Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 To Regulate Its Activity and Calcium Dependence

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca2+ and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was co-expressed with λ-phosphatase, and purified from bacteria in a three-step protocol using a calmodulin-affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca2+/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0–180 minutes. The following five major autophosphorylation sites were identified, Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500. In the presence of Ca2+/CaM, robust phosphorylation of Thr-348 occurs within seconds of adding MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348, and is associated with calcium-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K calcium-independent. Surprisingly, this calcium-independent activity requires the presence of calmodulin.

A Conserved Loop in the Catalytic Domain of Eukaryotic Elongation Factor 2 Kinase Plays a Key Role in Its Substrate Specificity

“…For eEF2K, this is an intramolecular reaction (14), and at extended times, several moles of phosphate are incorporated per mole of eEF2K protein (13,15). However, in our studies, only two major sites of autophosphorylation were evident (13), suggesting that further autophosphorylation involves lowlevel phosphorylation at multiple sites.…”

“…eEF2K and other ␣-kinases undergo autophosphorylation at multiple sites (10)(11)(12)(13)(14)(15). For eEF2K, this is an intramolecular reaction (14), and at extended times, several moles of phosphate are incorporated per mole of eEF2K protein (13,15).…”

Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases

“…The center of the polypeptide is predicted to be unstructured and may act as a flexible linker between the two main folded regions of the protein (Figure 1). eEF2K undergoes autophosphorylation, with a major site being Thr-348 (of the sequence of human eEF2K [9,10] ). This position corresponds to an autophosphorylation site in the related enzyme myosin heavy chain kinase (MHCK), which, when phosphorylated, docks into a binding pocket to allow the enzyme to adopt an active conformation [11] .…”

EEF2K (eukaryotic elongation factor 2 kinase)

“…Phosphorylation of eEF2 on Thr-56 disrupts the interaction between eEF-2 and the ribosome, leading to reduced protein synthesis. eEF-2K is regulated by phosphorylation by multiple signaling pathways and kinases at 11 different phosphorylation sides (Ryazanov et al, 1988;Carlberg et al, 1990;Abramczyk et al, 2011;Browne et al, 2004;Marshall et al, 2012;Chafouleas et al, 1981;Bowden et al, 2013). Hypoxia, nutrient deprivation and metabolic stress are all known to stimulate eEF-2K through activation of AMPK (Chafouleas et al, 1981).The activity of eEF-2K is increased in rapidly proliferating malignant cells and in cancer specimens, but is absent in normal adjacent tissues (Ashour et al, 2014b).…”