By studying calcineurin, we aim to discover and elucidate new Ca2+-regulated signaling pathways. The calcineurin phosphatase is activated by Ca2+ and calmodulin, and thus dephosphorylates proteins only 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). Currently, we are studying human calcineurin which is ubiquitously expressed and plays critical roles in the nervous, cardiac and immune systems. Calcineurin is best known for activating the adaptive immune response by dephosphorylating the NFAT transcription factors, and is the target of widely prescribed immunosuppressant drugs, FK506 (tacrolimus) and Cyclosporin A. However, these drugs cause many adverse effects due to inhibition of calcineurin in non-immune tissues, where the majority of calcineurin substrates and functions remain to be discovered. Our current strategies to elucidate human calcineurin signaling are described below.
Discovering the human calcineurinome
Protein phosphatases are essential for cell signaling, but have proved difficult to study at the systems-level. We are leveraging recent insights into how calcineurin recognizes substrates to systematically map its signaling network, the calcineurinome, in humans (Wigington et al 2019). 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 Cyclosporin A, which block LxVP binding. We used proteomic peptide phage display to systematically uncover 43 novel PxIxITs and LxVPs SLiMs in disordered regions of the human proteome, including an LxVP motif in the Notch1 intracellular domain. To validate our SLiM-based approach, we showed that Notch1 is directly dephosphorylated by calcineurin, and that mutation of the LxVP sequence disrupts this regulation. All validated human PxIxIT and LxVP instances were used to develop Position-Specific Scoring Matrices (PSSMs) for systematic discovery of motif instances in the proteome, and in calcineurin-proximal proteins identified in HEK293 cells using proximity biotinylation (BioID). Together, these analyses identified 486 proteins that make up the human calcineurin signaling network or calcineurinome ( see Calcineurin docking motif repository). The network, which is statistically enriched for known calcineurin-regulated processes, also suggests previously undiscovered functions for calcineurin at centrosomes, in neuronal and cardiac signaling as well as nucleocytoplasmic transport, a process whose regulation by Ca2+ signaling was unknown. We showed that calcineurin targets Nup153 and Nup50, components of the nuclear pore basket structure to reverse their phosphorylation by ERK and stimulate transport of NLS-containing cargo through the nuclear pore. Yeast nucleoporins, Nup1, Nup2 and Nup60 also contain PxIxIT motifs and are dephosphorylated by calcineurin, suggesting that regulation of nuclear transport by Ca2+ and calcineurin is evolutionarily conserved.
Ongoing studies in the lab continue to mine the calcineurinome to elucidate Ca2+-dependent regulation of critical cell cycle, cytoskeletal and membrane signaling events by calcineurin, using state of the art methods for mass spectrometry, live-cell imaging and proximity-labelling. Discovery of new calcineurin-regulated pathways may help explain the many adverse effects that result from prolonged use of calcineurin inhibitors as immunosuppressants, including onset of diabetes, hypertension, seizures and pain, and provide insights into genetic disorders, such as early onset epilepsy, that are caused by perturbations in calcineurin signaling.
The membrane-associated CNβ1 isoform has unique functions and regulation
Alternative 3’ end processing of the CNAβ gene (PPP3CB) generates CNAβ1 and CNAβ2: catalytic subunits that differ only at their C-termini and complex with the CNB regulatory subunit to form the calcineurin enzymes, CNβ1 and CNβ2. The CNβ1 C-tail gives this enzyme unique regulation and localization to membranes, where it accesses distinct substrates from other (cytosolic) calcineurin isoforms. We are discovering signaling by CNβ1, which is conserved in vertebrates and widely expressed, but has been little studied.
In vitro, we showed that CNAβ1 has surprising enzymatic properties due to the presence of an autoinhibitory ‘LxVP’ SLiM sequence in its C-terminus that blocks substrate binding and limits Ca2+/calmodulin-dependent activation of the enzyme (Bond et al. (2017)). In vivo CNβ1 is predominantly membrane-associated, in contrast to canonical, cytosolic calcineurin isoforms. This is due to lipidation (palmitoylation and prenylation) of CNβ1’s unique C-tail. Currently, we are studying how the dynamic palmitoylation-depalmitoylation of CNAβ1 regulates CNβ1 localization and activity in vivo. We are also identifying substrates for CNβ1, which are predominantly membrane proteins, including a highly conserved protein complex (PI4KIIIa, TTC7B, FAM126A/Hyccin, and EFR3B) which synthesizes phosphatidylinositol-4-phosphate (PI4P), a precursor of the critical signaling phospholipid, PI(4,5)P2, at the plasma membrane. During sustained signaling through G-protein coupled receptors or receptor tyrosine kinases, activation of this PI4P-kinase complex is required for sustained signaling. We are examining possible roles for CNβ1 in this regulation and are identifying additional functions and signaling pathways for this unique phosphatase.
Developing MRBLE-Pep for systematic and quantitative SLiM profiling
In vivo, rapid regulation of weak, transient protein-protein interactions is essential for dynamically shaping cellular responses. Most of these occur via binding of a globular domain to a short (3-10 amino acids long) peptide motif or SLiM (Short Linear Motif), found primarily in disordered protein regions. The low affinities of SLiMs for their receptor domain, sequence degeneracy, and the prevalence of PTMs render SLiMs difficult to identify using current techniques. This project applies MRBLE-Pep (Microspheres with Ratiometric Barcode Lanthenide Encoding coupled to Peptides), a technology recently developed by the Fordyce lab, to analyze SLiM binding to calcineurin, the conserved Ca2+/calmodulin dependent protein phosphatase and target of immunosuppressants (FK506 and Cys A). Our study systematically queried the contribution of PxIxIT sequence to calcineurin binding affinity and showed that residues flanking the core ‘PxIxIT’ sequence significantly influence binding strength, and should be considered when discovering PxIxIT sequences. Our strategy identified peptides with unprecedented affinity and ability to inhibit calcineurin signaling in vivo (Nguyen et al.(2019)). MRBLE-Pep employs spectrally-encoded microfluidic beads to create peptide libraries, where each optical code is coupled to a unique peptide sequence. MRBLE-Pep allows protein binding to hundreds of different peptides to be quantified in a single small volume incubation, thus providing many advantages over current approaches like ITC that require large amounts of purified protein and peptides. Current projects are using MRBL-Pep to analyze and discover critical binding determinants for a variety of human SLiMs.