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
• dimerizace MeSH
• fosforylace MeSH
• lidé MeSH
• proteinkinasy asociované se smrtí genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPK2 protein, human MeSH Prohlížeč
• proteinkinasy asociované se smrtí MeSH
• proteiny 14-3-3 MeSH
• YWHAG protein, human MeSH Prohlížeč

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 regulace MeSH
• fosforylace MeSH
• myši genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy Nedd4 genetika   metabolismus   MeSH
• vazba proteinů MeSH
• WW domény * MeSH
• zvířata MeSH
• Check Tag
• myši genetika   metabolismus   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Nedd4l protein, mouse MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• Sfn protein, mouse MeSH Prohlížeč
• ubikvitinligasy Nedd4 MeSH