Identification of a Novel Region Critical for Calcineurin Function in Vivo and in Vitro

Abstract

Calcineurin is a Ca2؉ /calmodulin-regulated protein phosphatase that plays critical functional roles in T-cell activation and other Ca 2؉ -mediated signal transduction pathways in mammalian cells. In Saccharomyces cerevisiae, calcineurin regulates the transcription of several genes involved in maintaining ion homeostasis (PMC1, PMR1, and PMR2) and cell wall synthesis (FKS2). In this paper, we report the identification and characterization of 11 single amino acid substitutions in the yeast calcineurin catalytic subunit Cna1p. We show that six substitutions (R177G, F211S, S232F, D258V, L259P, and A262P) affect the stability of calcineurin and that two substitutions (V385D and M400R) disrupt the interaction between Cna1p and the calcineurin regulatory subunit Cnb1p. We also identify three mutations (S373P, H375L, and L379S) that are clustered between the catalytic and the calcineurin B subunit-binding domains. These mutations do not significantly affect the ability of Cna1p to interact with Cnb1p, calmodulin, or Fkb1p (FK506-binding protein). However, these residue substitutions dramatically affect calcineurin activity both in vitro and in vivo. Thus, by using a random mutagenesis approach, we have shown for the first time that the linker region of the calcineurin catalytic subunit, as defined by the Ser 373 , His 375 , and Leu 379 residues, is crucial for its function as a phosphatase.Calcineurin, also known as PP2B, is a Ser/Thr-specific protein phosphatase (1). Calcineurin is tightly regulated by Ca 2ϩ / calmodulin, and it plays critical functional roles in many calcium-mediated signal transduction pathways. For example, calcineurin is required during T-cell activation for the transcriptional regulation of interleukin 2 (IL2) and other cytokine genes (2, 3). In human T-cells, calcineurin directly binds to and dephosphorylates the transcription factor NFAT.1 The dephosphorylated NFAT then translocates from the cytosol to the nucleus and activates the transcription of IL2 and other cytokine genes required for T-cell activation and proliferation (4, 5). The immunosuppressive drugs FK506 and cyclosporin A, in association with their respective cellular receptor proteins FKBP12 and cyclophilin A, bind to calcineurin and inhibit its phosphatase activity (6), thus preventing the calcineurin-dependent transcriptional activation of cytokine genes and T-cell activation (7).In the budding yeast Saccharomyces cerevisiae, calcineurindeficient strains exhibit normal growth under standard conditions (8 -10). However, calcineurin function is required for cell viability under some specific growth conditions. For example, mutations that disrupt calcineurin function are lethal in combination with several mutations that impair cell wall synthesis, including fks1⌬ (11-13). FKS1 and FKS2 are a pair of homologous genes responsible for the synthesis of ␤1, 3-glucan (11, 12, 14), which is a glucose homopolymer. Since ␤1,3-glucan is an essential cell wall component, yeast cells require both the FKS1 and FKS2 genes for viability. In a fks...

Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site

“…Using a directed yeast two-hybrid approach, we looked for an interaction between calcineurin (Cna1p) and Crz1p. AD-CRZ1 186-340 , the region that shows regulated nuclear localization, interacted with BD-CNA1 (Jiang and Cyert 1999), but AD-CRZ1 186-310 did not (Fig. 5C).…”

Regulation of Salt Tolerance byTorulaspora delbrueckiiCalcineurin Target Crz1p

“…Like calcineurin mutants, crz1-null cells show retarded growth at high concentrations of Li ϩ /Na ϩ and Mn 2ϩ cations (41, 61). We also tested whether the production of TdCrz1p could activate the expression of a 4ϫ CDRE::lacZ reporter, which contains four tandem copies of the 24-bp CDRE (38). Multiple copies of the CDRE increase the calcineurin-dependent transcriptional activation of the reporter gene (61).…”

Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the α or β catalytic subunit

“…Phospho-RII peptide, CaP, and BioMol Green reagent were purchased from BioMol. Phospho-DARPP-32 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) [LDPRQVE-MIRRRRPT(PO 4 )PAML] was purchased from American Peptide Company. Human CnAb cDNA was generously provided by M. M. Lai and S. Snyder (The Johns Hopkins University School of Medicine [9]).…”

“…The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the a or b catalytic subunit were compared using in vitro phosphatase assays. CaN containing the a isoform (CnAa) has lower K m and higher V max values than CaN containing the b isoform (CnAb) toward the PO 4 -RII, PO 4 -DARPP-32 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the a or b catalytic subunit isoform displayed identical calmodulin dissociation rates.…”

“…At low concentrations of FKBP12/FK506, CaN containing the a isoform is more sensitive to inhibition than CaN containing the b isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of b CaN using PO 4 -DARPP-32 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) as substrate. The functional differences conferred upon CaN by the a or b catalytic subunit isoforms suggest that the a:b and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types.…”