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J. Biol. Chem., Vol. 280, Issue 13, 12231-12238, April 1, 2005

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The Role of the Regulatory Subunit of Fission Yeast Calcineurin for in Vivo Activity and Its Relevance to FK506 Sensitivity^*

Susie O. Sio¶, Takafumi Suehiro, Reiko Sugiura||, Mai Takeuchi, Hideyuki Mukai**, and Takayoshi Kuno

From the 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, ^||Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka, 577-8502, Japan, and ^**Biosignal Research Center and Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan

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 (cnb1) exhibits identical phenotypes with ppb1 and that overexpression of Ppb1 failed to suppress the phenotypes of cnb1. Interestingly, overexpression of the C-terminal-deleted Ppb1 (Ppb1C), the constitutively active form of Ppb1, also failed to suppress the phenotypes of cnb1. FK506 caused MgCl₂ sensitivity to the wild-type cells in an FKBP12-dependentmanner. 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 Ppb1C alone had little effect on cell growth, co-overexpression of Ppb1C 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.

Received for publication, December 17, 2004

^* This work was supported by the 21st Century Center of Excellence Program and by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^¶ Present address: Dept. of Pharmacology and Toxicology, College of Medicine, University of the Philippines Manila, Manila 1000, Philippines.

To whom correspondence should be addressed. Tel.: 81-78-382-5440; Fax: 81-78-382-5459; E-mail: tkuno{at}med.kobe-u.ac.jp.

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