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Yeast holoenzyme of protein kinase CK2 requires both β and β′ regulatory subunits for its activity

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (α and/or α′) subunits and two regulatory β and β′ subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of αα′ββ′, α2ββ′, α′2ββ′ and a free α′-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory β subunit cannot substitute other β′ subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the β or β′ regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic α subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2β/β′ subunits in the regulation of holoenzyme assembly and phosphotransferase activity.

Isolation of a CK2α Subunit and the Holoenzyme from the Mussel Mytilus galloprovincialis and Construction of the CK2α and CK2β cDNAs

Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α′) and two regulatory (β) subunits, forming a differently assembled tetramer. The free and catalytically active α/α′ subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2β subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2β subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2β subunits have similar features to their mammalian orthologs.

Protein kinase CK2 cellular function in normal and disease states

Natural modulators of protein kinase CK2 activity might be divided into two groups. Substances of the fi rst one, polycations, like polyamines and polylysine are able to stimulate enzyme activity. On the other hand, compounds like heparin (polyanions) have inhibitory properties towards CK2 activity. The sequence of Asf1 possesses fi ve potential phosphorylation sites for CK2, but it seems that it does not underlie phosphorylation. Yeast Asf1 amino acid sequence contains a characteristic acidic fragment at its C-terminus. Such a sequence, called pseudosubstrate region, of regulatory subunits is present in several protein kinases, like PKA and PKG, and can be also found in the regulatory subunit of CK2. Experimental data suppose a modulating effect of Asf1 towards protein kinase CK2 in a different manner when comparing the infl uence on each catalytic subunits itself as well as the corresponding holoenzymes.

Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata

The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5′/3′ RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.

Yeast Asf1 Protein as Modulator of Protein Kinase CK2 Activity

Natural modulators of protein kinase CK2 activity might be divided into two groups. Substances of the first one, polycations, like polyamines and polylysine are able to stimulate enzyme activity. On the other hand, compounds like heparin (polyanions) have inhibitory properties towards CK2 activity. The sequence of Asf1 possesses five potential phosphorylation sites for CK2, but it seems that it does not underlie phosphorylation. Yeast Asf1 amino acid sequence contains a characteristic acidic fragment at its C-terminus. Such a sequence, called pseudosubstrate region, of regulatory subunits is present in several protein kinases, like PKA and PKG, and can be also found in the regulatory subunit of CK2. Experimental data suppose a modulating effect of Asf1 towards protein kinase CK2 in a different manner when comparing the influence on each catalytic subunits itself as well as the corresponding holoenzymes.

Sensitivity of Protein Kinase CK2 to ATP/GTP and Specific Inhibitors Depends on Complexity of Interacting Proteins

In yeast cells five isoforms of protein kinase CK2 may simultaneously exist composed of αα′ββ′, α2ββ′, α′2ββ′, and free catalytic α, α′. Each isoform exhibits properties typical for CK2, but they differ in their substrate specificity and sensitivity to specific modulators. Enzymes tested with protein substrates differently interacting with CK2 subunits were tested for their ATP/GTP binding capacity as well as with two commonly used ATP-competitive inhibitors TBB and TBI. Obtained results show that both ATP/GTP binding capacity and sensitivity to specific inhibitors are determined by the composition of CK2/substrate complexes. Both inhibitors, TBB and TBI, decrease cell growth to extend devoting interactions with different CK2 isoforms present in the cell; however, the presence of the regulatory ββ′ dimer has a high importance toward sensitivity. Conceivably, only selected CK2-mediated processes in the cell can be inhibited by a given inhibitor concentration.

Protein kinase CK2 from two higher eukaryotes of economical importance, the mussel Mytilus galloprovincialis and the medfly Ceratitis capitata

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase involved in cell cycle control, transcription, signal transduction and cell proliferation. It is upregulated in several diseases and by oxidative stress. CK2 is generally composed of two catalytic subunits and two regulatory subunits and utilizes either ATP or GTP as a phosphate donor. CK2 was isolated from the sea mussel Mytilus galloprovincialis, a biomarker of marine pollution, and the Mediterranean fly Ceratitis capitata, an insect capable of wreaking extensive damage to a wide range of fruit crops with great economical importance. The catalytic CK2α and regulatory CK2β subunits of M. galloprovincialis and C. capitata show similar properties. The mussel and fly catalytic subunits and holoenzymes were capable of phosphorylating the recombinant ribosomal stalk P1 protein, implying functional conservation. They also demonstrate the characteristics of a typical CK2: use of ATP and GTP as phosphate donors, inhibition by known modulators of CK2 activity (like benzotriazole derivatives and heparin), and stimulation by polycations. Both organisms seem to be ideal models for the analysis of CK2 in the control of gene expression in response to cellular stress.