IDENTIFICATION OF A 100-KDA PHOSPHOPROTEIN IN DEVELOPING MURINE EMBRYOS AS ELONGATION-FACTOR-2

Protein phosphorylation is a key regulatory mechanism for several func tions. Although the complex control of organogenesis and growth most l ikely includes such mechanisms, few reports have examined protein phos phorylation in the developing mammal. The identification and character ization of mammalian embryonic phosphoproteins will allow a greater un derstanding of the regulation and mechanisms of developmental processe s. Phosphorylation of the endogenous mouse proteins during development revealed a 100-kDa protein, located in the cytosolic fraction, to be the major substrate. The Ca2+-calmodulin kinase inhibitors, trifluoper azine and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic a cid, 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 phospho amino acid analysis indicated that phosphothreonine was the major phos phorylated 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 n-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 an alysis confirmed that the 100 kDa protein was EF-2. Our results of pho sphorylated EF-2 in the developing mouse are in agreement with those r eported in the avian embryo. However, our results differ in that phosp hotyrosine detected in avian embryos could not be detected in murine e mbryos. This is the first report to demonstrate EF-2 in the developing mammalian embryo and its specific phosphorylation pattern. Our data s uggest that the functional phosphoregulation of elongation factor 2 du ring protein synthesis in mammals is conserved from the developing emb ryo to the adult and thus emphasizes the importance of EF-2 in normal development and survival. (C) 1994 Academic Press, Inc.