Abstract
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.
Highlights
Using a variety of in vitro binding assays, two-hybrid analysis, and in vivo purification of affinity-tagged subunits, an extensive analysis of the subunit interactions within the yeast multifactor complex (MFC) has resulted in a provisional model of the yeast MFC (5)
The predicted binding domains from yeast appear to be conserved in plant initiation factors when analyzed by multiple sequence alignment; evidence of multifactor complex interactions in plants only comes from the anecdotal observations of co-purification (41)
This study presents data to show that plant initiation factors do form multifactor complexes similar to those in yeast
Summary
Expression, and Purification of Recombinant Translation Initiation Factors and CK2. EIF3c—To obtain recombinant wild-type wheat eIF3c without a His tag, the full-length cDNA for eIF3c was PCR-amplified from expressed sequence tag BJ257961 and cloned into pET23d(ϩ) using NcoI/SacI restriction sites. This construct was transformed into Arctic Express DE3(RIL) E. coli cells (Stratagene) and expressed as described previously for HiseIF3c (41). To express wild-type wheat eIF5, which did not possess a His tag, the eIF5 coding region of His-eIF5pET15b was cleaved with Nde/BamHI and cloned into pET23b(ϩ) This construct was transformed into BL21(DE3) E. coli, and wild-type wheat eIF5 was expressed and purified as described previously for A. thaliana eIF5 (41). Initiation factors containing the desired mutations were expressed in BL21(DE3) E. coli and purified as described previously for the wild-type forms of each protein (20, 21)
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