Elucidating calcineurin signaling mechanisms at membranes

Stanford University, Stanford CA

Investigators

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Abstract

PROJECT SUMMARY. Calcineurin (CN), the only Ca2+/calmodulin-regulated protein phosphatase, is a major effector of Ca2+ signaling. Best known for its regulation of adaptive immunity, CN is the target of immunosuppressants, FK506 and Cyclosporin A, which cause a wide range of adverse effects in patients by inhibiting CN outside the immune system. For >30 years, my work has elucidated CN signaling in yeast and human cells. With previous MIRA funding, we defined the human CN signaling network by systematically identifying proteins that harbor the CN- binding SLiMs (short linear motifs), PxIxIT and LxVP. This network reveals locations for CN signaling throughout the cell, including at nuclear pore complexes and centrioles/cilia where we have confirmed that it functions. These studies also show that many CN signaling pathways are yet to be studied, identifying a major gap in our knowledge of cellular regulation. Future studies will focus on the intersection of CN signaling with protein S- acylation (also called palmitoylation). S-acylation reversibly adds one or more fatty acid molecules (usually palmitate) to proteins to regulate their association with and/or function at membranes where Ca2+/CN signaling occurs. Dysregulation of S-acylation, which modifies up to 25% of human proteins, causes disease states ranging from neurological disorders to cancer, and our knowledge of the fundamental mechanisms that control S-acylation activity and specificity is incomplete. Thus, future efforts will focus on our discovery of two novel mechanisms that employ S-acylation to direct CN signaling to membranes. Project 1 focuses on elucidating the regulation and functions of a conserved CN isoform, CNAb1. S-acylation of the CNAb1 C-terminus promotes its direct association with membranes where it regulates PI4KIIIa, an essential enzyme complex that produces phosphatidylinositol-4-phosphate (PI4P) at the plasma membrane. Proposed studies will identify the enzymes that control CNAb1 S-acylation, which is key for understanding and manipulating its signaling pathways. We will examine regulation of PI4KIIIa by CNAb1, especially in physiological contexts such as oncogenic K-RAS signaling and store operated Ca2+ entry and pursue unbiased strategies to identify additional substrates for this isoform. Project 2 focuses on how calcimembrin (c16orf74), an uncharacterized S-acylated protein and CN substrate, regulates CN signaling by targeting the enzyme to membranes, particularly in the context of cancer. Calcimembrin contains an unusual composite LxVPxIxIT motif, which dictates unique biochemical properties including its dephosphorylation in multimeric complexes that confer sensitivity to local calcimembrin:CN ratios. Future studies aim to understand, mechanistically, how Clmb shapes CN signaling in breast cancer cells to regulate cellular processes such as estrogen receptor signaling and to test key elements of the model formulated from our in vitro findings. Together these projects, at the nexus of CN signaling and reversible S-acylation, will address multiple gaps in our knowledge of membrane-based signaling, and provide new insights into fundamental aspects of cellular regulation in both healthy and diseased cells.