Abstract
Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. However, the structural basis of 14-3-3-mediated DAPK2 regulation remains unclear. Here, we structurally and biochemically characterize the full-length human DAPK2:14-3-3 complex by combining several biophysical techniques. The results from our X-ray crystallographic analysis revealed that Thr369 phosphorylation at the DAPK2 C terminus creates a high-affinity canonical mode III 14-3-3-binding motif, further enhanced by the diterpene glycoside Fusicoccin A. Moreover, concentration-dependent DAPK2 dimerization is disrupted by Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca2+/CaM binding. Overall, our findings provide mechanistic insights into 14-3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti‐inflammatory therapies.
Highlights
Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3
Concentration-dependent DAPK2 dimerization is disrupted by Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser[318] against dephosphorylation and preventing Ca2+/CaM binding
DAPK2 is regulated in a Ca2+/calmodulin (Ca2+/CaM)dependent manner and consists of an N-terminal kinase domain (KD, residues 23–285), which shares 80% homology with the kinase domain of DAPK1, followed by an autoinhibitory domain (AID, residues 287–311), a Ca2+/CaM-binding domain (CBD, residues 312–330) and a C-terminal tail with unique properties, lacking all other C-terminal domains of DAPK1 involved in protein–protein interactions (Fig. 1a)[4,5,12]
Summary
Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. The crystal structure of the DAPK2 homodimer has shown that its dimerization interface covers most of the kinase domain and part of the CBD, indicating that homodimerization is likely responsible for DAPK2 inhibition[13,14] This alternative model presents the inactive DAPK2 as a tightly packed homodimer in which both protomers are phosphorylated at Ser[318], their active sites are blocked by AID, and their dimerization is mediated via KDs, including interactions between the basic loop in the kinase Nlobe, a unique feature of DAPKs, and the AID of the opposing protomer[14]
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