Abstract
Calcineurin (CaN) is the serine/threonine protein phosphatase (phosphatase 2B) that is activated by binding of Ca2+ to its B subunit and to calmodulin (CaM). This paper identifies residues between the catalytic region and the CaM-binding domain of the A subunit as the domain that binds the regulatory B subunit. A purified fusion protein containing residues 328-390 of the A subunit 1) binds CaN B subunit, and 2) inhibits (IC50 = 0.1 microM) the in vitro stimulation of CaN A phosphatase activity by purified CaN B subunit. A synthetic peptide corresponding to residues 341-360 blocked the binding of CaN B to residues 328-390 in the fusion protein, so 4 hydrophobic residues within this region (Val349-Phe350 and Phe356-Val357) were mutated to either Glu (E mutant) or Gln (Q mutant). The wild-type and mutant A subunits were expressed individually or coexpressed with B subunit in Sf9 cells, purified and characterized. The mutant A subunits were similar to wild-type A subunit in terms of basal phosphatase activity (1-3 nmol/min/mg) and activation by Mn2+/CaM. Addition of purified B subunit to purified wild-type A subunit at a 1:1 molar ratio gave a 40-fold increase in phosphatase activity whereas addition of B subunit to either of the mutant A subunits had no effect on phosphatase activity, even at a 3:1 molar excess of B subunit. Furthermore, when wild-type or mutant A subunits were coexpressed with B subunit and purified on CaM-Sepharose, the B subunit co-eluted with the wild-type A subunit but not with either mutant A subunit. These results demonstrate that residues 328-390 in the A subunit bind B subunit and that the mutated hydrophobic residues are essential.
Highlights
Terminal catalytic domain (residues [71-325], numbering based on the rat brain ao isoform (8)), the A subunit contains a central CaM-binding domain (9) and a COOH-terminal autoinhibitory region (10).2 The myristylated B subunit has four "EF" hand high affinity Ca2+-binding sites (2)
Construction and Expression ofthe CaN A 328 --390Fusion ProteinThe CaN 328-390 fragment was constructed by PCR from ",I)-CaN A plus engineered BamHI and EcoRI restriction sites. This fragment was subcloned into pGEX-2T vector which contains glutathione S-transferase (GST) (Pharmacia Biotech Inc.), and the fusion protein was expressed in Escherichia coli and purified by affinity chromatography on glutathione-Sepharose (Pharmacia Biotech Inc.)
Interaction of CaN B with CaN A328--390-CaN A subunit sequences from Drosophila to human brain show a highly conserved region of unknown function between the catalytic domain and the CaM-binding domain (Fig. lA)
Summary
Vol 270, No., Issue of January 6, pp. 456-460, 1995 Printed in U.S.A. Identification in the Calcineurin A Subunit of the Domain That Binds the Regulatory B Subunit*. Calcineurin (CaN) is the serine/threonine protein phosphatase (phosphatase 2B) that is activated by binding of Ca2+ to its B subunit and to calmodulin (CaM). This paper identifies residues between the catalytic region and the CaM-binding domain of the A subunit as the domain that binds the regulatory B subunit. When wild-type or mutant A subunits were coexpressed with B subunit and purified on CaM-Sepharose, the B subunit co-eluted with the wild-type A subunit but not with either mutant A subunit These results demonstrate that residues [328-390] in the A subunit bind B subunit and that the mutated hydrophobic residues are essential. Studies on limited proteolysis of CaN in the absence of Ca2+/ CaM demonstrated cleavage of the CaN A subunit NH2-terminal of the CaM-binding domain with retention of phosphatase activity and B subunit binding (12). Sitespecific mutagenesis and enzymology, this paper identifies this region as important for B subunit interaction
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