Phosphorylation by kinases governs key cellular and extracellular processes, such as transcription, cell cycle progression, differentiation, secretion and apoptosis. Precise kinase regulation is a prerequisite for normal cell functioning and kinase dysregulation often leads to disease. Many kinases are regulated through protein-protein interactions, often mediated by phosphorylated motifs and involving associations with the scaffolding and chaperon protein 14-3-3. Such mode of regulation has already been demonstrated for more than 30 kinases, and more than 170 kinases contain phosphorylated motifs closely resembling 14-3-3-binding sites. However, many details concerning these interactions, especially the exact role of 14-3-3 binding and the mechanism of action of 14-3-3-mediated kinase regulation, remain elusive because only a few 14-3-3:kinase complexes have been structurally characterized so far.
This project aims to study the 14-3-3 protein-dependent inhibition of Apoptosis signal-regulating kinase 1 (ASK1) and Death-associated protein kinase 2 (DAPK2).
ASK1 belongs to a group of mitogen-activated protein kinase kinase kinases (MAP3Ks). The aberrantly enhanced ASK1-MAPK signaling leads to many disorders and neurological diseases, such as amyotrophic lateral sclerosis, multiple sclerosis and Parkinson’s, Alzheimer´s, and Huntington´s disease. Under normal, non-stress conditions, inactive ASK1 interacts with several other proteins, including thioredoxin (TRX) and 14-3-3, forming a high molecular mass complex known as the ASK1 signalosome. However, the role of 14-3-3 in the inhibition of ASK1 is still unclear. Low-resolution structural analysis has indicated that 14-3-3 might inhibit ASK1 by modulating the structure of its active site, by sterically blocking the phosphorylation of the activation loop and by blocking the interactions between ASK1 and its substrates.
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 the 14-3-3 protein. The mechanism of the 14-3-3-mediated DAPK2 inhibition has not been elucidated yet. Our recent results have suggested that the 14-3-3 protein might stabilize the DAPK2 dimerization and protect regulatory phosphorylation sites of DAPK2 against dephosphorylation.
In both cases, the high-resolution structures are needed to fully understand the exact role of 14-3-3 in the regulation of these protein kinases. Therefore we propose to investigate the structural basis for the inhibition of these protein kinases by performing the structural analysis of their complexes with 14-3-3 by a combination of single particle cryo-electron microscopy (cryo-EM), protein crystallography and fluorescence spectroscopy. The acquired results will enable to decipher the molecular mechanism of the allosteric regulation of ASK1 and DAPK2 by 14-3-3 and aid in design of novel alternative strategies to suppress the activity of these kinases.
Specific aims are:
The structural biology of 14-3-3 protein complexes is a long-term research interest of prof. T. Obsil’s group and the proposed project builds on the results of previous research done by this group.
Funding and project approval:
Co-founding resources will be provided by the Dept. of Physical and Macromolecular Chemistry.
Required application materials:
How to submit application materials:
Please send email with the required application materials to Prof. Tomáš Obšil: firstname.lastname@example.org, Faculty of Science, Charles University, Prague.
The application deadline is July 23, 2021.
For more information please visit the webpage of the JUNIOR Fund project of the Charles University.