Partial Inhibition of Calcineurin Activity by Rcn2 as a Potential Remedy for Vps13 Deficiency.

Patrycja Wardaszka,Joanna Kaminska,Teresa Zoladek,Marzena Sienko,Piotr Soczewka

doi:10.3390/ijms22031193

Abstract

Regulation of calcineurin, a Ca2+/calmodulin-regulated phosphatase, is important for the nervous system, and its abnormal activity is associated with various pathologies, including neurodegenerative disorders. In yeast cells lacking the VPS13 gene (vps13Δ), a model of VPS13-linked neurological diseases, we recently demonstrated that calcineurin is activated, and its downregulation reduces the negative effects associated with vps13Δ mutation. Here, we show that overexpression of the RCN2 gene, which encodes a negative regulator of calcineurin, is beneficial for vps13Δ cells. We studied the molecular mechanism underlying this effect through site-directed mutagenesis of RCN2. The interaction of the resulting Rcn2 variants with a MAPK kinase, Slt2, and subunits of calcineurin was tested. We show that Rcn2 binds preferentially to Cmp2, one of two alternative catalytic subunits of calcineurin, and partially inhibits calcineurin. Rcn2 ability to bind to and reduce the activity of calcineurin was important for the suppression. The binding of Rcn2 to Cmp2 requires two motifs in Rcn2: the previously characterized C-terminal motif and a new N-terminal motif that was discovered in this study. Altogether, our findings can help to better understand calcineurin regulation and to develop new therapeutic strategies against neurodegenerative diseases based on modulation of the activity of selected calcineurin isoforms.

Highlights

Calcium ions (Ca2+ ) are important signaling molecules that regulate multiple processes
We show that overexpression of the RCN2 gene, which encodes a negative regulator of calcineurin, is beneficial for vps13∆ mutant cells, and we studied the molecular mechanisms underlying RCN2 action
We found that a plasmid containing the RCN2 gene, which encodes a negative regulator of calcineurin, restored the growth of the vps13∆

Summary

Introduction

Calcium ions (Ca2+ ) are important signaling molecules that regulate multiple processes. The calcineurin recognizes two types of linear motifs in its substrates and regulators, PxIxIT and LxVP, by two binding pockets—one for each motif. The LxVP-type motif binds at the junction of catalytic and regulatory subunits [6]. By contrast, blocking the binding site for the LxVP-type motif interferes with substrate binding and inhibits calcineurin activity. This site is used by the immunosuppressants FK506 and cyclosporin A, which block substrate binding to the active p site of phosphatase [6]. There is a group of proteins, known as the regulators of calcineurin (RCANs) [9], that inhibit phosphatase activity by occupying substrate-binding motifs.

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