Identification of a 100-kDa phosphoprotein in developing murine embryos as elongation factor 2

Protein phosphorylation is a key regulatory mechanism for several functions. Although the complex control of organogenesis and growth most likely includes such mechanisms, few reports have examined protein phosphorylation in the developing mammal. The identification and characterization of mammalian embryonic phosphoproteins will allow a greater understanding of the regulation and mechanisms of developmental processes. Phosphorylation of the endogenous mouse proteins during development revealed a 100-kDa protein, located in the cytosolic fraction, to be the major substrate. The Ca(2+)-calmodulin kinase inhibitors, trifluorperazine and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, inhibited this phosphorylation. Inhibitors of protein kinase C (H-7)- and cAMP-dependent protein kinase, as well as the tyrosine kinase inhibitor, genistein, had no effect. One- and two-dimensional phosphoamino acid analysis indicated that phosphothreonine was the major phosphorylated amino acid. To determine the identity of this protein, the 100-kDa band was isolated and submitted for amino acid analysis and N-terminal sequencing. The N-terminal sequence Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp-Lys, was identical to the N-terminal sequence of human, hamster and rat elongation factor 2 (EF-2). Western blotting analysis confirmed that the 100 kDa protein was EF-2. Our results of phosphorylated EF-2 in the developing mouse are in agreement with those reported in the avian embryo. However, our results differ in that phosphotyrosine detected in avian embryos could not be detected in murine embryos. This is the first report to demonstrate EF-2 in the developing mammalian embryo and its specific phosphorylation pattern. Our data suggest that the functional phosphoregulation of elongation factor 2 during protein synthesis in mammals is conserved from the developing embryo to the adult and thus emphasizes the importance of EF-2 in normal development and survival.