Calcium-regulated proteolysis of eEF1A

Eukaryotic elongation factor 1alpha (eEF1A) can be post-translationally modified by the addition of phosphorylglycerylethanolamine (PGE). [(14)C]Ethanolamine was incorporated into the PGE modification, and with carrot (Daucus carota L.) suspension culture cells, eEF1A was the only protein that incorporated detectable quantities of [(14)C]ethanolamine (Ransom et al., 1998). When 1 mM CaCl(2) was added to microsomes containing [(14)C]ethanolamine-labeled eEF1A ([(14)C]et-eEF1A), there was a 60% decrease in the amount of [(14)C]et-eEF1A recovered after 10 min. The loss of endogenous [(14)C]et-eEF1A was prevented by adding EGTA. Recombinant eEF1A, which did not contain the PGE modification, also was degraded by microsomes in a Ca(2+)-regulated manner, indicating that PGE modification was not necessary for proteolysis; however, it enabled us to quantify enodgenous eEF1A. By monitoring [(14)C]et-eEF1A, we found that treatment with phospholipase D or C, but not phospholipase A(2), resulted in a decrease in [(14)C]et-eEF1A from carrot microsomes. The fact that there was no loss of [(14)C]et-eEF1A with phospholipase A(2) treatment even in the presence of 1 mM Ca(2+) suggested that the loss of membrane lipids was not essential for eEF1A proteolysis and that lysolipids or fatty acids decreased proteolysis. At micromolar Ca(2+) concentrations, proteolysis of eEF1A was pH sensitive. When 1 microM CaCl(2) was added at pH 7.2, 35% of [(14)C]et-eEF1A was lost; while at pH 6.8, 10 microM CaCl(2) was required to give a similar loss of protein. These data suggest that eEF1A may be an important downstream target for Ca(2+) and lipid-mediated signal transduction cascades.