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 eIF2alpha, 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 (alpha1 and alpha2) and regulatory (beta1, beta2, beta3, and beta4) subunits of CK2 were cloned and expressed in Escherichia coli. Recombinant A. thaliana CK2beta subunits spontaneously dimerize and assemble into holoenzymes in the presence of either CK2alpha1 or CK2alpha2 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 eIF2alpha, eIF2beta, 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 beta-subunits. Analysis of A. thaliana mutants either lacking or overexpressing CK2 subunits showed that the amount of eIF2beta protein present in extracts was affected, which suggests that CK2 phosphorylation may play a role in eIF2beta stability. These results provide evidence for a potential mechanism through which the expression and/or subcellular distribution of CK2 beta-subunits could participate in the regulation of the initiation of translation and other physiological processes in plants.
Highlights
The acidophilic nature of CK2 separate it biochemically from other protein kinases [1]
Several other proteins co-purified with wheat CK2␣ were identified as 60 S ribosomal protein S7 (28 kDa, band 3), 40 S ribosomal protein S19 (19 kDa, band 4), two isoforms of 60 S ribosomal protein L3 (16/17 kDa, bands 5a/5b), and one band that could not be identified
Mammalian ribosomal protein S19 (RPS19) was found to interact with mammalian orthologs (60 S ribosomal protein L3, 60 S ribosomal protein S7, and CK2␣) of three of the plant proteins identified in this work by mass spectrometry of the purified wheat germ CK2 preparation
Summary
Wheat germ kinase was purified from S30 [24, 25] using DEAE-cellulose (Whatman DE52) and phosphocellulose (Whatman P11) as described previously [23]. The protein, eluted from the phosphocellulose column containing the peak of activity (8 ml containing 9.3 mg of protein), was concentrated to ϳ1 ml using an Amicon Ultra-4 centrifugal filter (Millipore). Three fractions containing the peak kinase activity were pooled and concentrated by centrifugal filtration to a final volume of 200 l. The purified kinase was divided into 25-l aliquots, quick frozen in dry ice, and stored at Ϫ80 °C for further analysis. A 48-kDa protein termed T-substrate, which was previously identified as an endogenous phosphoryl acceptor for the wheat germ kinase, was purified as described previously [21]
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