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Systematic elucidation of calcineurin phosphatase signaling in humans

系统阐明人类钙调神经磷酸酶信号传导

基本信息

批准号：
10596473
负责人：
Martha S. Cyert
金额：
$ 54.91万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10596473
关键词：
Address Affinity Bar Codes Biochemical Biotin C-terminal Calcineurin Calcineurin inhibitor Calmodulin Cardiovascular system Cell physiology Cells Centrosome Coupled Cyclosporine Diabetes Mellitus Disease Enzymes Epilepsy Eukaryota Explosion FK506 Future Heart Diseases Human Hypertension Immune System Diseases Immune system Immunosuppressive Agents Knowledge Label Ligase Location Malignant Neoplasms Maps Mediating Membrane Methods Microspheres Nervous System Nuclear Pore Complex Peptides Pharmaceutical Preparations Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Process Protein Dephosphorylation Protein Isoforms Protein Kinase Protein phosphatase Proteins Regulation Research Role Saccharomyces cerevisiae Seizures Signal Pathway Signal Transduction Tissues Work adaptive immune response calcineurin phosphatase in silico in vivo insight new technology novel strategies novel therapeutic intervention nuclear factors of activated T-cells palmitoylation phosphoproteomics protein aminoacid sequence ratiometric research study sensor transcription factor

项目摘要

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.

项目总结