Tyrosine phosphorylation of elongation factor 2 in chick embryonic cells

Abstract

Protein Phosphorylation and dephosphorylation have been generally accepted as one of the important mechanisms in the regulation of diverse cellular reactions. Although the mechanisms of action remain still equivocal, tyrosine phosphorylation of proteins have been reported to be particularly implicated in cell proliferation and differention. This assumption is based on the observation that several retroviral oncogenes are related to tyrosine specific protein kinases and that some growth factor receptors also show tyrosine kinase activities. To understand the role of tyrosine phosphorylation, it is essential to find critical target proteins of tyrosine phosphorylation and to characterize their biochemical properties. To this end, 32P-labeled tyrosine phosphorylated proteins have been searched in chick embryo. An endogenous 95 KDa chick embryo cytosolic protein with pI 6.8-7.0 was phosphorylated in vitro in the presence of [$\gamma$ -32P]ATP. Phosphorylation of the protein was prominent in the embryos of early developmental stage. While the 95 KDa protein was present in the cytosol, the kinase activity for the protein was mostly associated with the particulate fraction. Hydrolysis of the phosphorylated protein yielded phosphotyrosine in addition to phosphothreonine and phosphoserine. Native 95 KDa protein was also phosphorylated on its tyrosine residue. The tyrosine-phosphorylated 95 Kda protein was purified by DEAE-Sephacel chromatography and immunoprecipitation with antiphosphotyrosine antibody and its amino acid sequence was determined. The obtained N-terminal sequence, Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala- Ile-Met-Asp-Lys-Lys-Asn-Ile-Arg-Asn-Met-, was found to be identical to that of elongation factor 2 (EF-2) of both rat and hamster. Our results show that EF-2 in chick embryo is phosphorylated on its tyrosine residue and suggest the presence of other EF-2 kinase in chick embryo cells than the previously reported $Ca^{2+}$/calmodulin-dependent protein kinas...