Phosphorylation of Plant Translation Initiation Factors by CK2 Enhances the in Vitro Interaction of Multifactor Complex Components

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 eIF2␣, eIF2␤, eIF5, and wheat eIF3c. Native wheat eIF5 and eIF2␣ 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.Translation initiation is a critical, rate-limiting step in eukaryotic gene expression, with a key step early in the pathway being the assembly of the 43 S preinitiation complex (1, 2). This 43 S preinitiation complex is composed of the small 40 S ribosomal subunit, eIF1, eIF1A, the eIF2-GTP-Met-tRNA i Met ternary complex, eIF3, and eIF5. Biochemical data from yeast suggest that a multifactor complex (MFC) 2 consisting of eIF3-eIF1-eIF5-eIF2-GTP-Met-tRNA i Met exists free of the ribosome allowing these factors to be simultaneously loaded onto the 40 S ribosomal subunit (3). Mutations that disrupt the interaction of factors within this complex result in substantial reductions in translation initiation (3, 4). 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 MFC has resulted in a provisional model of the yeast MFC (5). The yeast eIF3c (NIP1) N-terminal domain plays a particularly important role in binding to other initiation factors in the context of the multifactor complex (6). The NIP1 N-terminal domain was found to bind directly to both eIF1 and eIF5 (7,8), and it is suspected that this binding coordinates the interaction between eIF1 and eIF5 that is responsible for the inhibition of GTP hydrolysis at non-AUG codons (6). The C-terminal domain (CTD) of yeast eIF5 simultaneously binds to both the N terminus of eIF3c, the N-terminal K-boxes of eIF2␤, and eIF1 (3, 9). This creates an indirect link between eIF3c and eIF2␤,...

Phosphorylation by CK2 Enhances the Rapid Light-induced Degradation of Phytochrome Interacting Factor 1 in Arabidopsis

“…These include translation initiation factors (e.g. eIF2␣, eIF2␤, eIF3c, eIF4B, and eIF5) (40,41), a chromatin remodeling enzyme (histone deacetylase 2B) (40), circadian clock components (e.g. CCA1 and LHY) (33,36,37,46), HMBG proteins from maize and Arabidopsis (47), abscisic acid responsive protein Rab17 in maize (48), and positively acting transcription factors (e.g.…”

“…Effect of Different Subunit Composition on the Phosphorylation of PIF1-Because the Arabidopsis genome encodes four ␣ and four ␤ subunits of CK2 (39), we investigated whether different CK2 ␣ and ␤ subunit combinations have differential phosphorylation activity toward PIF1 as described (40,41). Results show that all the ␣␤ holoenzyme combinations had stronger activity than either of the two ␣ subunits alone.…”

“…Mapping CK2 Phosphorylation Sites in PIF1-The identification of CK2 phosphorylation sites in PIF1 was essentially performed as described (41). Briefly, the phosphopeptides were enriched using an iron-based affinity resin (Phos-Select, Sigma), followed by analysis on a MALDI-TOF/TOF mass spectrometer (4700 proteomics analyzer, ABSciex, Foster City, CA).…”

“…The vector was transformed into an Arctic DE3 cell for protein expression and purification using the His tag as described (40,41). CK2 phosphorylation assays were performed as described (40,41).…”

Novel insights into the architecture and protein interaction network of yeast eIF3

“…In yeast, eIF3, eIF2, eIF5, and eIF1 are shown to bind to each other to form a multifactor complex (MFC). MFC formation was also observed in plants (Dennis et al 2009) and in humans (Sokabe et al 2012). This preassembled complex then binds to the 40S ribosome to form the 43S preinitiation complex (43S PIC) (Asano et al 2000).…”