Differential Phosphorylation of Plant Translation Initiation Factors by Arabidopsis thaliana CK2 Holoenzymes

Abstract

A previously described wheat germ protein kinase (Yan, T. F., and Tao, M. (1982) J. Biol. Chem. 257, 7037-7043) was identified unambiguously as CK2 using mass spectrometry. CK2 is a ubiquitous eukaryotic protein kinase that phosphorylates a wide range of substrates. In previous studies, this wheat germ kinase was shown to phosphorylate eIF2␣, eIF3c, and three large subunit (60 S) ribosomal proteins (Browning, K. S., Yan, T. F., Lauer, S. J., Aquino, L. A., Tao, M., and Ravel, J. M. (1985) Plant Physiol. 77, 370 -373). To further characterize the role of CK2 in the regulation of translation initiation, Arabidopsis thaliana catalytic (␣1 and ␣2) and regulatory (␤1, ␤2, ␤3, and ␤4) subunits of CK2 were cloned and expressed in Escherichia coli. Recombinant A. thaliana CK2␤ subunits spontaneously dimerize and assemble into holoenzymes in the presence of either CK2␣1 or CK2␣2 and exhibit autophosphorylation. The purified CK2 subunits were used to characterize the properties of the individual subunits and their ability to phosphorylate various plant protein substrates. CK2 was shown to phosphorylate eIF2␣, eIF2␤, eIF3c, eIF4B, eIF5, and histone deacetylase 2B but did not phosphorylate eIF1, eIF1A, eIF4A, eIF4E, eIF4G, eIFiso4E, or eIFiso4G. Differential phosphorylation was exhibited by CK2 in the presence of various regulatory ␤-subunits. Analysis of A. thaliana mutants either lacking or overexpressing CK2 subunits showed that the amount of eIF2␤ protein present in extracts was affected, which suggests that CK2 phosphorylation may play a role in eIF2␤ stability. These results provide evidence for a potential mechanism through which the expression and/or subcellular distribution of CK2 ␤-subunits could participate in the regulation of the initiation of translation and other physiological processes in plants.

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

“…HY5 and HFR1) involved in light signaling pathways (34,35). However, phosphorylation by CK2 has been shown to either stabilize or modulate the activity of these factors (34,35,40). In contrast, our data show that phosphorylation by CK2 promotes the light-induced degradation of PIF1 through the ubiquitin/26 S proteasomal pathway (Fig.…”

“…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.…”

“…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).…”

“…We purified recombinant His-tagged PIF1, two CK2 ␣ (␣1 and ␣2) subunits, and four ␤ (␤1-4) subunits and performed in vitro kinase assays as described (40). Results show that PIF1 is weakly phosphorylated by the ␣1 subunit of CK2.…”