Alignment of eukaryotic translation initiation factor 5A (eIF5A) sequences has shown, for plants, in contrast to most other eukaryotes, the presence of N-terminal serine residue (Ser2) which could be phosphorylated by CK2. Using point directed mutagenesis, we demonstrate here that in recombinant maize ZmeIF5Awt Ser2 is exclusively phosphorylated by catalytic subunit of CK2 (CK2α), whereas its mutated variant Ser2Ala is not phosphorylated. To shed light on the physiological significance of this Ser2 phosphorylation, transient expression of fluorescence-labeled proteins was performed in maize protoplast. Wild-type ZmeIF5A was distributed evenly between nucleus and cytoplasm, but the replacement of Ser2 by aspartic acid, which mimics the phosphorylated serine, influences its intracellular localization. We postulate that phosphorylation of Ser2 in maize eIF5A, and most probably in other plant cells, plays a role in specific regulation of nuclear export of eIF5A-bound mRNAs.
Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with the 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 S2A and S4A were expressed in Escherichia coli and purified. Of these recombinant proteins, only ZmeIF5A-S2A was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A-S2A 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 Ser 2 in ZmeIF5A is indeed phosphorylated. To find a role of the Ser 2 phosphorylation, ZmeIF5A and its variants mutated at Ser 2 (S2A and S2D) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. Although wild-type ZmeIF5A and its S2A variant were distributed evenly between the nucleus and cytoplasm, the variant with Ser 2 replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser 2 plays a role in regulation of nucleocytoplasmic shuttling of eIF5A in plant cells. Protein kinase CK22 (formerly known as casein kinase 2) is a ubiquitously expressed serine/threonine protein kinase present in all eukaryotes that displays extraordinary evolutionary conservation (for review, see Ref. 1). In contrast to most other protein kinases, CK2 is constitutively active, not regulated by a second messenger, and can use both ATP and GTP as phosphate donors. CK2 is most often present as a tetramer composed of two catalytic ␣ and two regulatory  subunits (for review, see Ref.2). CK2 is a pleiotropic kinase with a growing list of more than 300 substrates (for review, see Ref.3). A characteristic feature of CK2 phosphorylation sites are multiple acidic residues located mostly downstream of the phosphorylatable amino acid (4). The striking variety of its cellular targets and its absolute requirement for cell viability suggest that CK2 plays essential role(s); it has been implicated in many cellular functions with special reference to proliferation, gene expression, and RNA and protein synthesis (for review, see Ref.3).Studies concerning functions of plant CK2s have shown that this enzyme is involved in light-regulated gene expression and growth control (5), cell division, and cell expansion (6 -8). CK2 phosphorylates transcription factors (G-box-binding and bZiP factors) involved in photomorphogenesis (9 -11). Opaque2, a basic leucine zipper transcriptional activ...
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