Calcineurin subunit interactions: mapping the calcineurin B binding domain on calcineurin A.
Recombinant forms of the A and B subunits of the protein phosphatase calcineurin were produced in Escherichia coli, reconstituted into a heterodimer and purified to homogeneity. The reconstituted heterodimer exhibited properties like that of bovine brain calcineurin. This included calmodulin-stimulated activity and a subunit stoichiometry and Stokes radius consistent with native-like structure. In order to map the region on the A subunit where calcineurin B binds, a series of overlapping 20-residue peptides corresponding to this putative domain were synthesized. Using isolated calcineurin A and B subunits, an assay that relied upon peptide inhibition of calcineurin B stimulation of calcineurin A activity was developed. All five peptides, but not a control peptide, inhibited calcineurin B-dependent stimulation of calcineurin A although with different potencies. The three most effective inhibitory peptides spanned calcineurin A residues 338-377. These three peptides also altered the electrophoretic mobility of the isolated calcineurin B subunit during native polyacrylamide gel electrophoresis indicating a direct interaction between these peptides and calcineurin B. The peptide corresponding to residues 348-367 was also able to block binding of calcineurin B to the catalytic subunit.
Sikkink
R
R
Section of Hematology Research, Mayo Clinic & Foundation, Rochester, Minnesota 55905, USA.
Haddy
A
A
MacKelvie
S
S
Mertz
P
P
Litwiller
R
R
Rusnak
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005169:Factor VIII / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D005169:Factor VIII; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases