Kinetic Mechanism Of TTK Kinase And The Effects Of Its Carboxyl Terminal Tail (D Domain) On Phosphorylation

Cell cycle control is of paramount importance in the cells of living organisms, regulating growth, differentiation, and apoptosis. If aberrant control of the cell cycle develops within cells, a number of serious consequences may occur, potentially leading to cancer. One such defect is caused by faulty mitotic progression leading to genome instability and chromosome missegregation. While it is known that mitotic progression is controlled by a number of protein kinases, including the series of Cyclin dependant kinases (Cdks), the precise signaling pathways and molecular mechanisms involved in regulating this process stand to be resolved. Therefore, mechanistic studies of the kinases involved in mitotic regulation are critical and will further our understanding for the roles of mitotic kinases in cancer.One kinase which has been implicated in regulation of mitotic progression is TTK coded by the human MPS1 (MonoPolar Spindle 1) gene. It is an essential dual specificity protein kinase, capable of phosphorylation on serine, threonine and tyrosine residues. TTK is involved in the spindle assemble checkpoint, the kinetochore localization of key checkpoint proteins and correction of improper chromosome attachments. Checkpoint signal transduction from the kinetochore depends on TTK except several other kinases, and the maintenance of ploidy during mitosis and survival of human cancer cells also required the kinase activity of TTK. Because of these central roles of TTK in many crucial processions central to mitotic fidelity such as cellular growth, survival, and proliferation, TTK has recently emerged as an evolutionary conserved critical regulator of genetic stability in eukaryotes. The loss of TTK function in the cell cycle causes meiotic errors, aneuploidy and developmental defects, suggesting a relationship between this kinase and cancer. These observations have raised the possibility that Mpsl can be exploited as a novel therapeutic strategy to cancer. Nevertheless the success of this novel therapeutic approach will require a more detailed interrogation of its phosphorylation mechanism. Conversely, the kinetic mechanism for TTK kinase activity is still unclear and the characterization of the enzymology of it has yet to be thoroughly explored.In this work, the molecularity of TTK autophosphorylation and a kinetic mechanism of TTK transphosphorylation were studied using traditional radioactive kinase assay after purification of this protein from high5 insect cells.Firstly, the hMpsl was cloned and expressed in insect high five cells as fusion proteins using baculovirus expression vector system. To purify the full length TTK, a GST-tag was added to the C-terminus of the construct before a precission protease cleavage site and a 6xHis-tag was present at the N-terminus of the construct after a thrombin cleavage site. Expressed TTK was purified by two step-affinity chromatography including glutathione-sepharose and nickel-NTA agarose. Then 3C and thrombin proteases were separately used to remove the GST-tag and 6xHis-tag from the purified protein. In addition, to investigate the roles of carboxyl terminal tail (also named D domain) of TTK in its kinase activity, a mutant TTK-d was constructed by deletion the D domain of TTK and purified from insect cells using the same method as TTK wild type. Then properties of the purified full length TTK-wt and TTK TTK-d were further studied.Subsequently, to find out if TTK protein level was regulated by cell cycle progressions, I tracked the level of endogenous TTK and another two crucial mitotic kinases Cdk/CyclinB through the cell cycle in mitotic Hela cells and measured the absolute abundance of these proteins at various points during cell cycle using quantitative immunoblotting. The levels of Mpsl are low in interphase cells but high in mitosis or activation of spindle checkpoint and the cellular concentration of TTK is increased transiently at the G1/S transition, formed a peak at the G2/M transition and then returns to basal levels at G1 phase to reminder the next cell cycle. Its change curve is consistent with that of cyclinB/Cdkl. This profile of protein level suggested that the stability of TTK is likely regulated by cell cycle progression in vivo and TTK is required for centrosome duplication because its elevation is coincident with the timing of centrosome duplication.Thirdly, I have determined the molecularity of TTK autophosphorylation. My result showed that the overall rate of TTK autophosphorylation reaction was linearly dependent on enzyme concentrations, implying that its autophosphorylation mechanism is not intramolecular but an intermolecular reaction. I have also presented evidence that TTK autophosphorylation is required for full kinase activity. This phosphorylation of activation loop residues is a common mechanism for the activation of protein kinases and it transits the kinase from a low-activity to a high-activity state.Lastly, to better understand the enzymatic mechanism of the TTK, I proceeded to characterize two-substrate steady-state kinetics of TTK. Varying both the concentration of ATP and TTKA in the same assay gave Km values of 54?M and 74?M for the TTKA and ATP substrates respectively and revealed that TTK employs a ternary complex mechanism with a negative interaction between two substrates binding. In addition, comparison the kinase activity between TTK-wt and TTK-d showed that removal of d region causes a 3-5 fold decrease in TTK substrate phosphorylation both in vivo and in vitro, suggesting that the carboxyl terminal tail is dispensable for autophoshorylation of kinase but essential for trans-phosphorylation. To further explore the effect of the D domain (aa 792-853) in transphosphorylation, I also identified two-substrate steady-state kinetics of purified TTK-d. The results revealed that TTK-d has a similar Km value for ATP (80?M) but a higher Km value for TTKA (130?M) than TTK-wt, suggesting that residues in D domain assistant kinase activity by interacting with kinase domain to benefit TTKA substrate binding. Overall, these findings provide the first detailed kinetic description of the TTK enzyme mechanism and underscore the importance of the D domain of TTK in kinase activity.