Elongation factor-2 kinase: immunological evidence for the existence of tissue-specific isoforms

Abstract

eEF-2 kinase is a ubiquitous Ca 2+/calmodulin-dependent protein kinase that is specific for protein synthesis elongation factor-2 (eEF-2). This study describes an improved procedure for the purification of eEF-2 kinase from rabbit reticulocyte lysate. The eEF-2 kinase preparation was used to raise polyclonal antibodies, which immunoprecipitated eEF-2 kinase protein and activity from rabbit reticulocyte lysate. The antibodies recognized a single 103 kDa band in extracts from several cell lines including NIH 3T3, PC12, C6 glioma, HeLa, and MCF-7 breast carcinoma. However, there was no immunoreactivity in extracts of rabbit or bovine liver or rabbit kidney despite the presence of abundant eEF-2 kinase activity in these tissues. Exposure of PC12 cells to nerve growth factor (NGF) resulted in rapid down-regulation of eEF-2 kinase activity and a decrease in immunoreactivity. After 24 h of incubation with NGF, the activity of the kinase recovered to 80% of initial values. In contrast, the immunoreactivity of eEF-2 kinase continued to decrease. These data suggest that tissue-specific isoforms of eEF-2 kinase may exist and that these isoforms may be regulated by growth factors.