Phosphorylation of maize eukaryotic translation initiation factor 5A (eIF5A) by casein kinase 2: identification of phosphorylated residue and influence on intracellular localization of eIF5A.
Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with the catalytic alpha 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.
Łebska
Maja
M
Institute of Biochemistry and Biophysics, Polish Academy of Science, Pawińskiego 5a, 02-106 Warsaw, Poland.
Ciesielski
Arkadiusz
A
Szymona
Lidia
L
Godecka
Luiza
L
Lewandowska-Gnatowska
Elzbieta
E
Szczegielniak
Jadwiga
J
Muszynska
Grazyna
G
D017360:Arabidopsis / Q000378:metabolism*; D001665:Binding Sites; D047390:Casein Kinase II / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012639:Seeds; D012694:Serine / Q000378:metabolism; D003313:Zea mays / Q000378:metabolism; D000097574:Eukaryotic Translation Initiation Factor 5A
D047390:Casein Kinase II; D010448:Peptide Initiation Factors; D010940:Plant Proteins; D016601:RNA-Binding Proteins; D012694:Serine; D000097574:Eukaryotic Translation Initiation Factor 5A