Tousled like kinases:phosphorylation and function during the cell cycle

The Tousled Like Kinases (TLKs) are cell cycle regulated kinases with highest activity during S-phase of the cell cycle. In response to DNA damage the activity of the TLKs is suppressed during S and G2 phases of the cell cycle. The suppression of TLK activity in response to DNA damage requires a functional signalling cascade requiring ATM, NBS1 and Chk1. Although the regulatory pathways controlling the suppression of these kinases is well understood in mammalian cells, very little is known about their function during the cell cycle. In this thesis, I investigate the function of the TLKs during both S-phase and mitosis. The function of the TLKs is studied using a chicken B lymphocyte DT40 cell line deficient in TLK1 (TLK1-/-) and RNAi targeting both TLK1 and TLK2 in human HeLa cells. In DT40 cells, TLK1 is not required for survival or timely completion of the cell cycle. In HeLa cells, both TLK1 and TLK2 depleted cells are able to incorporate the synthetic nucleoside BrdU and progress through S-phase. A slight delay in late S-phase was observed when TLK1 is depleted. During mitosis, I show that depletion of either protein increased the percentage of cells displaying anaphase bridges. Using time-lapse microscopy analysis, I show that these anaphase bridges occur in cells, which display only a minor delay in anaphase onset, which is not due to problems aligning chromosomes. A role for the TLKs in chromosome segregation is presented and a specific requirement for the TLKs in the correction of merotelic attachments is suggested. In the second half of this thesis I study the phosphorylation of TLK1 at serine 159 (S159). TLK1 S159 phosphorylation is examined in detail using a phospho-specific antibody raised in this study. I show that phosphorylation of S159 is cell cycle dependent with the highest level of phosphorylation achieved from late G2 until completion of mitosis. TLK1 S159 phosphorylation is induced during prolonged prometaphase arrest and phosphorylation of S159 can be achieved in vitro by Cyclin B/Cdk1 and Cyclin A/Cdk2. Using green fluorescence protein (GFP) tagged wild type TLK1 and S159A and S159D mutants I demonstrate that S159 phosphorylation does not regulate the localisation of TLK1 during mitosis but does cause a phosphorylation dependent arrest in G1