Ca2+ binding site 2 in calcineurin-B modulates calmodulin-dependent calcineurin phosphatase activity.
Calcineurin is the Ca(2+)- and calmodulin-dependent Ser/Thr phosphatase. Human calcineurin-Aalpha and wild-type or mutated calcineurin-Bs were coexpressed in Escherichia coli and purified by calmodulin-Sepharose affinity chromatography. Four calcineurin-B mutants were studied. Each had a single conserved Glu in the 12th position of one EF-hand Ca(2+) binding site replaced by a Lys, resulting in the loss of Ca(2+) binding to that site. Phosphatase activities of the enzymes toward a (32)P-labeled phosphopeptide substrate were measured. Inactivating Ca(2+) binding sites 1, 2, or 3 in calcineurin-B reduced Ca(2+)-dependent phosphatase activity of the enzymes in the absence of calmodulin with the site 2 mutation being most effective. Inactivating Ca(2+) binding site 4 did not change enzyme activity or sensitivity to Ca(2+) in either the absence or presence of calmodulin. The calmodulin-dependent phosphatase activity of the enzymes containing site 1, 2, or 3 mutations in calcineurin-B was also decreased compared to enzyme with wild-type calcineurin-B. Of these enzymes, the one with the site 2 mutation was most profoundly affected as determined by the magnitude of the shift in Ca(2+) concentration dependence. Binding of a fluorescein-labeled calmodulin to the wild-type and the site 2 mutant enzymes was examined using fluorescence polarization measurements. The decrease in Ca(2+) sensitivity for the enzyme with calcineurin-B site 2 inactivated is apparently due to a decrease in the affinity of that enzyme for calmodulin at low Ca(2+) concentrations. These data support a role for Ca(2+) binding site 3 in the carboxyl half of calcineurin-B in transmitting the Ca(2+) signal to calcineurin-A and indicate that site 2 in the amino half of calcineurin-B is critical for enzyme activation.
Feng
B
B
Department of Pediatrics, College of Medicine, 986255 Nebraska Medical Center, Omaha, Nebraska 68198-6255, USA.
Stemmer
P M
PM
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism / Q000502:physiology*; D020134:Catalytic Domain / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D019793:Fluorescein / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics
D000345:Affinity Labels; D002147:Calmodulin; C120817:calcineurin phosphatase; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium; D019793:Fluorescein