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 phosphastase. 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, calcipressin1) 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 GSK-3 phosphorylation site was not required for inhibition, suggesting 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 of canonical RCANs in facilitating calcineurin signaling, but they may secondarily inhibit calcineurin signaling by interfering with substrate interactions and enzymatic activity.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|