Regulation of Elongation Factor-2 Kinase by pH

Abstract

Elongation factor-2 kinase (eEF-2K) is a Ca(2+)/calmodulin-dependent protein kinase that phosphorylates and inactivates eEF-2 and that can regulate the rate of protein synthesis at the elongation stage. Here we report that a slight decrease in pH, within the range observed in vivo, leads to a dramatic activation of eEF-2K. The activity of eEF-2K in mouse liver extracts, as well as the activity of purified recombinant human eEF-2K, is low at pH 7.2-7.4 and is increased by severalfold when the pH drops to 6.6-6.8. eEF-2K requires calmodulin for activity at neutral as well as acidic pH. Kinetic studies demonstrate that the pH does not affect the K(M) for ATP or eEF-2 and activation of eEF-2K at acidic pH is due to an increase in V(max). To analyze the potential role of eEF-2K in regulating protein synthesis by pH, we constructed a mouse fibroblast cell line that expresses eEF-2K in a tetracycline-regulated manner. Overexpression of eEF-2K led to a decreased rate of protein synthesis at acidic pH, but not at neutral pH. Our results suggest that pH-dependent activation of eEF-2K may play a role in the global inhibition of protein synthesis during tissue acidosis, which accompanies such processes as hypoxia and ischemia.

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

“…Furthermore, pharmacological inducers of ER stress such as Tm and thapsigargin (Tg), an inhibitor of the ER-resident Ca 2 þ -dependent ATPase, induced eEF-2 phosphorylation with kinetics similar to those of sal (Supplementary Figure 3). EEF-2 phosphorylation has been observed in response to several stimuli, [26][27][28][29][30][31][32][33] but never in the context of ER stress. Our observations suggest that eEF-2 might be regulated by phosphorylation during ESR.…”

“…[22][23][24] The phosphorylation of eEF-2 is catalyzed by eEF-2 kinase (eEF-2K), an unusual calcium/calmodulin-dependent enzyme belonging to the alpha kinase family of atypical protein kinases. 25 EEF-2 phosphorylation has been shown to play an important role in coupling protein synthesis to energy metabolism in response to calcium flux, 26 cyclic adenosine monophosphate (AMP)/protein kinase A and AMP-activated protein kinase signaling, [27][28][29][30] amino acid or glucose limitation, 31,32 and cytoplasmic pH changes, 33 but no role for eEF-2 phosphorylation in ER stress has been reported.…”

Responses to hypoxia and recovery: repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid,Astronotus ocellatus

“…The reduction of protein synthesis has been linked to an increase in recombinant elongation factor 2 kinase (EF2K) caused by exposure to low pH (Dorovkov et al, 2002). The significant reduction of protein synthesis in liver, brain, heart and gill tissues in hypoxia-exposed A. ocellatus, observed in the present study, combined with the decreases in extracellular and intracellular pH in A. ocellatus exposed to comparable hypoxic conditions (Richards et al, 2007) strengthens the argument of Richards et al, that pH may have a direct effect on protein synthesis and therefore, metabolic rate in A. ocellatus.…”

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

“…Two novel genes identified in humans and mouse were shown to carry an ion channel subdomain fused to an ␣-kinase catalytic domain and were termed channel kinases (ChaK1 and ChaK2) (Dorovkov et al, 2002), also referred to recently as transient receptor potential (TRP)M6 and TRPM7 (Montell et al, 2002). The channel portions of ChaK1 and ChaK2 are homologous to the TRP family of ion channels, and these novel kinase/ion channels have received considerable attention in recent years as divalent cation gates with possible roles in regulation of calcium and magnesium homeostasis, neuronal function, and control of anoxic cell death (Aarts et al, 2003;Montell, 2003;Drennan and Ryazanov, 2004;Chubanov et al, 2004).…”

“…Other human ␣-kinase genes include a series of cDNA clones that have been named based on tissue source, including Lymphocyte ␣-kinase, Heart ␣-kinase, and Muscle ␣-kinase (Ryazanov, 2002). A mouse ␣-kinase closely related to the human "muscle ␣-kinase," named Midori, has been reported to be nuclear in localization and participates in gene expression control during murine heart development (Hosoda et al, 2001).Two novel genes identified in humans and mouse were shown to carry an ion channel subdomain fused to an ␣-kinase catalytic domain and were termed channel kinases (ChaK1 and ChaK2) (Dorovkov et al, 2002) ChaK2 are homologous to the TRP family of ion channels, and these novel kinase/ion channels have received considerable attention in recent years as divalent cation gates with possible roles in regulation of calcium and magnesium homeostasis, neuronal function, and control of anoxic cell death (Aarts et al, 2003;Montell, 2003;Drennan and Ryazanov, 2004;Chubanov et al, 2004). There seems to be no genes encoding ␣-kinases in the genome of Drosophila, Arabidopsis, Saccharomyces cerevisiae, or Schizosaccharomyces pombe.…”