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.
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.
AbstractProtein 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.
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