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Molecular and Cellular Biology, May 2009, p. 2777-2793, Vol. 29, No. 10
0270-7306/09/$08.00+0     doi:10.1128/MCB.01197-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Domain Architecture of the Regulators of Calcineurin (RCANs) and Identification of a Divergent RCAN in Yeast ^,

Sohum Mehta,¹ Huiming Li,^2,3 Patrick G. Hogan,³ and Kyle W. Cunningham^1*

Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, Maryland 21218,¹ Department of Pathology,² Immune Disease Institute, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115³

Received 30 July 2008/ Returned for modification 25 September 2008/ Accepted 27 February 2009

Regulators of calcineurin (RCANs) in fungi and mammals have been shown to stimulate and inhibit calcineurin signaling in vivo through direct interactions with the catalytic subunit of the phosphatase. The dual effects of RCANs on calcineurin were examined by performing structure-function analyses on yeast Rcn1 and human RCAN1 (a.k.a. DSCR1, MCIP1, and calcipressin 1) proteins expressed at a variety of different levels in yeast. At high levels of expression, the inhibitory effects required a degenerate PxIxIT-like motif and a novel LxxP motif, which may be related to calcineurin-binding motifs in human NFAT proteins. The conserved glycogen synthase kinase 3 (GSK-3) phosphorylation site was not required for inhibition, suggesting that RCANs can simply compete with other substrates for docking onto calcineurin. In addition to these docking motifs, two other highly conserved motifs plus the GSK-3 phosphorylation site in RCANs, along with the E3 ubiquitin ligase SCF^Cdc4, were required for stimulation of calcineurin signaling in yeast. These findings suggest that RCANs may function primarily as chaperones for calcineurin biosynthesis or recycling, requiring binding, phosphorylation, ubiquitylation, and proteasomal degradation for their stimulatory effect. Finally, another highly divergent yeast RCAN, termed Rcn2 (YOR220w), was identified through a functional genetic screen. Rcn2 lacks all stimulatory motifs, though its expression was still strongly induced by calcineurin signaling through Crz1 and it competed with other endogenous substrates when overexpressed, similar to canonical RCANs. These findings suggest a primary role for canonical RCANs in facilitating calcineurin signaling, but canonical RCANs may secondarily inhibit calcineurin signaling by interfering with substrate interactions and enzymatic activity.

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* Corresponding author. Mailing address: Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218. Phone: (410) 516-7844. Fax: (410) 516-5213. E-mail: kwc{at}jhu.edu

Published ahead of print on 9 March 2009.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, May 2009, p. 2777-2793, Vol. 29, No. 10
0270-7306/09/$08.00+0     doi:10.1128/MCB.01197-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.