Regulation of Calcineurin by Calcium-Binding Cousins

Abstract

Calcineurin (CaN), a heterodimeric, calcium-activated phosphatase (PP2B), dephosphorylates members of the NF-AT family of transcription factors. This promotes nuclear localization of NF-AT, which initiates transcription of genes controlling immune responses and regulates key steps in eukaryotic development, especially cardiac muscle. Calcium partially activates CaN by binding to CaNB, its intrinsic calcium-binding subunit, but maximal CaN activity requires binding of calmodulin (CaM), a ubiquitous 4-EF-Hand cousin of CaNB. The activity of CaN changes based on intermediate occupancy of the N- and C-domains of CaNB and CaM. To probe how CaNB controls the phosphatase activity of CaN, we compared CaN regulated by mutant forms of CaNB in which one or more calcium-binding sites were “knocked out”. The double knockouts in each domain reduced activity to basal level. Knockouts of individual sites within each domain were not equivalent, despite their sequence similarity and cooperativity in WT CaN. In comparison, in CaM, individual mutations of the N-domain calcium-binding sites had a larger effect on enzymatic activity of CaN than individual mutations of the C-domain sites, although the affinity of the isolated C-domain for the CaMBD is almost 10-fold higher than that of the isolated N-domain. These studies were consistent with our prior thermodynamic measurements of the affinity of the same site-knockout CaM mutants for the canonical CaMBD sequence embedded in a YFP-CFP biosensor. They support a model in which calcium binding to the N-domain of CaM is a critical trigger for the transition from medium to high activity. Supported by AHA 12GRNT12050395 & NIH R01 GM57001.