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 activation is dependent on Ca2+/calmodulin (CaM) and involves an intrinsically disordered regulatory loop.
• Unlike other CaM-dependent kinases, eEF-2K stabilizes an active state upon CaM binding, with T348 autophosphorylation being crucial for full activity.

Purpose of the Study:

• To investigate the role of S500 phosphorylation in eEF-2K regulation.
• To elucidate the synergistic effects of T348 and S500 phosphorylation on eEF-2K activity and CaM binding.
• To understand how these modifications integrate upstream signals.

Main Methods:

• Site-directed mutagenesis (S500D phosphomimetic mutant).
• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe conformational changes.
• In vitro kinase assays to measure eEF-2K activity and CaM binding.
• Cell-based assays to assess eEF-2 phosphorylation.

Main Results:

• The S500D mutation enhanced CaM binding in T348-phosphorylated eEF-2K and increased CaM-independent activity.
• Phosphorylation at both T348 and S500 synergistically stabilized an active-like conformation, increasing CaM responsiveness.
• Deletion of residues near S500 mimicked S500D effects, suggesting an inhibitory role for this segment.
• HDX-MS revealed CaM-dependent conformational changes near S500, indicating relief of inhibitory constraints.

Conclusions:

• Phosphorylation at T348 and S500 synergistically regulate eEF-2K by stabilizing an active conformation and lowering the Ca2+/CaM activation threshold.
• This dual phosphorylation allows eEF-2K to integrate Ca2+, cAMP/PKA, and metabolic signals for precise control of translational elongation.
• S500 phosphorylation may also play a role in eEF-2K degradation, suggesting a dual regulatory function.