The role of drosophila protein kinase doubletime in circadian period determination, morning and evening oscillators and tauopathy

Abstract

In this dissertation, I used the GAL4-UAS binary expression method to overexpress mutant and wild type forms of the circadian protein kinase DBT in order to address several basic questions about DBT's biological functions. Different dbt mutations either shorten or lengthen the circadian period, although they all possess lower kinase activity in vitro. Therefore, I first addressed whether these period-altering mutations of DBT act independently of any effects on DBT's intrinsic kinase activity by analyzing them in a kinase inactive background (DBTK/R) in cis. All three double mutants shortened the DBTK/R period in cis and enhanced PER oscillations, supporting our hypothesis. Next, I addressed whether DBT has different roles in the cytoplasm and the nucleus with opposite effects in these two compartments (lengthening period in the cytoplasm and shortening it in the nucleus). I mutated the putative nuclear localization sequence (DBTWTNLS-) and added a strong NLS (DBTWT stNLS) to make DBT cytoplasmic or nuclear, respectively. In contrast to my predictions, the DBTWTNLS- shortened period, while DBTWT stNLS did not alter period. The NLS- mutation affected amino acids that are part of a proposed phosphate-binding domain. Additional mutations in the vicinity of the NLS shortened period, while mutations that were not close lengthened period. The shortening of period by some mutations suggests that they affect a TAU-like domain involved in a protein-protein interaction that lengthens period. A third question addressed was whether DBT has specific roles in the brain morning and evening oscillator cells. I investigated these cellular interactions by expression of DBTK/R in different subsets of cells to damp just one oscillator. Our experiments suggest the evening oscillator is necessary for suppressing the morning startle response, in addition to its role in production of evening activity. The final part of my thesis employed a fly eye model for tauopathy to investigate whether DBT might have a role in neurodegeneration. Overexpression of the dominant negative DBTK/R enhanced the eye neurodegeneration produced by expression of human TAU in the fly eye. Our investigation suggests that DBT phosphorylates a caspase (DRONC), thereby preventing it from cleaving TAU and thus preventing its toxicity.