文摘
英文文摘
Chapter Ⅰ ： Introduction
1.1.The TTK protein kinase family
1.2.The roles for Mps1
1.2.1.Mps1p in spindle pole duplication
1.2.2.Mps1 in the spindle assembly checkpoint
1.2.3.Mps1 corrects chromosome attachment errors
1.2.4.Other functions of Mps 1 kinase
1.3.The SAC kinases Mps1 in cancer
1.4.Regulation of Mps1
Chapter Ⅱ Cloning,expression and purification of recombinant proteins
2.1 Material and methods
2.1.1.Cell cultures
2.1.2 Chemicals and buffers
2.1.3.Mammalian expression constructs
2.1.4.Amplification of recombinant baculovirus encoding TTK-wt and TTK-d
2.1.5.Baculovirus expression of TTK-wt and TTK-d
2.1.6.Protein purification of TTK-wt and TTK-d
2.1.7.Western blot
2.1.8.3C protease expression and purification
2.1.9.Protein TTKA expression and purification
2.1.10.In vitro kinase assay
2.2 Results
2.2.1 Expression and purification of 3C protease
2.2.2 Expression and purification of TTKA
2.2.3 Purification of TTK-wt and TTK-d
2.2.4 Time course experiment of TTK kinase activity
2.2.5.Assay for different substrates phosphorylation by TTK
2.2.6.Comparison of kinase activity between TTK-wt and TTK-d
2.3 Discussion
Chapter Ⅲ Measuring the absolute abundance of the crucial protein kinases during cell cycle in Hela cells
3.1.Materials and methods
3.1.1.Materials and reagents
3.1.2.Cell lines
3.1.3.Cell density and viability estimations
3.1.4.Cell synchronization
3.1.5.Cell cycle analysis
3.1.6.Cell culture and nocodazole treatment
3.1.7.Cell lysis and sample preparation
3.1.8.Quantification of cellular concentrations of TTK and Cdks
3.1.9.Densitometry using ImageJ
3.1.10.Data analysis and calculations for Coomassie-stained gels using Microsoft Excel
3.1.11.Data analysis and calculations for immunoblot data using Microsoft Excel
3.2 Results
3.2.1.The TTK level in Hela cells and sw480 cells
3.2.2.Analysis for cell cycle status by flow cytometry(FACScan)
3.2.3.Measuring GST-TTK protein standard concentration
3.2.4.Measuring GST-Cdk and CyclinB protein standards concentration
3.2.5.Determine cellular abundance of Mps1,Cdk1 and Cyclin B
3.2.6.Data analysis
3.3 Discussion
3.3.1.A method for accurately quantifying the absolute number of biological molecules
3.3.2.Cell cycle-dependent regulation of the steady state level of TTK in vivo
3.3.3.Accumulation of Mps1 at centrosomes is essential for centrosome duplication
3.3.4.Mitosis might require a higher threshold of TTK than centrosome duplication
3.3.5.Cdk attenuates degradation of the TTK at centrosome duplication
Chapter Ⅳ Kinetic study of TTK and the modulation of its kinase activity by the carboxyl terminal tail
4.1.Materials and Method
4.1.1.Autokinase assay
4.1.2.In vitro kinase assays
4.1.3.Quantitating gels using Molecular dynamics densitometer and Image quant 5.0
4.1.4.Which one is suitable substrate for kinetics studies?
4.1.5.Is product formation linear with time?
4.1.6.Does autophosphorylation increase transphosphorylation?
4.1.7.Is autophosphorylation of TTK intermolecular or intramolecular action?
4.1.8.How the reaction rate varied with enzyme concentration
4.1.9.Is there a product inhibition?
4.1.10.Is D domain of TTK responsible for its kinase activity?
4.1.11.Two-substrate kinetic analysis for TTK catalytic mechanism
4.1.12.What is role of D domain in TTK transphophorylation?
4.1.13.Substrate Analog Inhibitors Inhibition Analysis
4.2.Results
4.2.1.Suitable substrate for kinetics study
4.2.2.A Lag-phase in TTK Product Formation
4.2.3.Autophosphorylation enhances transphophorylation
4.2.4.Autophosphorylation is an intermolecular reaction
4.2.5.The reaction rate is varied with enzyme concentration
4.2.6.Interpretation of product inhibition experiments
4.2.7.D domain is critical for substrate phosphorylation of TTK
4.2.8.Two-substrate Kinetic Analysis for TTK-wt and TTK-d
4.2.9.Investigation of the TTK kinase activity using substrate analog inhibitor
4.3 Discussion
4.3.1.A lag-phase in TTK product formation and auto-phosphorylation enhances activity
4.3.2.Self association or clustering of Mps1 molecules may enhance autophosphorylation and activate its kinase activity
4.3.3.TTK catalysis transpires through a sequential kinetic mechanism
Conclusions
References
Acknowledgements
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