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.

• MeSH
• Dimerization MeSH
• Phosphorylation MeSH
• Humans MeSH
• Death-Associated Protein Kinases genetics   metabolism   MeSH
• 14-3-3 Proteins genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DAPK2 protein, human MeSH Browser
• Death-Associated Protein Kinases MeSH
• 14-3-3 Proteins MeSH
• YWHAG protein, human MeSH Browser

Neural precursor cell expressed developmentally down-regulated 4 ligase (Nedd4-2) is an E3 ubiquitin ligase that targets proteins for ubiquitination and endocytosis, thereby regulating numerous ion channels, membrane receptors and tumor suppressors. Nedd4-2 activity is regulated by autoinhibition, calcium binding, oxidative stress, substrate binding, phosphorylation and 14-3-3 protein binding. However, the structural basis of 14-3-3-mediated Nedd4-2 regulation remains poorly understood. Here, we combined several techniques of integrative structural biology to characterize Nedd4-2 and its complex with 14-3-3. We demonstrate that phosphorylated Ser342 and Ser448 are the key residues that facilitate 14-3-3 protein binding to Nedd4-2 and that 14-3-3 protein binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Overall, our findings provide the structural glimpse into the 14-3-3-mediated Nedd4-2 regulation and highlight the potential of the Nedd4-2:14-3-3 complex as a pharmacological target for Nedd4-2-associated diseases such as hypertension, epilepsy, kidney disease and cancer.

• MeSH
• Down-Regulation MeSH
• Phosphorylation MeSH
• Mice genetics   metabolism   MeSH
• 14-3-3 Proteins genetics   metabolism   MeSH
• Ubiquitination MeSH
• Nedd4 Ubiquitin Protein Ligases genetics   metabolism   MeSH
• Protein Binding MeSH
• WW Domains * MeSH
• Animals MeSH
• Check Tag
• Mice genetics   metabolism   MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nedd4l protein, mouse MeSH Browser
• 14-3-3 Proteins MeSH
• Sfn protein, mouse MeSH Browser
• Nedd4 Ubiquitin Protein Ligases MeSH