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

Abstract

A cDNA encoding a pea nuclear apyrase was previously cloned. Overexpressions 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) binding and phosphorylation studies. Fusion protein apyrase but not the C-terminus of apyrase can be recognized by polyclonal antibody pc480. This suggested that the motif recognized by pc480 was located in the N-terminal region of apyrase. The recombinant apyrase protein also showed an activity 70 times higher than that of endogenous apyrase using ATP as a substrate. The recombinant apyrase has a preference for ATP more than other nucleoside triphosphate 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 the 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-binding domain. This segment 293 FNKCKNTIRKALKLNY 308 has a basic amphiphilic-helical structure, which shows the predominance of basic residues on one side and hydrophobic residues on the other when displayed on a helical wheel plot. Using the gel mobility shift binding assay, this synthetic peptide was shown to bind to CaM, indicating that it is the CaM-binding domain. Both recombinant apyrase and the C-terminus of apyrase can be phosphorylated by a recombinant human protein kinase CKII. Phosphorylation does not affect CaM binding to recombinant apyrase. However, CaM does inhibit CKII phosphorylation of recombinant apyrase and this inhibition can be blocked by 5 mM EGTA.