Regulation of a recombinant pea nuclear apyrase by calmodulin and casein kinase II.
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.
Hsieh
H L
HL
Section of Molecular Cell and Developmental Biology, University of Texas, Austin, 78712-1187, USA.
Song
C J
CJ
Roux
S J
SJ
D000595:Amino Acid Sequence; D001081:Apyrase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001665:Binding Sites; D002147:Calmodulin / Q000494:pharmacology*; D047390:Casein Kinase II; D002467:Cell Nucleus / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics
D002147:Calmodulin; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D001081:Apyrase