Regulation of a recombinant pea nuclear apyrase by calmodulin and casein kinase II

A cDNA encoding a pea nuclear apyrase was previously cloned. Overexpression s of a full-length and a truncated cDNA have been successfully expressed in Escherichia coli BL21(DE3). The resulting fusion proteins, apyrase and the C-terminus (residues 315-453) of apyrase, were used for calmodulin (CaM) b inding and phosphorylation studies. Fusion protein apyrase but not the C-te rminus of apyrase can be recognized by polyclonal antibody pc480. This sugg ested that the motif recognized by pc480 was located in the N-terminal regi on of apyrase. The recombinant apyrase protein also showed an activity 70 t imes higher than that of endogenous apyrase using ATP as a substrate. The r ecombinant apyrase has a preference for ATP more than other nucleoside trip hosphate substrates. CaM can bind to recombinant apyrase, but not to the C- terminus of apyrase. This implies that the CaM-binding domain must be in th e first 315 amino acids of the N-terminal region of apyrase. We found that one segment from residue 293 to 308 was a good candidate for the CaM-bindin g domain. This segment 293 FNKCKNTIRKALKLNY 308 has a basic amphiphilic-hel ical structure, which shows the predominance of basic residues on one side and hydrophobic residues on the other when displayed on a helical wheel plo t. Using the gel mobility shift binding assay, this synthetic peptide was s hown to bind to CaM, indicating that it is the CaM-binding domain. Both rec ombinant apyrase and the C-terminus of apyrase can be phosphorylated by a r ecombinant human protein kinase CKII. Phosphorylation does not affect CaM b inding to recombinant apyrase. However, CaM does inhibit CKII phosphorylati on of recombinant apyrase and this inhibition can be blocked by 5 mM EGTA. (C) 2000 Elsevier Science B.V. All rights reserved.