Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Eukaryotic elongation factor-2 kinase (eEF-2K) regulates protein synthesis by phosphorylating eEF-2.
• eEF-2K activity is modulated by Ca2+/calmodulin (CaM) and upstream signaling pathways like PKA and mTOR.
• Serine 500 (S500) is a key phosphorylation site on eEF-2K, but its functional impact was unclear.

Purpose of the Study:

• To investigate the functional consequences of S500 phosphorylation on eEF-2K activity.
• To elucidate the interplay between S500 phosphorylation, T348 phosphorylation, and CaM binding.
• To understand how S500 modification influences eEF-2K conformation and activation.

Main Methods:

• Site-directed mutagenesis (S500D) to mimic phosphorylation.
• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to study conformational changes.
• Enzyme activity assays to measure kinase function.
• Deletion mutagenesis to assess the role of S500 and surrounding residues.

Main Results:

• S500 phosphorylation (mimicked by S500D) enhances eEF-2K intrinsic activity, synergizing with T348 phosphorylation.
• CaM binding induces conformational changes near S500, enhancing kinase activity.
• Deletion of S500 and adjacent residues also promotes CaM-independent activity.
• S500 phosphorylation increases binding affinity for both apo-CaM and Ca2+/CaM.

Conclusions:

• S500 phosphorylation, similar to CaM binding, likely relieves an inhibitory constraint on eEF-2K.
• Phosphorylation at both T348 and S500 stabilizes the active conformation of eEF-2K.
• S500 phosphorylation may prime eEF-2K for rapid activation by Ca2+ transients.