Phosphorylation of maize eif5a by ck2: identification of phosphorylated residue and influence on intracellular localization of eif5a.

Abstract

Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with catalytic  subunit of protein kinase CK2 and is phosphorylated by this enzyme. Phosphorylated ZmeIF5A was also identified after separation of maize leaf proteins by two-dimensional electrophoresis. Multiple sequence alignment of eIF5A proteins showed that in monocots, in contrast to other eukaryotes, there are two serine/threonine residues that could potentially be phosphorylated by CK2. To identify the phosphorylation site(s) of ZmeIF5A, the serine residues potentially phosphorylated by CK2 were mutated. ZmeIF5A and its mutated variants Ser2Ala and Ser4Ala were expressed in E. coli and purified. Of these recombinant proteins, only ZmeIF5ASer2Ala was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A Ser2Ala mutants were not phosphorylated despite effective phosphorylation of wild-type variants. A newly developed method exploiting the specificity of thrombin cleavage was used to confirm that Ser2 in ZmeIF5A is indeed phosphorylated. To find a role of the Ser2 phosphorylation, ZmeIF5A and its variants mutated at Ser2 (Ser2Ala and Ser2Asp) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. While wild-type ZmeIF5A and its Ser2Ala variant were distributed evenly between the nucleus and the cytoplasm, the variant with Ser2 replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser2 plays a role in the regulation of nucleocytoplasmic shuttling of eIF5A in plant cells.