Dioletis, E.; (2012) Biochemical and biophysical characterisation of the death domain of death associated protein kinase. Doctoral thesis , UCL (University College London).

Abstract

Death associated protein kinase (DAPk) is a multidomain protein kinase that plays a key role in the promotion of programmed cell death. A number of different stimuli such as TΝF-α, TGF-β, IFN-γ, ceramide, c-Myc oncogenic transformation and matrix detachment are all known to activate DAPk, suggesting that DAPk lies at an important convergent point for the different cell death signalling pathways. DAPk-induced cell death is not entirely dependent on the presence of functional p53 or caspases and is highly conserved in C. elegans, rodents and human. The pro-apoptotic functions of DAPk are dependent on its kinase domain as well as the death domain (DAPk-DD). The structure of the kinase domain has been solved, but no experimental structural information is available for the DAPk-DD. The death domain belongs to the death fold superfamily that also includes the death effector domain, the caspase recruitment domain, and the Pyrin domain. These domains all share a common architecture of a six α-helix bundle arranged in an antiparallel manner. The death domain is a protein-protein interaction motif found in different signalling proteins involved in apoptosis, inflammation and NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) signalling. The role of the death domain is to promote the assembly of large multimember protein complexes, such as the DISC and the apoptosome, required for signal transduction purposes. This project aimed to tackle the specific structural parameters of the DAPk-DD; an important step in understanding the biological function of this enzyme. An efficient protocol was developed for the production of milligram quantities of human DAPk-DD in a stable and soluble form. Purification and buffer optimization provided the necessary sample homogeneity and high concentration solubility required for downstream applications, respectively. The multimeric state of DAPk-DD was assessed hydrodynamically, while biophysical techniques such as NMR, CD spectroscopy and fluorimetry were used to explore the 3D shape of DAPk-DD. Further analysis was performed on the in vitro interaction of the DAPk-DD with a newly identified partner, extracellular signal-regulated kinase (ERK2). GST pull-down assays highlighted the necessity of an intact DAPk-DD for interaction with ERK2, while ITC estimated the ratio and affinity of the complex. CD spectroscopy was also performed on the complex to monitor structural changes upon mutual binding. Overall, the DAPk-DD proved to have an unusual structural behaviour not previously seen in other members of the death domain superfamily that puts into question its classification as a classic DD.

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