Research Projects
Overview
The Cyert lab is discovering new Ca2+-regulated signaling pathways mediated by the calcineurin phosphatase. Calcineurin dephosphorylates proteins when Ca2+ signaling is triggered by a hormone, growth factor, neurotransmitter etc. In yeast, we discovered how calcineurin allows cells to survive environmental stress (Goldman et al, 2014). Now we study human calcineurin, which is ubiquitously expressed and plays critical roles in the nervous, cardiac and immune systems (Wigington et al. 2020), and are particularly interested in calcineurin signaling at membranes (Ulengin-Talkish et al. 2021,Bradburn et al, 2025). Calcineurin is best known for dephosphorylating the NFAT transcription factors to activate the immune response, and is the target of widely prescribed immunosuppressant drugs, FK506 (tacrolimus) and Cyclosporin A. However, these drugs cause many adverse effects by inhibiting calcineurin in non-immune tissues, where the majority of calcineurin substrates and functions remain to be discovered. Our current projects are described below.
Discovering the human calcineurinome
Calcineurin interacts with its substrates and regulators by binding to two Short Linear Motifs (SLiMs) termed PxIxIT and LxVP, which occur in intrinsically disordered domains, have low affinity for calcineurin and are degenerate in sequence. These peptides bind to conserved surfaces on calcineurin that are targeted by inhibitors, including the immunosuppressants, FK506 and Cyclospoin A (Grigoriu et al, 2013). We mapped 486 proteins to the human calcineurin signaling network or calcineurinome by leveraging SLiM discovery (Wigington et al 2020). This network revealed functions for calcineurin at centrosomes and cilia (Tsekitsidou et al), and at the nuclear pore complex, where it regulates nucleocytoplasmic transport. Discovery of new calcineurin functions may help explain adverse effects caused by prolonged clinical use of calcineurin inhibitors, such as diabetes, hypertension, seizures and pain, and provide insights and treatments for genetic disorders caused by calcineurin mutations (see the PPP3CA Hope Foundation).
A pool of calcineurin that localizes to the centriole revealed by expansion microscopy. See Tsekitsidou et al 2023
Calcineurin regulates nuclear transport
Calcineurin dephosphorylates nuclear pore complex (NPC) proteins in vivo and in vitro, and promotes nuclear accumulation of an NLS-containing cargo (see below) (Wigington et al 2020). Calcineurin’s role at the NPC is conserved, as yeast nucleoporins are also calcineurin substrates. Now we are determining how calcineurin regulates critical transport pathways through the NPC.
CNβ1-a calcineurin isoform regulated by S-acylation
CNβ1 is an, understudied membrane-associated calcineurin isoform that regulates the essential lipid kinase, phosphatidylinositol 4-kinase alpha (Ulengin-Talkish et al. 2021). CNβ1 has unique localization, substrate specificity and regulation due to S-acylation (palmitoylation) of two conserved cysteine residues in its C-tail. Current studies are identifying how palmitoyl acyltransferases (PATs) regulate CNβ1 localization and activity and are discovering additional functions for this isoform.
Calcimembrin/C16orf74 regulates calcineurin at membranes
Calcineurin signaling occurs in microdomains, where it co-localizes with substrates and Ca2+ signals (Ulengin-Talkish and Cyert, 2023). Calcimembrin (CLMB/C16orf74) is a disordered microprotein that binds calcineurin via an unusual composite LxVPxIxIT SLiM and associates with membranes via lipidation, especially reversible palmitoylation (Bradburn et al, 2025). High levels of CLMB correlate with poor prognosis for melanoma, pancreatic and head and neck cancer. We are currently investigating how CLMB regulates calcineurin signaling in pancreatic and breast cancer.
Mapping calcineurin signaling pathways that mediate pancreatitis
Pancreatitis is an excruciatingly painful medical emergency caused by inflammation of the pancreas, a banana-shaped organ that is located behind the stomach and that produces insulin and a slew of pancreatic digestive enzymes. There are no targeted therapies for pancreatitis. The disease state is caused by a prolonged pathological rise in Ca2+ that induces calcineurin signaling, which is critical for development of the disease as shown by Sohail Husain, Department of Pediatrics/Gastroenterology, Stanford Medicine. We are currently collaborating with the Husain lab to systematically identify calcineurin-regulated signaling pathways and phosphoproteins during pancreatitis using unbiased phosphoproteomic analyses. The goal of this work is to uncover fundamental information that may lead to the development of focused therapies for pancreatitis.