Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit

Yasuo Watanabe, Brian A. Perrino, Thomas R. Soderling

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subnit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. We have previously demonstrated that interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobia residues within the CaN A sequence 328-390 [Watanabe Y., Perrino, B. A., Chang, B. H., & Soderling, T. R. (1995) J. Biol. Chem. 270, 456-460]. In the present study, selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bound 45Ca2+ normally. Mutants BE-2 and BQ-2 interacted with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390), and they stimulated the phosphatase activity of the CaN A subunit in an in vitro reconstitution assay. Mutant BE-1 had a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gave very little stimulation of CaN A phosphatase activity. We conclude that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects [i.e., decrease Km and increase Vmax; Perrino, B. A., Ng, L. Y., & Soderling, T. R. (1995) J. Biol. Chem. 270, 340-346] of B subunit Ca2+ binding to stimulation of CaN A phosphatase activity.

Original languageEnglish (US)
Pages (from-to)562-566
Number of pages5
JournalBiochemistry
Volume35
Issue number2
DOIs
StatePublished - Jan 16 1996

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit'. Together they form a unique fingerprint.

Cite this