# Systematic elucidation of calcineurin phosphatase signaling in humans

> **NIH NIH R35** · STANFORD UNIVERSITY · 2024 · $118,365

## Abstract

Project Summary
All eukaryotes use phosphorylation-based signaling networks, composed of protein kinases and
phosphatases, to regulate cellular processes. While global information about kinase signaling has
exploded, knowledge of phosphatases has lagged behind. The lack of systematic, unbiased approaches
to analyze phosphatase signaling leaves a major gap in our understanding of cellular regulatory
networks. For >25 years, my research on calcineurin, the conserved Ca2+/calmodulin-regulated
phosphatase, in S. cerevisiae and humans has directly addressed this issue. Calcineurin is ubiquitously
expressed and plays critical regulatory roles in the cardiovascular, nervous and immune systems (1);
however, only ~70 proteins are currently established as calcineurin substrates (2). Calcineurin
dephosphorylates the Nuclear Factor of Activated T-cells (NFAT) transcription factors to activate the
adaptive immune response (3), and calcineurin inhibitors, FK506 and cyclosporin A, are commonly
prescribed as immunosuppressants (4). However, these drugs cause unwanted effects, including
hypertension, diabetes, and seizures by inhibiting calcineurin in non-immune tissues (5), highlighting the
need to map human calcineurin signaling pathways systematically. My work elucidates calcineurin
signaling through novel approaches based on the discovery of short linear motifs (SLiMs): short
degenerate peptide sequences found within regions of intrinsic disorder that determine specific, low-
affinity interactions that are essential for signaling (6). Using experimental and in silico SLiM-based
methods, we recently defined the human calcineurin signaling network (2). This work uncovered
conserved regulation of nuclear pore complexes by calcineurin and showed unexpected calcineurin
proximity to centrosomes (2). Future efforts will elucidate Ca2+ and calcineurin signaling at these
compartments using a combination of methods that include phosphoproteomics and proximity labeling
with faster-labeling biotin ligases (miniTurbo (33), Split TurboID). Fluorescent sensors will be used to
probe Ca2+ signals at these locations in vivo. Calcineurin functions at membranes will be analyzed by
focusing on CNb1, a little-studied isoform with distinct localization and regulation conferred by its unique,
lipidated C-terminal sequences (10). Regulation of CNb1 activity via dynamic palmitoylation will be
examined, and CNb1-specific signaling pathways, including regulation of PI4P synthesis, will be
established to achieve a comprehensive and mechanistic understanding of this enzyme. Finally, the
novel technology, MRBLE-PEP (Microspheres Ratiometric Barcode Lanthenide Encoding coupled to
PEPtides) (11), will be developed for quantitative analysis and discovery of SLiMs. Overall this research
aims to map human calcineurin signaling pathways systematically and to advance our understanding of
SLiM-based signaling more broadly.

## Key facts

- **NIH application ID:** 11046029
- **Project number:** 3R35GM136243-05S1
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Martha S. Cyert
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $118,365
- **Award type:** 3
- **Project period:** 2020-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11046029

## Citation

> US National Institutes of Health, RePORTER application 11046029, Systematic elucidation of calcineurin phosphatase signaling in humans (3R35GM136243-05S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/11046029. Licensed CC0.

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