Enzyme activity is regulated by several mechanisms, including phosphorylation. Phosphorylation is a key signal transduction process in all eukaryotic cells and is thus crucial for virtually all cellular processes. In addition to its direct effect on protein structure, phosphorylation also affects protein-protein interactions, such as binding to scaffolding 14-3-3 proteins, which selectively recognize phosphorylated motifs. These interactions then modulate the catalytic activity, cellular localisation and interactions of phosphorylated enzymes through different mechanisms. The aim of this mini-review is to highlight several examples of 14-3-3 protein-dependent mechanisms of enzyme regulation previously studied in our laboratory over the past decade. More specifically, we address here the regulation of the human enzymes ubiquitin ligase Nedd4-2, procaspase-2, calcium-calmodulin dependent kinases CaMKK1/2, and death-associated protein kinase 2 (DAPK2) and yeast neutral trehalase Nth1.

• MeSH
• Phosphorylation MeSH
• Humans MeSH
• 14-3-3 Proteins * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• 14-3-3 Proteins * MeSH

Ca2+ /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca2+ /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca2+ /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.

• Keywords
• 14-3-3 proteins, CaMKK, SAXS, calcium/calmodulin-dependent protein kinase, fluorescence spectroscopy, hydrogen/deuterium exchange coupled to MS, protein-protein interaction,
• MeSH
• X-Ray Diffraction MeSH
• Phosphorylation MeSH
• Catalytic Domain MeSH
• Calcium-Calmodulin-Dependent Protein Kinase Kinase * chemistry   metabolism   MeSH
• Scattering, Small Angle MeSH
• 14-3-3 Proteins * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Calcium-Calmodulin-Dependent Protein Kinase Kinase * MeSH
• 14-3-3 Proteins * MeSH

Phosphorylation by kinases governs many key cellular and extracellular processes, such as transcription, cell cycle progression, differentiation, secretion and apoptosis. Unsurprisingly, tight and precise kinase regulation is a prerequisite for normal cell functioning, whereas kinase dysregulation often leads to disease. Moreover, the functions of many kinases are regulated through protein-protein interactions, which in turn are mediated by phosphorylated motifs and often involve associations with the scaffolding and chaperon protein 14-3-3. Therefore, the aim of this review article is to provide an overview of the state of the art on 14-3-3-mediated kinase regulation, focusing on the most recent mechanistic insights into these important protein-protein interactions and discussing in detail both their structural aspects and functional consequences.

• Keywords
• 14-3-3, ASK1, CaMKK2, LRRK2, PI4KB, PKC, RAF kinase, kinase, phosphorylation,
• MeSH
• Allosteric Regulation genetics   MeSH
• Apoptosis genetics   MeSH
• Phosphorylation genetics   MeSH
• Humans MeSH
• p38 Mitogen-Activated Protein Kinases genetics   MeSH
• Protein Kinases genetics   MeSH
• 14-3-3 Proteins genetics   MeSH
• Signal Transduction genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• p38 Mitogen-Activated Protein Kinases MeSH
• Protein Kinases MeSH
• 14-3-3 Proteins MeSH