Domain architecture of the regulators of calcineurin (RCANs) and identification of a divergent RCAN in yeast.
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.
Mehta
Sohum
S
Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Li
Huiming
H
Hogan
Patrick G
PG
Cunningham
Kyle W
KW
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D019143:Evolution, Molecular; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C080443:CDC4 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D044783:F-Box Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D050778:NFATC Transcription Factors; C108612:RCAN1 protein, human; C486302:RCN1 protein, S cerevisiae; C541019:Rcn2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases; D019703:Calcineurin