Calcium-regulated proteolysis of eEF1A

Eukaryotic elongation factor 1 alpha (eEF1A) can be post-translationally mo dified by the addition of phosphorylglycerylethanolamine (PGE). [C-14]Ethan olamine was incorporated into the PGE modification, and with carrot (Daucus carota L.) suspension culture cells, eEF1A was the only protein that incor porated detectable quantities of [C-14]ethanolamine (Ransom et al., 1998). When 1 mM CaCl2 was added to microsomes containing [C-14]ethanolamine-label ed eEF1A ([C-14]et-eEF1A), there was a 60% decrease in the amount of [C-14] et-eEF1A recovered after 10 min. The loss of endogenous [C-14]et-eEF1A was prevented by adding ECTA. Recombinant eEF1A, which did not contain the PGE modification, also was degraded by microsomes in a Ca2+-regulated manner, i ndicating that PGE modification was not necessary for proteolysis; however, it enabled us to quantify enodgenous eEF1A. By monitoring [C-14]et-eEF1A, we found that treatment with phospholipase D or C, but not phospholipase A( 2), resulted in a decrease In [C-14]et-eEF1A from carrot microsomes. The fa ct that there was no loss of [C-14]et-eEF1A with phospholipase A(2) treatme nt even in the presence of 1 mM Ca2+ suggested that the loss of membrane li pids was not essential for eEF1A proteolysis and that lysolipids or fatty a cids decreased proteolysis. At micromolar Ca2+ concentrations, proteolysis of eEF1A was pH sensitive. When 1 mu M CaCl2 was added at pH 7.2, 35% of [C -14]et-eEF1A was lost; while at pH 6.8, 10 mu M CaCl2 was required to give a similar loss of protein. These data suggest that eEF1A may be an importan t downstream target for Ca2+ and lipid-mediated signal transduction cascade s.