title: Competitive inhibition of calcineurin phosphatase activity by its autoinhibitory domain.
abstract: Calcineurin (protein phosphatase 2B), a calmodulin- and calcium-dependent serine/threonine phosphatase, appears to be regulated by a C-terminal autoinhibitory domain. A 25 amino acid peptide derived from this domain inhibits calcineurin phosphatase activity in vitro. Here we show that a 97 amino acid fragment of the calcineurin A alpha C-terminus is approx. 8-fold more potent than the shorter peptide in calcineurin inhibition experiments. Mutation of an evolutionarily conserved Asp to Asn, previously shown to disrupt calcium-dependent signalling and calcineurin regulation in T-lymphocytes, greatly reduced inhibition by the autoinhibitory domain in vitro. Kinetic analysis of wild-type and mutated autoinhibitory domains show that both are competitive inhibitors of calcineurin phosphatase activity with Ki values of 5.0 +/- 0.2 microM and 36.0 +/- 3.7 microM respectively. This suggests intrasteric regulation of calcineurin, with the autoinhibitory domains interacting at the active site of the enzyme. The competitive behaviour of the autoinhibitory domains contrasts with the mechanism of calcineurin inhibition by immunosuppressant-immunophilin complexes, which have been shown to bind to calcineurin at a region removed from the active site.
authors.affiliation: Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Sagoo
authors.forename: D A
authors.initials: DA
authors.lastname: Fruman
authors.forename: S
authors.initials: S
authors.lastname: Wesselborg
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
authors.forename: B E
authors.initials: BE
authors.lastname: Bierer
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases