Phosphorylation of maize eukaryotic translation initiation factor 5A (eIF5A) by casein kinase 2: identification of phosphorylated residue and influence on intracellular localization of eIF5A.

Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with the catalytic alpha subunit of protein kinase CK2 and is phosphorylated by this enzyme. Phosphorylated ZmeIF5A was also identified after separation of maize leaf proteins by two-dimensional electrophoresis. Multiple sequence alignment of eIF5A proteins showed that in monocots, in contrast to other eukaryotes, there are two serine/threonine residues that could potentially be phosphorylated by CK2. To identify the phosphorylation site(s) of ZmeIF5A, the serine residues potentially phosphorylated by CK2 were mutated. ZmeIF5A and its mutated variants S2A and S4A were expressed in Escherichia coli and purified. Of these recombinant proteins, only ZmeIF5A-S2A was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A-S2A mutants were not phosphorylated despite effective phosphorylation of wild-type variants. A newly developed method exploiting the specificity of thrombin cleavage was used to confirm that Ser(2) in ZmeIF5A is indeed phosphorylated. To find a role of the Ser(2) phosphorylation, ZmeIF5A and its variants mutated at Ser(2) (S2A and S2D) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. Although wild-type ZmeIF5A and its S2A variant were distributed evenly between the nucleus and cytoplasm, the variant with Ser(2) replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser(2) plays a role in regulation of nucleocytoplasmic shuttling of eIF5A in plant cells.

Phosphorylation of maize eukaryotic translation initiation factor on Ser2 by catalytic subunit CK2.

Alignment of eukaryotic translation initiation factor 5A (eIF5A) sequences has shown, for plants, in contrast to most other eukaryotes, the presence of N-terminal serine residue (Ser2) which could be phosphorylated by CK2. Using point directed mutagenesis, we demonstrate here that in recombinant maize ZmeIF5Awt Ser2 is exclusively phosphorylated by catalytic subunit of CK2 (CK2α), whereas its mutated variant Ser2Ala is not phosphorylated. To shed light on the physiological significance of this Ser2 phosphorylation, transient expression of fluorescence-labeled proteins was performed in maize protoplast. Wild-type ZmeIF5A was distributed evenly between nucleus and cytoplasm, but the replacement of Ser2 by aspartic acid, which mimics the phosphorylated serine, influences its intracellular localization. We postulate that phosphorylation of Ser2 in maize eIF5A, and most probably in other plant cells, plays a role in specific regulation of nuclear export of eIF5A-bound mRNAs.

A novel splicing variant encoding putative catalytic alpha subunit of maize protein kinase CK2.

A cDNA highly homologous to the known catalytic alpha subunit of protein kinase CK2 was cloned from maize (Zea mays). It was designated ZmCK2alpha-4 (accession no. AAF76187). Sequence analysis shows that ZmCK2alpha-4 and the previously identified ZmCK2alpha-1 (accession no. X61387) are transcribed from the same gene, ZmPKCK2AL (accession no. Y11649), but at different levels in various maize organs and at different stages of development. The cDNA encoding ZmCK2alpha-4 has three potential translation initiation sites. The three putative variants of ZmCK2alpha-4 were expressed in Escherichia coli as GST-fusion proteins and purified from bacterial extracts. In contrast to the previously characterized ZmCK2alphas, the obtained GST:ZmCK2alpha-4 proteins were catalytically inactive as monomers or in the presence of equimolar amounts of the human CK2beta. However, GST:ZmCK2alpha-4 did phosphorylate casein in the presence of a large excess of the beta subunit. The activity of ZmCK2alpha-4 toward casein could also be stimulated by increasing ATP concentration. Modeling studies have shown that there is no interaction between the N-terminal segment of ZmCK2alpha-4 and the activation loop responsible for constitutive catalytic activity of CK2alpha. Preliminary results suggest that ZmCK2alpha-4 may function as a negative regulator of other CK2s, and at certain circumstances as a holoenzyme which catalytic activity is stimulated by specific regulatory subunit(s).

Synthesis of new analogs of benzotriazole, benzimidazole and phthalimide--potential inhibitors of human protein kinase CK2.

New derivatives of 4,5,6,7-tetrabromo-1H-1,2,3-benzotriazole (TBBt), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), and N-substituted tetrabromophthalimides were synthesized and their effect on the activity of human protein kinase CK2 was examined. The most active were derivatives with N-hydroxypropyl substituents (IC(50) in 0.32-0.54 microM range) whereas derivatives of phthalimide were almost ineffective.

Relative role of halogen bonds and hydrophobic interactions in inhibition of human protein kinase CK2α by tetrabromobenzotriazole and some C5-substituted analogues.

To examine the relative role of halogen bonding and hydrophobic interactions in the inhibition of human CK2alpha by 4,5,6,7-tetrabromobenzotriazole (TBBt), we have synthesized a series of 5-substituted benzotriazoles (Bt) and the corresponding 5-substituted 4,6,7-tribromobenzotriazoles (Br3Bt) and examined their inhibition of human CK2alpha relative to that of TBBt. The various C(5) substituents differ in size (H and CH3), electronegativity (NH2 and NO2), and hydrophobicity (COOH and Cl). Some substituents were halogen bond donors (Cl, Br), while others were fluorine bond donors (F and CF3). Most of the 5-substituted analogues of Br3Bt (with the exception of COOH and NH2) exhibited inhibitory activity comparable to that of TBBt, whereas the 5-substituted analogues of the parent Bt were only weakly active (Br, Cl, NO2, CF3) or inactive. The observed effect of the volume of a ligand molecule pointed to its predominant role in inhibitory activity, indicating that presumed halogen bonding, identified in crystal structures and by molecular modeling, is dominated by hydrophobic interactions. Extended QSAR analysis additionally pointed to the monoanion and a preference for the N(1)-H protomer of the neutral ligand as parameters crucial for prediction of inhibitory activity. This suggests that the monoanions of TBBt and its congeners are the active forms that efficiently bind to CK2alpha, and the binding affinity is coupled with protomeric equilibrium of the neutral ligand.

New inhibitors of protein kinase CK2, analogues of benzimidazole and benzotriazole.

Derivatives of 4,5,6,7-tetrabromobenzotriazole (TBBt) and 4,5,6,7-tetrabromobenzimidazole (TBBi) with IC50 in the low micromolar range and with high selectivity belong to the most promising inhibitors of protein kinase CK2 (casein kinase 2). Treatment of various cell lines with TBBt, TBBi or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) affected cell viability with simultaneous induction of apoptosis. The inhibitory activity of newly synthesized hydroxyalkyl derivatives of TBBi and TBBt depends on the length of the alkyl chain. The hydroxypropyl substituted derivatives show higher or similar inhibitory activity than the parent compounds when tested with human protein kinase CK2. To test the distribution of this class of compounds in mammals, [14C] TBBi was synthesized.