Regulator of calcineurin 1 (RCAN1) binds to calcineurin through the PxIxIT motif, which is evolutionarily conserved. SP repeat phosphorylation in RCAN1 is required for its complete function. The specific interaction between RCAN1 and calcineurin is critical for calcium/calmodulin-dependent regulation of calcineurin serine/threonine phosphatase activity. In this study, we investigated two available deletion
Regulator of CAlcineuriN 1 (RCAN1) is a highly conserved protein, first identified in fungi and yeasts as an inhibitory regulator to calcineurin, a calcium/calmodulin-dependent phosphatase (Gorlach et al., 2000; Kingsbury and Cunningham, 2000). RCAN1 is highly expressed in the striated muscle, the nervous system and induced, during pathological conditions such as neurodegeneration (Crawford et al., 1997; Fuentes et al., 2000; Wiese et al., 1995; Yang et al., 2000). RCAN1 suppresses dephosphorylation of a multi-functional transcription factor NFAT, which is a well-known substrate for calcineurin, and enhanced calcineurin activity induces RCAN1 expression via negative feedback (Fuentes et al., 2000; Yang et al., 2000). Therefore, RCAN1 overexpression due to trisomy 21 in Down syndrome plays a critical role in the pathology, because of RCAN1’s inhibitory interaction with calcineurin (Ermak et al., 2001; Fuentes et al., 1995; 1997). However, there is evidence that RCAN1 is also required for optimal calcineurin activity. Low calcineurin activity was observed in RCAN1 null mutant yeast, accompanied by phenotypes similar to RCAN1-overexpressing cells (Kingsbury and Cunningham, 2000). In addition, relatively low RCAN1 expression in physiological conditions activates the calcineurin pathway, when RCAN1 is functionally phosphorylated at highly conserved serine sites (Hilioti et al., 2004; Kingsbury and Cunningham, 2000; Li et al., 2015). Therefore, RCAN1’s regulatory modes on calcineurin is complicated, depending on the cellular and developmental contexts of organisms, and the biochemical and molecular properties of RCAN1.
Down syndrome model transgenic mice overexpressing RCAN1 exhibit a variety of phenotypic features that mimic Down syndrome such as cranial structure malformation, learning and memory deficits, disorganized neuronal differentiation and tumorigenesis suppression (Dierssen et al., 2011; Kurabayashi and Sanada, 2013; Martin et al., 2012; Reynolds et al., 2010). Interestingly, RCAN1 knock-out mice also show phenotypes similar to transgenic mice, such as impaired spatial learning and memory, and abnormal long-term potentiation (Hoeffer et al., 2007). In addition, both RCAN1 knock-out mice and RCAN1-1S TG mice displayed reduced exocytosis levels (Keating et al., 2008). Neuronal differentiation is also defective in transgenic flies overexpressing RCAN1 and loss-of-function mutant flies, presumably due to disturbed axonal extension associated with actin dynamics dysregulation (Chang and Min, 2009; Chang et al., 2003; Wang et al., 2012; 2016). In
RCAN1 binds to calcineurin through specified binding motifs. The PxIxIT motif is required for binding to calcineurin, which is also present in many calcineurin substrates such as a transcripttion factor NFAT (nuclear factor of activated T-cells) (Aramburu et al., 1998; Li et al., 2015; Mehta et al., 2009). RCAN1 also harbors SP repeats containing conserved serines whose phosphorylation is required for calcineurin stimulatory regulation in yeasts (Hilioti et al., 2004; Kingsbury and Cunningham, 2000). We previously reported the highly conserved PxIxIT motif, not the less conserved PKIIQT motif, is critical for full RCAN1 inhibitory regulation on
In this study, we investigated two available
Wild type N2 and
pPD95.77 and pPD49.26 were generous gifts from Andy Fire. To make a RCAN-1 translational expression construct, a
Immunostaining against RCAN-1 was performed as previously described (Park et al., 2001b).
Serotonin stimulated egg-laying was conducted as previously described with some modification (Trent et al., 1983). Worms were placed in 96-well plates containing M9 buffer either with or without 12.5 mM serotonin. After 90 min, the number of eggs laid by each worm was counted.
Worms were synchronized at L4, and then adult body sizes were measured using a Zeiss microscope and Axio Image software.
Worms overexpressing RCAN-1 exhibited pleiotrophic phenotypes similar to
The predicted RCAN-1 translational product in
RCAN1 has been widely shown to be an inhibitory calcineurin regulator, but several lines of studies have shown that phosphorylated RCAN1 is able to stimulate calcineurin at low expression levels (Genesca et al., 2003; Hilioti et al., 2004; Kishi et al., 2007). Also, both knock-out and overexpression lines in various models show similar malfunction in different phenotypes. This study showed that
Previously considered to be null mutants, both