Phosphorylation of plant translation initiation factors by CK2 enhances the in vitro interaction of multifactor complex components.
CK2 phosphorylates a wide variety of substrates, including translation initiation factors. A mass spectrometric approach was used to identify residues phosphorylated by CK2, which may regulate the activity of initiation factors during the translation initiation process in plants. CK2 in vitro phosphorylation sites were identified in wheat and Arabidopsis thaliana eIF2alpha, eIF2beta, eIF5, and wheat eIF3c. Native wheat eIF5 and eIF2alpha were found to have phosphorylation sites that corresponded to some of the in vitro CK2 phosphorylation sites. A large number of the CK2 sites identified in this study are in conserved binding domains that have been implicated in the yeast multifactor complex (eIF1-eIF3-eIF5-eIF2-GTP-Met-tRNA(i)(Met)). This is the first study to demonstrate that plant initiation factors are capable of forming a multifactor complex in vitro. In addition, the interaction of factors within these complexes was enhanced both in vitro and in native extracts by phosphorylation of one or more initiation factors by CK2. The importance of CK2 phosphorylation of eIF5 was evaluated by site-directed mutagenesis of eIF5 to remove CK2 phosphorylation sites. Removal of CK2 phosphorylation sites from eIF5 inhibits the CK2-mediated increase in eIF5 interaction with eIF1 and eIF3c in pulldown assays and reduces the eIF5-mediated stimulation of translation initiation in vitro. These results suggest a functional role for CK2 phosphorylation in the initiation of plant translation.
Dennis
Michael D
MD
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, USA.
Person
Maria D
MD
Browning
Karen S
KS
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D047390:Casein Kinase II / Q000378:metabolism*; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism*; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D016415:Sequence Alignment; D013379:Substrate Specificity; D014908:Triticum / Q000378:metabolism*
D046912:Multiprotein Complexes; D050505:Mutant Proteins; D010448:Peptide Initiation Factors; D010455:Peptides; D047390:Casein Kinase II