Abstract
Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent ‘D-motif’ located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.
Highlights
The expression of the gene encoding death-associated protein kinase (DAPk; HUGO Gene Nomenclature Committee identifier: DAPK1) was originally identified as being essential for the execution of apoptosis induced by IFN2c [1]
Rather we found that the protein was poorly behaved and that it was a requirement to append the DAPk-death domain (DD) with a solubility enhancement tag in order to recover protein sufficiently tractable for biophysical analysis
The GB1DAPk-DD fusion proteins were soluble, size exclusion chromatography analysis showed that the DAPk-DD fusion constructs exhibited a tendency to reversible self-association, and that this was attributable to the DAPk-DD component
Summary
The expression of the gene encoding death-associated protein kinase (DAPk; HUGO Gene Nomenclature Committee identifier: DAPK1) was originally identified as being essential for the execution of apoptosis induced by IFN2c [1]. Research has shown that DAPk acts as a positive mediator of cell death induced by a range of stimuli including tumor necrosis factor-a (TNF-a), anti-CD95/Fas agonists, transforming growth factor-b (TGF-b), the dependence receptor UNC5H2 in the absence of netrin-1, short-chain acyl ceramide derivatives, endoplasmic reticulumstress [2], detachment from the extracellular matrix and oncogeneinduced hyperproliferative signals [3,4,5,6,7,8,9,10] Together these observations suggest that DAPk functions at an important point of integration for different cell death signaling pathways. Cell death was attenuated in HEK 293 cells co-transfected with the expression constructs DAPk/DCaM and DAPk-DD (comprising just the death domain residues), suggesting that the over-expressed DAPk-DD can act in a dominant negative fashion perhaps by sequestering factors required for endogenous DAPk activity [4]
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