THE ROLES OF DROSOPHILA PROTEIN KINASE DOUBLETIME IN CIRCADIAN PERIOD DETERMINATION, MORNING AND EVENING OSCILLATORS AND TAUOPATHY A DISSERTATION IN Molecular Biology and Biochemistry and Cell Biology and Biophysics Presented to the faculty of the University of Missouri-Kansas City in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY By ANANDAKRISHNAN VENKATESAN B.Sc., University of Chennai, 2000 M.Sc., University of Chennai, 2002 M.S., University of Missouri-Kansas City, 2007 Kansas City, Missouri 2012 THE ROLES OF DROSOPHILA PROTEIN KINASE DOUBLETIME IN CIRCADIAN PERIOD DETERMINATION, MORNING AND EVENING OSCILLATORS AND TAUOPATHY Anandakrishnan Venkatesan, Candidate for the Doctor of Philosophy Degree University of Missouri Kansas City, 2012 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 i 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. ii APPROVAL PAGE The faculty listed below, appointed by the Dean of School of Graduate Studies have examined a dissertation titled “The role of Drosophila Protein Kinase Doubletime in Circadian Period Determination, Morning and Evening Oscillators and Tauopathy “ presented by Anandakrishnan Venkatesan, candidate for the Doctor of Philosophy degree, and certify that in their opinion it is worthy of acceptance. Supervisory committee Jeffrey L Price, Ph.D., Committee chair Division of Molecular Biology and Biochemistry Leonard Dobens, Ph.D. Division of Molecular Biology and Biochemistry Michael Plamann Ph.D. Division of Cell Biology and Biophysics Saul Honigberg Ph.D. Division of Cell Biology and Biophysics Stephen J King Ph.D. Division of Molecular Biology and Biochemistry iii CONTENTS ABSTRACT ......................................................................................................................... i LIST OF ILLUSTRATIONS ...............................................................................................v LIST OF TABLES ............................................................................................................ vii ABBREVIATIONS ......................................................................................................... viii ACKNOWLEDGEMENTS .................................................................................................x Chapter 1. BACKGROUND AND SIGNIFICANCE ...........................................................1 2. MATERIALS AND METHODS .......................................................................25 3. DROSOPHILA DBT PERIOD ALTERING MUTANTS IN-CIS SHORTEN PERIODS PRODUCED BY EXPRESSION OF A KINASE INACTIVE DBT..39 4. A MUTATIONAL ANALYSIS OF DBT REVEALS A UNIQUE DOMAIN THAT PRODUCES SHORT PERIODS ...............................................................63 5. EXPRESSION OF DBTK/R IN THE CLOCK CELLS AFFECTS PER IN A CELL-AUTONOMOUS MANNER BUT REVEALS A NEED FOR A FUNCTIONAL CIRCADIAN CLOCK IN THE EVENING CELLS TO PRODUCE HIGH LEVELS OF EVENING ACTIVITY AND SUPPRESS THE MORNING ACTIVITY.........................................................................................90 6. A ROLE OF DBT IN TAU PROCESSING AND TOXICITY .......................109 7. BDBT IS NECESSARY FOR THE DESTABILIZATION OF PER IN THE LATERAL NEURONS OF ADULT BRAINS ...................................................140 REFERENCES ................................................................................................................143 VITA ................................................................................................................................151 iv ILLUSTRATIONS Figure Page 1. Basic circadian clock model ............................................................................................4 2. Actograms are used to analyze the circadian behavioral activity of model organisms ...8 3. The molecular mechanism of the Drosophila circadian clock .......................................11 4. The use of the UAS-GAL4 binary expression system technique to study the effects that DBT’s kinase activity confers on circadian rhythms .........................................................33 5. Representation of double mutants of DBTK/R ................................................................42 6. An alignment of Drosophila DBT with various vertebrate CKIε/δs .............................44 7. Averaged actogram and periodograms of double mutants ............................................48 8. Detection of PER and DBT-MYC from various double mutants ..................................51 9. Difference between PER mobilities at ZT1 and ZT13 ..................................................52 10. Comparison of DBT-MYC expression levels for different timGAL4>UAS-DBT- MYC lines ..........................................................................................................................54 11. Detection of endogenous DBT levels ..........................................................................57 12. Conserved DBT residues mutated in the vicinity of the TAU mutation......................66 13. Localization of NLS mutants in S2 cells .....................................................................69 14. Localization of DBT-myc in larval brains ...................................................................70 15. Nuclear score for localization of DBTWT and DBTK/R .......................................................................71 16. Detection of PER and DBT-MYC from various NLS mutants ...................................74 17. Localization of DBT-myc consturcts in larval and adult brains ..................................76 18. Crystal structure of CK1δ around the TAU mutation ..................................................78 19. Schematic representation of the list of Mutations in the TAU domain of DBT ..........80 v 20. Averaged actogram and periodogram for TAU domain mutants ................................81 21. Detection of DBT-MYC from various TAU domain mutants .....................................83 22. Averaged activity of wildtype (Canton S) flies in LD .................................................92 23. A pictorial representation of fly brain and the distribution of clock neurons ..............93 24. Cross scheme to disrupt different subsets of cells in the fly brain...............................97 25. LD assays of different driver/responder combinations ..............................................102 26. Localization of PER in adult brains from flies expressing DBTK/R in different oscillators .........................................................................................................................105 27. List of drivers and DBT constructs used for TAU expression ...................................112 28. Expression of Drosophila TAU in S2 cells ................................................................116 29. Eye phenotypes with co-expression of hTAU with DBTWT or DBTK/R .............................. 119 30. Detection of phosphorylated TAU with AT8 and PHF1 antibodies ..........................124 31. Quantification of phosphorylated TAU with AT8 and PHF1 ....................................125 32. Detection of armadillo in head extracts .....................................................................128 33. Detection of armadillo in 3rd instar larval imaginal discs ..........................................129