Elongation factor 2 as the major substrate for Ca2+/calmodulin-dependent protein kinase in rat adrenal glomerulosa...

We have previously shown the existence of the major substrate protein of Mr 100,000 (substrate 100 K protein) for Ca2+/calmodulin (CaM)-dependent protein kinase in rat adrenal glomerulosa cells. In the present study, the identity of the substrate 100 K protein to elongation factor 2 (EF-2) was investigated. In a 105,000 g-supernatant fraction (cytosol), the protein of Mr 100,000 with the pI (isoelectric point) value of 6.7 was phosphorylated in the presence of calcium and CaM. The optical densities of this phosphorylated band were greatly enhanced in the presence of the EF-2 purified from pig liver (1 microgram) [20-23-fold, n = 5] when compared with those in the absence of the component. In the presence of the purified EF-2, the phosphorylation of Mr 100,000 was detected only in the presence of calcium alone or calcium plus CaM. This phosphorylation in the presence of calcium alone was completely inhibited in the presence of the CaM antagonist pimozide (500 microM), showing the existence of endogenous CaM in the cytosol. In the same fraction, the ADP-ribosylated protein of Mr 100,000 was detected in the presence of diphtheria toxin (fragment A) and (adenylate-32P) NAD, indicating the presence of EF-2 in the cytosol from rat adrenal glomerulosa cells. These results suggest that the substrate 100 K protein may be identical to EF-2 in rat adrenal glomerulosa cells.