Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.
Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.
Perrino
B A
BA
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Fong
Y L
YL
Brickey
D A
DA
Saitoh
Y
Y
Ushio
Y
Y
Fukunaga
K
K
Miyamoto
E
E
Soderling
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007313:Insecta; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007527:Isoenzymes; D046911:Macromolecular Substances; D010455:Peptides; D011994:Recombinant Proteins; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases