Structural and Functional Characterization of the Recombinant Death Domain from Death-Associated Protein Kinase

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.

Tumor-suppressor NFκB2 p100 interacts with ERK2 and stabilizes PTEN mRNA via inhibition of miR-494

“…The protein-protein interaction has also been reported to participate in the modulation of ERK2 phosphorylation. 32 – 34 To test this possibility that p100 interacts with ERK2 to inhibit its phosphorylation, we therefore performed co-immunoprecipitation (Co-IP) assay by using HEK293T cells that expressed HA-ERK2 and Flag-p100. Intriguingly, HA-tagged ERK2 did present in the immunoprecipitates following anti-Flag antibodies pull down of Flag-tagged p100 ( Fig.…”

“…This finding is consistent with previous reports that the members of death domain superfamily, PEA-15 and DAPK, interact with ERK2 and modulate ERK2 phosphorylation. 32 – 34 To further investigate the physiological consequence of this physical interaction between p100 and ERK2 in cells upon serum stimulation, we incubated NFκB2+/+ cells in 20% FBS for 30 min, and performed co-immunoprecipitation assay to pull down endogenous p100 from these cellular extracts by using anti-NFκB2 antibodies. The results showed that serum stimulation led to a substantial decrease in p100-associated ERK2 protein in NFκB2+/+ cells, whereas anti-NFκB2 antibodies failed to capture endogenous ERK2 in NFκB2−/− cells under the same experimental conditions ( Fig.…”

Death‐associated protein kinase (DAPK) family modulators: Current and future therapeutic outcomes

“…Unlike other DDs that consist of six amphipathic α‐helices, DAPK's DD did not adopt the classical compact fold. It is anticipated that when DAPK's DD has been expressed apart from the other DAPK1 domains and in solution, it possesses low‐ordered secondary structure content and had no stable globular fold required to mediate its protein‐protein interaction function, yet this low‐ordered form retained its ability to bind extracellular signal‐regulated kinase (ERK) . DD mediates DAPK1 most protein‐protein interactions and details regarding those interactions shall be discussed in the context of DAPK1 cellular functions.…”

“…The DD only away from other DAPK1 domains was enough for binding to ERK. This binding results in DAPK1 phosphorylation at Ser735, hence increasing the catalytic ability of DAPK1 to phosphorylate MLC . The molecular mechanism behind this elevated catalytic activity through phosphorylation of the distant Ser735 is still largely unknown.…”

The Role of Death Domains Superfamily in Multiple Sclerosis Pathogenesis

“…The primary function of the death domains superfamily is to mediate protein-protein interaction by forming predominantly homotypic associations, thereby underpinning the formation of multi-subunit signaling complexes [34]. Most of the interactions observed in DD complexes seem to fall into one of three topologically similar arrangements as follows: type I, negatively charged residues of H2 and H3 of one DD interact with positively charged residues of H1 and H4 of another DD; type II, H4 and the H4-H5 loop of one DD interact with the N-terminal end of H6 of another DD; and type III, H3 of one DD interacts with the H1-H2 and H3-H4 loops of a second DD [35].…”