Identifying New Substrates and Functions for an Old Enzyme: Calcineurin
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Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Identifying New Substrates and Functions for an Old Enzyme: Calcineurin Jagoree Roy and Martha S. Cyert Department of Biology, Stanford University, Stanford, California 94305-5020 Correspondence: [email protected] Biological processes are dynamically regulated by signaling networks composed of protein kinases and phosphatases. Calcineurin, or PP3, is a conserved phosphoserine/phospho- threonine-specific protein phosphatase and member of the PPP family of phosphatases. Calcineurin is unique, however, in its activation by Ca2+ and calmodulin. This ubiquitously expressed phosphatase controls Ca2+-dependent processes in all human tissues, but is best known for driving the adaptive immune response by dephosphorylating the nuclear factor of the activated T-cells (NFAT) family of transcription factors. Therefore, calcineurin inhibi- tors, FK506 (tacrolimus), and cyclosporin A serve as immunosuppressants. We describe some of the adverse effects associated with calcineurin inhibitors that result from inhibition of calcineurin in nonimmune tissues, illustrating the many functions of this enzyme that have yet to be elucidated. In fact, calcineurin has essential roles beyond the immune system, from yeast to humans, but since its discovery more than 30 years ago, only a small number of direct calcineurin substrates have been shown (∼75 proteins). This is because of limitations in current methods for identification of phosphatase substrates. Here we discuss recent insights into mechanisms of calcineurin activation and substrate recognition that have been critical in the development of novel approaches for identifying its targets systematically. Rather than comprehensively reviewing known functions of calcineurin, we highlight new approaches to substrate identification for this critical regulator that may reveal molecular mechanisms un- derlying toxicities caused by calcineurin inhibitor-based immunosuppression. STRUCTURE AND ACTIVATION OF human genes: PPP3CA, PPP3CB (alternatively CALCINEURIN spliced to generate β1 and β2), and PPP3CC. Of these α, β2, and γ are highly homologous canon- Canonical Calcineurin Isozymes ical isoforms that, despite some divergence at alcineurin is a heterodimer composed of their amino and carboxyl termini, share domain Ccatalytic (CN-A) and regulatory subunits architectures and activation mechanisms that (CN-B) (Fig. 1). Calcineurin is a metalloen- are widely conserved in calcineurin enzymes zyme, and CN-A contains two cofactors (Zn2+ across eukaryotes (Thewes 2014). CN-A con- and Fe2+) that coordinate a water molecule dur- tains a globular catalytic domain that is highly ing catalysis (Rusnak and Mertz 2000). CN-A related to other PPP phosphatases, followed by isoforms, α, β1, β2, and γ, are encoded by three an α-helical region that binds CN-B (Fig. 1A; Editors: Geert Bultynck, Martin D. Bootman, Michael J. Berridge, and Grace E. Stutzmann Additional Perspectives on Calcium Signaling available at www.cshperspectives.org Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a035436 1 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press J. Roy and M.S. Cyert A CN-A: Canonical isoforms: CN-Aα, β2, γ NH2- Catalytic domain BBH CBD AID -COOH AIS Alternate CN-Aβ1 carboxyl terminus NH2- Catalytic domain BBH CBD LAVP MLS -COOH CN-B: AIS NH2- EF1 EF2 EF3 EF4 -COOH Catalytic cleft B AID CN-A AIS CN-B Figure 1. Overview of calcineurin structure. (A) Schematic of canonical (α, β2, and γ) and β1 CN isoforms. BBH, CN-B binding helix; CBD, calmodulin-binding domain; AIS, autoinhibitory sequence; AID, autoinhibitory domain; MLS, membrane localization sequence; EF, calcium-binding helix-loop-helix domain (EF1, 2 are lower affinity calcium-binding domains and EF3, 4 are higher affinity calcium-binding domains). (B) Ribbon diagram of CN heterodimer from PDB entry 4ORC (Li et al. 2016). CN-A subunit is in gray and CN-B subunit is in wheat. Calcium ions are colored in green, AIS in magenta, and AID in red. The catalytic site is represented by zinc (magenta) and iron (lime) metal ions. CN-B-binding helix (BBH)), and a calmodulin- pockets in the absence of Ca2+ and Ca2+–cal- binding domain, which binds one molecule of modulin to silence the enzyme (see below) Ca2+-bound calmodulin when activated (Fig. (Fig. 1B; Li et al. 2016). Finally, the carboxyl 1A, CBD; Aramburu et al. 2000; Rusnak and termini of canonical CN-A subunits (α, β2, Mertz 2000). In the inactive enzyme, this region and γ) contain an autoinhibitory domain is largely unstructured, but becomes α-helical (AID), which forms two short α helices that on binding Ca2+–calmodulin and contains ad- directly block the catalytic site under basal con- ditional sequences that stabilize the calmodu- ditions (i.e., nonsignaling, cytosolic Ca2+ con- lin–calcineurin interaction (Shen et al. 2008; centrations [<100 nM]) (Fig. 1B); a peptide en- Rumi-Masante et al. 2012; Dunlap et al. 2013). coding the AID sequence inhibits calcineurin in Immediately carboxy terminal to the CBD is the vivo and in vitro (Hashimoto et al. 1990; Kissin- recently defined autoinhibitory sequence (AIS) ger et al. 1995). CN-B, the regulatory subunit of that occludes one of two substrate-binding calcineurin, encoded by PPP3R1 and PPP3R2, is 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a035436 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press New Substrates and Functions of Calcineurin 2+ 2+ a highly conserved Ca -binding protein that trations ([Ca ]cyto <100 nM), only the high- contains two lobes with a pair Ca2+-binding affinity Ca2+-binding sites of CN-B are EF-hands (Fig. 1); one pair has low affinity occupied, and the enzyme is inactive. Elevation – fi 2+ (10 50 µM range) and the other high af nity in [Ca ]cyto on physiological stimulation 2+ 2+ fi (submicromolar) for Ca (Stemmer and Klee ([Ca ]cyto >1 µM) occupies the low-af nity sites 1994). Myristoylation of the CN-B amino ter- on CN-B, causing a conformational change in minus promotes thermal stability of the enzyme CN-A that promotes Ca2+–calmodulin binding (Aitken et al. 1982). and decreases the Km for substrates (Stemmer and Klee 1994; Yang and Klee 2000). Further binding of Ca2+–calmodulin to CN-A displaces Activation Mechanism the AIS from the substrate-binding pocket and Ca2+ activates calcineurin first by binding to the AID from the catalytic active site to achieve CN-B, and subsequently through the interaction maximal catalytic activity (Fig. 2; Stemmer and of Ca2+–calmodulin with CN-A (Stemmer and Klee 1994; Perrino et al. 1995; Li et al. 2016). Klee 1994). Under basal cytosolic Ca2+ concen- Thus, Ca2+ binding to CN-B and Ca2+–cal- CN-B CN-B CN-A CaM CN-A α, β2, γ α, β2, γ CaM AID AID CN-activating conditions Basal conditions CN-B CN-B CaM CN-A LAVP CN-A LAVP β CaM β1 1 Caspases/ calpain CN-B CaM Membrane N-B localization C LAVP CN-A CN-A β1 Constitutively activated CN Figure 2. Schematic of proposed activation mechanism for canonical and calcineurin-β1 isozymes. Ca2+ ions binding CN-B and CaM (calmodulin) are in white. The magenta box represents the autoinhibitory sequence (AIS) sequence. For canonical calcineurin isozymes, Ca2+ binding to CN-B and calmodulin binding to CN-A relieves autoinhibition by the AIS and autoinhibitory domain (AID). For the calcineurin β1 isozyme, activation by calcium and calmodulin entail removal of the AIS and LAVP sequence from the substrate-binding groove. Activation may also involve localization of calcineurin-β1 to membranes via lipid modifications at the carboxy- terminal end. For all calcineurin isozymes, cleavage of the carboxy-terminal domains by calpain or caspases results in constitutively active enzyme. Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a035436 3 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press J. Roy and M.S. Cyert modulin binding to CN-A cooperatively activate pocket, even after displacement of the AIS calcineurin by overcoming autoinhibition at two (Fig. 2). The unique carboxyl terminus of CN- sites, resulting in a large dynamic response, as Aβ1 also promotes distinct protein interactions well as rapid inactivation on termination of Ca2+ and targets the enzyme to intracellular mem- signaling. In contrast to this reversible regula- branes, including the Golgi apparatus (Felkin tion, cleavage of the entire carboxy terminal et al. 2011; Gómez-Salinero et al. 2016). This CN-A domain to remove the AIS and AID irre- membrane association is mediated by lipidation versibly activates calcineurin. CN-A is cleaved of conserved cysteines, which may also contrib- by the Ca2+-dependent protease, calpain, to ute to enzyme activation in vivo (Fig. 2; I Ulen- constitutively activate calcineurin independent gin-Talkish and MS Cyert, unpubl.). The few of Ca2+–calmodulin in several pathophysiolog- studies that focus on calcineurin-β1 show that ical contexts, and can also be proteolyzed by its physiological functions are distinct from caspase-3, which contributes to synaptic dys- those of canonical calcineurin isozymes. For in- function in mouse models of Alzheimer’s dis- stance, in mice, CN-Aβ1 is highly expressed in ease (Fig. 2; Mukerjee et al. 2001; Wu et al. 2007; regenerating muscle and stem cells (Lara-Pezzi D’Amelio et al. 2011). Calcineurin is irreversibly et al. 2007). In contrast to CN-Aβ2, CN-Aβ1is inactivated by oxidation, both in vivo and in cardioprotective rather than hypertrophic when vitro (Wang et al. 1996; Sommer et al. 2000; overexpressed in the heart, an effect attributed Namgaladze et al.