Phosphorylation of plant translation initiation factors by CK2
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Protein kinase CK2 phosphorylates wheat eIF2, eIF3, eIF4B, eIF5 and three 60S ribosomal proteins. The substrate specificity of CK2[alpha] toward various plant initiation factor substrates was altered in vitro through holoenzyme formation in the presence of regulatory [beta]-subunits. This presents a potential mechanism through which the differential expression and sub-cellular distribution of CK2 [beta]-subunits could regulate phosphorylation of various CK2 substrates in plants. Our analysis of initiation factor phosphopeptides produced by in vitro phosphorylation identified 20 CK2 phosphorylation sites in eIF2[alpha], eIF2[beta], eIF3c, eIF4B, and eIF5. Native wheat eIF5 was prepared in the presence of phosphatase inhibitors and analyzed by mass spectrometry. Native wheat eIF5 was determined to be a phosphoprotein containing at least 3 phosphorylation sites. The C-terminal CK2 site (S451) of native eIF5 was completely phosphorylated, and tryptic fragments containing the other in vitro CK2 two sites (S209, T240) also appear to be partially phosphorylated. Many of the CK2 phosphorylation sites identified are in conserved binding domains of the yeast multifactor complex (eIF1/eIF3/eIF5/eIF2/GTP/Met-tRNAi[superscript Met). This observation lead to the hypothesis that CK2 phosphorylation may regulate the formation of plant multifactor complexes. The results presented here suggest that plant initiation factors are capable of forming complexes similar to those previously reported in yeast. The in vitro interaction of initiation factors within these complexes appears to be enhanced by phosphorylation of eIF2, eIF3c, and eIF5 by CK2. Site-directed mutagenesis of eIF5 to remove CK2 phosphorylation sites not only prevents the CK2 mediated increase in interaction with eIF1, but also resulted in reduced stimulation of translation initiation in vitro.