding of the NLS to nuclear import machinery.
In Aim 2 I will use in and in vitro binding assays to determine which of the candidate tyrosine kinases that interact with PKC6 bind to it via the PxxP site, and assess the role of this interaction in the context of nuclear import of PKC5 using knockdown of the tyrosine kinase. We believe that our findings here will greatly contribute to public contribute to public as understanding the mechanism regulating nuclear import of PKC5 will give insight into mechanism by which PKC6 regulates apoptosis in the salivary epithelial cells which is crucial for the development of therapeutic strategies to protect against salivary gland apoptosis.
