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

Abstract

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.

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).…”