The role of the regulatory subunit of fission yeast calcineurin for in vivo activity and its relevance to FK506 sensitivity.
Calcineurin, a protein phosphatase required for Ca2+ signaling in many cell types, is a heterodimer composed of catalytic and regulatory subunits. The fission yeast genome encodes a single set of catalytic (Ppb1) and regulatory (Cnb1) subunits, providing an ideal model system to study the functions of these subunits in vivo. Here, we cloned the cnb1+ gene and showed that the cnb1 knock-out (Deltacnb1) exhibits identical phenotypes with Deltappb1 and that overexpression of Ppb1 failed to suppress the phenotypes of Deltacnb1. Interestingly, overexpression of the C-terminal-deleted Ppb1 (Ppb1DeltaC), the constitutively active form of Ppb1, also failed to suppress the phenotypes of Deltacnb1. FK506 caused MgCl2 sensitivity to the wild-type cells in an FKBP12-dependent manner. Co-overexpression of Ppb1 and Cnb1 suppressed the FK506-induced MgCl2 sensitivity, but the suppression was only partial, suggesting that an excess amount of the Ppb1-Cnb1 complex cannot compete out the FKBP12-FK506 complex. Although overexpression of Ppb1DeltaC alone had little effect on cell growth, co-overexpression of Ppb1DeltaC and Cnb1 caused a distinct growth defect. FK506 suppressed the growth defect when Cnb1 was co-expressed using the attenuated nmt1 promoter, but it failed to suppress the defect when Cnb1 was co-expressed using the wild-type nmt1 promoter. Knock-out of the prz1+ gene, encoding a downstream target transcription factor of calcineurin, suppressed the growth defect irrespective of the promoter potency. These results suggest that Cnb1 is essential for the activation of calcineurin and that the activated calcineurin is the pharmacological target of the FKBP12-FK506 complex in vivo.
Sio
Susie O
SO
Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Suehiro
Takafumi
T
Sugiura
Reiko
R
Takeuchi
Mai
M
Mukai
Hideyuki
H
Kuno
Takayoshi
T
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D019703:Calcineurin / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D017353:Gene Deletion; D005821:Genetic Techniques; D016681:Genome, Fungal; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007477:Ions; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016559:Tacrolimus / Q000494:pharmacology*; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism
D000199:Actins; D007166:Immunosuppressive Agents; D007477:Ions; D015636:Magnesium Chloride; D049452:Green Fluorescent Proteins; D019703:Calcineurin; D022061:Tacrolimus Binding Protein 1A; D002118:Calcium; D016559:Tacrolimus