We  study phosphatases and Ca2+-dependent signaling networks
The calcineurin signaling network in S. cerevisiae is composed of 39 proteins that define many new functions for the enzyme (Goldman et al, 2014). These proteins give rise to phosphopeptides whose abundance increases in calcineurin-deficient cells, and contain a short peptide motif, termed PxIxIT, that binds to calcineurin and is a determining feature of calcineurin substrates. By studying this network we learned that PxIxIT sequences are rapidly gained and lost during evolution, which allows the calcineurin signaling network to acquire new functions. Although calcineurin itself is highly conserved, the constellation of calcineurin-regulated proteins divergesbetween even closely related yeast species. Only a few proteins, such as RCAN, the calcineurin substrate and regulator, and synaptojanin, are retained in the calcineurin signaling network in yeast and humans. These proteins, conserved as calcineurin targets, make up the “core” network, with other interactions determining more variable components.
Calcineurin interacts with at least two distinct peptide in its substrates, which are examples of Short Linear Motifs, or SLiMs, that occur in disordered protein  domains and mediate millions of specific protein-protein interactions (Roy and Cyert, 2009). Each substrate contains a conserved sequence, termed the PxIxIT motif, which determines its affinity for calcineurin. For Crz1, the calcineurin dependent yeast transcription factor, changing the affinity of the PxIxIT motif for calcineurin changes the Ca2+ concentration dependence of calcineurin/Crz1-dependent gene expression in vivo (Roy et al, 2007). Furthermore, calcineurin and MAPK directly compete for access to some substrates, including Dig2 from yeast and JunB from mammals, via a composite docking sequence that encodes both a PxIxIT motif and a MAPK docking site (D-site) (Goldman et al, 2014). We are currently studying the regulatory properties that are conferred by this competitive binding between a kinase and phosphatase. Substrates contain a second, “LxVP” motif that binds to a conserved hydrophobic groove in calcineurin. Immunosuppressants, FK506 and cyclosporin A, bind to this LxVP-docking pocket and block substrate interaction with calcineurin (Grigoriu et al, 2013).
Evolution of calcineurin signaling networks
Calcineurin recognizes substrates via Short linear interaction motifs (SLiMs)
Systems-level analyses of phosphorylation-based signaling networks has transformed our understanding of kinase function, but knowledge of phosphatase signaling has lagged behind, primarily because global approaches to identify phosphatase substrates are lacking. Calcineurin, the conserved Ca2+/calmodulin-dependent protein phosphatase and target of immunosuppressants, FK506 and Cyclosporin A, is ubiquitously expressed, and critically regulates Ca2+-dependent processes in the immune system, heart, and brain. However, in the literature only 27 substrates are attributed to calcineurin. Systematic identification of calcineurin targets is now feasible due to insights into its conserved mechanism of substrate recognition. Calcineurin acts on phosphosites with little primary sequence similarity; thus specificity is not encoded within regions contiguous to the phosphosite. Rather, the enzyme binds to short linear motifs (SLiMs),“PxIxIT” and “LxVP”, which can occur hundreds of residues away from dephosphorylation sites. CsA , FK506 and the viral A238L protein inhibit calcineurin by blocking SLiM binding to conserved surfaces on the enzyme. SLiMs are a growing class of sequences that localize within intrinsically disordered regions, i.e. flexible protein domains that lack a defined structure. SLiMs mediate most protein-protein interactions in cells and evolve rapidly to mediate rewiring of signaling networks, including that of calcineurin. However, degenerate sequences and low affinities for their target domains make SLiMs challenging to identify. We are using novel experimental and computational approaches to identify calcineurin-binding SLiMs systematically in the human proteome. In collaboration with Ylva Ivarsson, Uppsala Unversity, Proteome peptide Phage Display (ProP-PD) was used to directly select calcineurin-binding sequences of the PxIxIT and LxVP types from predicted disordered regions of the human proteome. We also developed a novel computational tool to predict PxIxIT sequences, which makes use of their characteristic structural features (i.e. intrinsic disorder and beta strand formation), and predicts binding to the conserved PxIxIT-docking surface on CNA, the calcineurin catalytic subunit. Sequences identified either experimentally or computationally are validated using a high throughput calcineurin-binding assay, and their parent proteins tested for co-immunoprecipitation with calcineurin in HEK-293 cells. These studies have identified a new calcineurin substrate, C16Orf74, which is a marker for invasive bladder cancer and defined PxIxIT sites in calcineurin substrates, KSR2 and amphiphysin. Furthermore, more than 50 new targets for calcineurin have been identified, including ion channels, kinases, transcription factors and receptors, that reveal points of cross-talk between calcineurin and other signaling pathways in human cells. This basic approach can be broadly applied to systematic characterization of any SLiM-based signaling network.
Calcineurin is tightly controlled by Ca2+ and calmodulin, which activate the enzyme by relieving auto-inhibition of the active site, and revealing a critical binding pocket for "LxVP" substrate motifs. We are studying regulation of a conserved splice variant of the human CNAβ gene, CNAβ1, which promotes cardiac regeneration in vivo. The CNAβ1 C-terminus contains an LxVP sequence, which we showed auto-inhibits phosphatase activity by blocking substrate engagement. CNAβ1 has distinct enzymatic properties, and is relatively independent of calmodulin. Functional studies are identifying and characterizing unique protein partners for CNAβ1 and its substrates in cardiovascular and endocrine signaling.
Current projects