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

Abstract

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

“…The cDNAs of CK2α and CK2β subunits of M. galloprovincialis (Mg) and C. capitata (Cc) were isolated and the recombinant proteins were purified after expression in E. coli [14,15] (Figure 2). The free catalytic subunits and the reconstituted α 2 β 2 holoenzymes from both species possess properties typical of protein kinase CK2 [14,15].…”

“…The free catalytic subunits and the reconstituted α 2 β 2 holoenzymes from both species possess properties typical of protein kinase CK2 [14,15]. The phosphorylating acivities were measured and compared with the ones from other species like human, maize or yeast.…”

“…Sequence conservation is translated into conservation of function as well, as shown between human, nematode and yeast, in which the human or the nematode Caenorhabditis elegans CK2α catalytic subunit was shown to substitute for the yeast catalytic subunits [13]. We have recently studied CK2 in the Mediterranean mussel M. galloprovincialis and the medfly C. capitata [14,15]. Cloning and sequence analysis of the cDNAs and the predicted polypeptides showed that both, CK2α and CK2β subunits from M. galloprovincialis and C. capitata are similar in their theoretical molecular weight and pI compared to their homologues ( Table 1).…”

Site-Directed Mutagenesis in the Research of Protein Kinases - The Case of Protein Kinase CK2

“…The CK2 tetramer exhibits constitutive activity that can be easily detected in most cellular or tissue extracts in the absence of any stimulatory compounds. In many organisms, distinct isoenzymic forms of the catalytic subunit of CK2 have been identified (Glover, 1998;Kolaiti et al, 2011;Kouyanou-Koutsoukou et al, 2011a, b;Maridor et al, 1991;Litchfield et al, 1990;Shi et al, 2001). In humans, only a single regulatory CK2 subunit has been identified; but multiple forms of CK2 have been identified in other organisms, such as Saccharomyces cerevisiae (Glover, 1998).…”

“…The CK2 regulatory subunit is remarkably conserved among species, but it does not have homology with the regulatory subunits of other protein kinases (Bibby & Litchfield, 2005). The amino acid sequence of the CK2 regulatory subunit is almost identical in Homo sapiens, Drosophila melanogaster, Ceratitis capitata (Mediterranean fruit fly), Danio rerio (zebrafish), Ciona intestinalis (sea squirt), and Mytilus galloprovincialis (Mediterranean mussel) (Kouyanou-Koutsoukou et al, 2011a, b;Kolaiti et al, 2011). It is completely identical in birds and mammals (Maridor et al, 1991;Wirkner et al, 1994).…”