Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

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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 inhibitors, 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 underlying 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;

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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

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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.

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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. 2005). Originally, this was to activation of serine one-carbon metabolism thought to occur by oxidation of Fe2+ to Fe3+, (Felkin et al. 2011; Padrón-Barthe et al. 2018). but recent results suggest oxidation of key me- Mice lacking CN-Aβ1 (but not CN-Aβ2) are thionine residues as the mechanism (Zhou et al. viable and fertile, but develop cardiac hypertro- 2014). phy with age, and display increased hypertrophy in response to pressure overload (Padrón- Barthe et al. 2018). Thus, in vivo, this isoform Calcineurin-β1 Isozyme exhibits unique characteristics by failing to acti- A distinct activation mechanism is exhibited by vate NFAT, and instead regulates signaling path- the calcineurin-β1 isozyme consisting of a con- ways such as AKT/mTOR. Direct substrates for served and widely expressed variant of CN-A this isoform remain to be identified. Further- (the CN-Aβ1 isoform), which is generated by more, alternative splicing may be a more com- alternative 30-end processing of the PPP3CB mon mechanism than previously appreciated transcript (Lara-Pezzi et al. 2007; Bond et al. for modifying calcineurin properties in vivo. 2017), complexed with CN-B. The CN-Aβ1 iso- Splicing of CN-A isoforms changes during de- form is identical to CN-Aβ2 through the CBD, velopment of mouse skeletal muscle, and ex- but then diverges to encode a unique carboxyl pression of fetal splice forms of CN-A in adult terminus that lacks the AID, and instead con- tissue impacts its contractile and Ca2+-handling tains a substrate-like sequence, LAVP (Fig. 1A). properties (Brinegar et al. 2017). This sequence autoinhibits calcineurin-β1by binding strongly to the same substrate-binding SLiMS MEDIATE CALCINEURIN SUBSTRATE pocket that is occluded by the AIS (Bond et al. RECOGNITION 2017). The absence of an AID results in higher basal activity and significant activation of calci- Short linear motifs (SLiMs), short degenerate neurin-β1byCa2+ in the absence of calmodulin. peptide sequences that occur in disordered re- Furthermore, with LxVP-containing substrates gions of proteins critically mediate dynamic, (i.e., RII peptide or proteins), maximal activity low-affinity protein–protein interactions during of calcineurin-β1 in the presence of Ca2+ and signaling (Tompa et al. 2014). Calcineurin rec- Ca2+–calmodulin is significantly lower than ognizes two SLiMs in its substrates, PxIxIT and that of canonical calcineurin-β2 because the LxVP, enabling specificity (Fig. 3; Roy and Cyert LAVP sequence persists in the substrate-binding 2009; Nygren and Scott 2016). These SLiMs may

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New Substrates and Functions of Calcineurin

be hundreds of residues away from sites of de- of new calcineurin substrates, anchoring pro- phosphorylation and are found at variable dis- teins and regulators. tances and orientations with respect to each oth- Structures of PxIxIT–calcineurin complexes er (Grigoriu et al. 2013). PxIxIT and LxVP show this motif forming a β strand (Li et al. sequences were first defined in NFAT, and sub- 2007, 2012; Grigoriu et al. 2013) that contacts sequently in multiple calcineurin substrates a hydrophobic groove in CN-A formed by two β from humans and fungi (Saccharomyces cerevi- sheets: β11 and β14. This docking groove is siae) (Rodríguez et al. 2009; Roy and Cyert 2009; remote from the catalytic cleft and the car- Li et al. 2011; Goldman et al. 2014; Nygren and boxy-terminal CN-B and calmodulin-binding Scott 2016; Sheftic et al. 2016; Gibson et al. regions, allowing PxIxIT sequences to interact 2019). The surfaces on calcineurin that bind equally with the active and inactive forms of the these SLiMs are evolutionarily conserved, and enzyme (Fig. 3A). Thus, PxIxIT is critical for mediate a characteristic mode of substrate rec- recruiting calcineurin to substrates or regula- ognition that allows human calcineurin to func- tors, such as the AKAP79/150 scaffold protein tionally substitute for the yeast enzyme (Li et al. (Li et al. 2012), but high-affinity PxIxIT peptide 2004; Grigoriu et al. 2013; Bond et al. 2017). inhibitors do not interact with the catalytic center, or interfere with dephosphorylation of small molecules or substrates that lack a PxIxIT site The PxIxIT Motif (Aramburu et al. 1998). Mutations in a con- PxIxIT motifs are required in most, but not all, served sequence (330NIR332 to AAA in human calcineurin substrates or regulators. PxIxIT mo- CN-Aα) interfere with PxIxIT binding, and se- tifs are degenerate in sequence, but typically verely reduce enzyme function in vivo while share a core consensus sequence: [PI][IVLF] having no effect on the catalytic active site per [IVLF][TSHDEQNKR]. Compromising the se (Li et al. 2004; Roy et al. 2007). PxIxIT sequence or adding a peptide or small molecule that competes with native PxIxIT se- The LxVP Motif quences for binding to calcineurin inhibits dephosphorylation of many substrates in vitro and The LxVP motif is dominated by two hydropho- in vivo (Aramburu et al. 1999; Roy and Cyert bic residues, L and V, which are buried in a hy- 2009; Matsoukas et al. 2015; Nguyen et al. 2018). drophobic channel formed at the intersection of PxIxITs vary widely in their affinity for calci- CN-A and CN-B when bound to calcineurin neurin, with motifs from five different yeast sub- (Fig. 3A; Rodríguez et al. 2009; Grigoriu et al. ∼ strates having Kds ranging from 15 to 250 µM 2013; Sheftic et al. 2016). Structures of two (Roy et al. 2007). Changing the PxIxIT affinity LxVP:calcineurin complexes show this sequence alters the Ca2+-concentration dependence of binding in an extended conformation, and re- substrate dephosphorylation, showing that veal additional hydrogen bonds formed by ami- fine-tuning of this single interaction can modu- no acids at the two positions (-1, -2) preceding late the output of Ca2+ signaling in vivo (Czirják the leucine (Grigoriu et al. 2013; Sheftic et al. and Enyedi 2006; Roy et al. 2007; Muller et al. 2016). Two AISs in CN-A also bind this site: 2009). Recent analyses of PxIxIT peptide affinity The AIS, 416ARVFSVLR423 fills the LxVP-bind- using a novel microfluidic technology identified ing pocket with FSVL, overlaying the LxVP se- residues outside the PxIxIT core that influence quence from calcineurin-interacting partners affinity for calcineurin: basic residues at position (Li et al. 2016), preventing substrates from ac- 2 increase affinity; hydrophobic or acidic resi- cessing the LxVP-binding groove under basal dues at position -1 increase or decrease affinity, conditions (Fig. 2). A unique AIS in CN-Aβ1, respectively; and acidic or phosphorylated resi- 460MQLAVP465, also binds this region (Bond dues at position 9 increase affinity (Nguyen et et al. 2017). Finally, in combination with their al. 2018). These insights promise to improve immunophilin-binding partners, the immuno- PxIxIT discovery and, ultimately, identification suppressants, FK506 and CysA, specifically

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J. Roy and M.S. Cyert

A Catalytic cleft CN-A

LxVP-SLiM

PxlxIT-SLiM A238L peptide CN-B

B FK506-FKBP12 CN-A

CN-B

Figure 3. Calcineurin interacts with substrates via SLiMs, PxIxIT, and LxVP. (A) Space-filled diagram of calcineurin heterodimer complexed with the calcineurin-inhibiting domain from the viral protein A238L (PBD entry 4FOZ; Grigoriu et al. 2013). CN-A subunit is in gray and CN-B subunit is in wheat. The A238L peptide is in green with its PxIxIT-SLiM colored in orange and the LxVP-SLiM colored in purple. The calcineurin catalytic site is marked in red. (B) Space-filled diagram of calcineurin heterodimer complexed with FK506-FKBP12 (PDB entry ITCO; Griffith et al. 1995). CN-A and -B subunits and the calcineurin catalytic site are colored and in A. The FK506-FKBP12 complex is in magenta and occupies the same groove as the LxVP-SLiM in A.

block this docking site, indicating that LxVP under both basal and signaling conditions that 2+ binding is essential for dephosphorylation of elevate [Ca ]cyto, PxIxIT motifs target calci- protein substrates (Figs. 3B and 4; Grifﬁth neurin to substrates/regulators or to protein et al. 1995; Kissingeret al. 1995; Jin and Harrison complexes that contain substrates. For example, 2002; Rodríguez et al. 2009; Grigoriu et al. 2013). the human scaffold protein, AKAP79/150, co- binds calcineurin and two substrates (the L-type Ca2+ channel and the protein kinase A (PKA) Coordination of PxIxIT and LxVP during RII regulatory subunit) that lack PxIxITs but Substrate Dephosphorylation contain LxVP motifs (Murphy et al. 2014). In PxIxIT and LxVP docking are required for de- contrast, LxVP motifs bind calcineurin only af- phosphorylation of most substrates. The viral ter activation by Ca2+ and Ca2+–calmodulin, protein, A238L effectively and competitively in- and may orient the phosphosite toward the cat- hibits yeast and human calcineurin by blocking alytic center of calcineurin for dephosphoryla- both SLiM-binding surfaces without engaging tion (Fig. 4), as shown by computational mod- the active site (Fig. 3A; Grigoriu et al. 2013). eling of calcineurin in complex with the LxVP- However, PxIxIT and LxVP motifs play distinct containing peptide derived from the PKA RII roles during dephosphorylation. By binding regulatory subunit (Grigoriu et al. 2013). This

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New Substrates and Functions of Calcineurin

CN-B CN-B

CN-A CN-A LxVP α β γ CaM , 2, α, β2, γ CaM AID P PxIxIT PxIxIT AID

LxVP

P IS IMPH CN-B

IS CN-A AIS IMPH α, β2, γ CaM PxIxIT-SLiM

PxIxIT LxVP-SLiM AID LxVP P -PO4 group P

Figure 4. Schematic for proposed mechanism of substrate engagement by calcineurin. The substrate is shown as a dark brown line with PxIxIT (yellow) and LxVP (purple) SLiMs and the phosphate group to be removed (green circle). The PxIxIT SLiM can interact with calcineurin under nonactivating conditions, whereas the LxVP SLiM engages with calcineurin only after enzyme activation by Ca2+ and calmodulin. The immunosuppressants (IS) complexes, either CsA-cyclophilin or FK506-FKBP (both denoted as IS, red) with immunophilin (IMPH, orange) occlude the LxVP pocket to inhibit substrate binding.

model predicts that calcineurin dephosphory- LxVP, whereas NFATc1 contains a lower-affin- lates sites that are 9–15 residues carboxy termi- ity PxIxIT coupled with a high-affinity LxVP nal to the LxVP (Grigoriu et al. 2013). However, (Martínez-Martínez et al. 2006). only a subset of substrates currently fit this par- adigm (PKA RII, RCAN1-3), perhaps because of SLiMs Mediate Rapid Evolution of Calcineurin challenges in identifying functional PxIxIT and/ Signaling Networks or LxVP sequences that are particularly degenerate/low affinity. For example, the conserved SLiMs are more rapidly evolving than globular calcineurin regulator from yeast, Rcn1, contains protein domains and may be acquired de novo a very low-affinity PxIxIT motif lacking a P (Davey et al. 2015). Thus, the combination of (GAITID) (Mehta et al. 2009), which works in PxIxIT and LxVP motifs of differing strengths combination with a canonical LxVP sequence allows a wide variety of proteins to be potential (Rodríguez et al. 2009). Similarly, in substrates calcineurin substrates. Although calcineurin is with a high-affinity PxIxIT, a degenerate hydro- highly conserved in structure between yeast and phobic sequence may be sufficient to engage the mammals, the calcineurin network has diverged LxVP-binding pocket. NFAT isoforms also il- significantly between these two species because lustrate this theme: NFATc2 contains a compar- of the evolving nature of calcineurin-binding atively high-affinity PxIxIT and a lower-affinity SLiMs (Goldman et al. 2014). Many PxIxITs

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in S. cerevisiae proteins are not conserved even eukaryotes that will not be discussed here (Lee among closely related species and, as shown for et al. 2013; Juvvadi et al. 2014; Thewes 2014; Liu the Elm1 protein kinase, determine whether or et al. 2015; Paul et al. 2015; Daane et al. 2018). not the protein is regulated by calcineurin (Goldman et al. 2014). Thus, SLiMs direct sur- Phosphoproteomics prisingly dynamic changes in the calcineurin signaling network. Substantial rewiring also Advances in phosphoproteomics are facilitating occurs between the yeast and worm SH3- calcineurin substrate identification. Studies in SLiM-mediated interactome (Xin et al. 2013), yeast showed that 699 phosphorylated peptides suggesting that evolvability is a general feature were more abundant in calcineurin-deficient of SLiM-based PPI networks. versus calcineurin-proficient cell extracts, and that an equivalent number of phosphorylated peptides (660) decreased in abundance when STRATEGIES TO IDENTIFY HUMAN calcineurin was mutated or inhibited (Goldman CALCINEURIN SUBSTRATES et al. 2014). Therefore, to identify direct targets, Substrates are inherently more difficult to iden- 65 proteins whose phosphorylation increased tify for phosphatases relative to kinases, as under calcineurin-deficient conditions and con- proteins must be appropriately phosphorylated tained putative PxIxIT motifs were examined. before examining their dephosphorylation, and Candidates were tested for interaction with because incorporation of a labeled PO4 residue calcineurin and, after phosphorylation with co- is easier to detect than its loss. Calcineurin in purifying kinases, examined for in vitro dephos- yeast was first shown to mediate response to phorylation by calcineurin. This study identified extracellular stresses through its substrate, the the first signaling network for calcineurin in any transcription factor Crz1 (Stathopoulus and Cy- organism, made up of 39 yeast proteins that re- ert 1997). Strategies such as two-hybrid inter- vealed new calcineurin functions (Alvaro et al. action were able to identify additional yeast 2014; Goldman et al. 2014; Ly and Cyert 2017). calcineurin substrates, leading to the study of Integrated analyses of T-cell receptor (TCR) and calcineurin-dependent regulation of endocyto- Ca2+-calcineurin-mediated signaling in mouse sis, intracellular trafficking, response to high pH thymocytes used isobaric labeling to reveal stress, the mating response and cell-cycle con- new calcineurin substrates including Itpkb, a trol (Piña et al. 2011; Alvaro et al. 2014; Gold- calmodulin-regulated inositol trisphosphate 3- man et al. 2014; Arsenault et al. 2015; Guiney kinase (Hatano et al. 2016). Interestingly, these et al. 2015; Ly and Cyert 2017). In humans, a studies also identified multiple proteins that did handful of substrates for calcineurin were first not show obvious alterations in phosphoryla- identified via in vitro assays using well-charac- tion during TCR signaling because they were terized proteins with known kinases (Klee et al. concurrently dephosphorylated by calcineurin 1988). The demonstration that FK506 and CysA and phosphorylated by ERK. Thus, phospho- specifically inhibit calcineurin (Liu et al. 1991) proteomic analyses may fail to identify direct revolutionized investigations of calcineurin calcineurin targets, and are limited by the choice function, and use of these inhibitors remains of tissue and signaling conditions. central to calcineurin substrate identification to this day. Currently, ∼75 human calcineurin Interaction-Based Approaches substrates have been identified, although many more likely remain to be discovered, as illustrat- Affinity purification coupled to mass spectrom- ed by the broad physiological consequences of etry (AP-MS) is an alternative strategy that calcineurin inhibitor-based immunosuppres- has identified calcineurin-interacting proteins, sion (see below) (Li et al. 2011, 2013; Sheftic some of which are substrates. Inp53, yeast syn- et al. 2016). Calcineurin is also highly conserved, aptojanin, was identified as a conserved sub- and plays critical roles in a diversity of lower strate of calcineurin by identifying proteins

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New Substrates and Functions of Calcineurin

that interact with yeast calcineurin specifically containing a match to the “πϕLxVP” regular ex- under osmotic stress conditions, when calci- pression, which was compiled from published neurin is active (Guiney et al. 2015). Fifteen cal- sequences and structural predictions, plus a cineurin-interacting partners, including known PxIxIT consensus match, predicted a large net- substrates GSK-3β, and Rb (Qiu and Ghosh work of 567 potential calcineurin substrates 2008; Kim et al. 2009) were identified in Jurkat (Sheftic et al. 2016). This approach, however, is T-ALL cells expressing constitutively active, limited to finding only exact matches to the reg- truncated CN-Aα (Tosello et al. 2016). On the ular expression, and treats information from all whole, however, AP-MS studies have revealed positions equally. Additional data is needed to relatively few calcineurin-interacting proteins, strengthen in silico approaches for identification and are biased toward identifying high affinity, of calcineurin-binding SLiMs. First, informa- stable interactors such as regulators and scaf- tion about how specific sequence elements ei- folds rather than dynamic SLiM-mediated sub- ther positively or negatively affect affinity of strate interactions (Sundell and Ivarsson 2014; these motifs would improve evaluation of po- St-Denis et al. 2016; Huttlin et al. 2017). In con- tential motif instances. A recent quantitative trast, yeast two-hybrid assays and related meth- study of PxIxIT-calcineurin affinities revealed ods are sufficiently sensitive to detect transient the impact of nonconsensus residues, and pro- SLiM-mediated protein–protein interactions, vides valuable insights that can be applied to and multiple calcineurin substrates from yeast proteome-wide PxIxIT identification (see above; and humans have been identified using this Nguyen et al. 2018). Second, discovery of more technique (Liu et al. 2006; O’Donnell et al. calcineurin-binding peptides of both PxIxIT 2010; Piña et al. 2011; Arsenault et al. 2015; and LxVP types would expand the repertoires Nakamura et al. 2017). However, yeast two-hy- of these SLiMs and allow construction of a stat- brid analyses are also prone to false positives, istical model for each motif (i.e., a position- and can be applied only to a subset of the pro- specific scoring matrix (PSSM)), which includes teome (Mrowka et al. 2001; Stynen et al. 2012). weighted information for each residue in the Recently developed methods that capture motif. This approach provides a scoring method transient interactions such as proximity-depen- that ranks each instance in the proteome based dent biotinylation coupled with mass spec on its degree of similarity to the PSSM, allowing (PDB-MS) offer a new strategy to identify dy- sequences that are similar to but do not match namic calcineurin–substrate interactions, espe- the regular expression to be identified (Krystko- cially in combination with calcineurin mutants wiak et al. 2018). Currently, however, fewer than whose interaction with either PxIxIT or LxVP 15 examples each of human PxIxITs or LxVPs motifs is compromised (Gingras et al. 2019; are in the literature, and each set includes related Wigington et al. 2019). Mutations in the con- motifs from four NFAT isoforms. Proteomic served catalytic site have also been used to “trap” peptide phage display (ProP-PD), which uses a phosphatase–substrate interactions and suc- phage library expressing tiled 16-mers from all cessfully identify substrates for PPI, a strategy predicted disordered regions in the human pro- that has yet to be applied to calcineurin (Wu teome, is an ideal method to experimentally et al. 2018). identify SLiM instances (Sundell and Ivarsson 2014), and was used to discover 20 additional PxIxIT and 25 additional LxVP sequences in the In Silico Strategies for SLiM Identification human proteome (Wigington et al. 2019). These To date, computational strategies for PxIxIT or human peptides directly identified new calci- LxVP discovery have relied on identifying neurin substrates, including Notch1 and matches to a consensus sequence (i.e., a regular Nup153, and allowed construction and testing expression) in combination with disorder or ac- of robust PSSMs for PxIxIT and LxVP, contain- cessibility predictions (Goldman et al. 2014; ing 12 and 7 positions, respectively, that were Sheftic et al. 2016). Recently, human proteins used for motif discovery in the proteome

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(Wigington et al. 2019). A major strength of in direct connection to known NFAT targets. Rath- silico SLiM-based strategies is the ability to iden- er, these studies highlight the many functions tify putative calcineurin targets comprehensive- that calcineurin serves throughout the body, ly (i.e., without experimental limitations such as and the need to identify physiologically relevant protein level and accessibility or tissue/cell-type- substrates that mediate these responses. specific expression). Using this approach, we discovered that calcineurin regulates transport Kidney Dysfunction and Hypertension through the nuclear pore by dephosphorylating multiple nucleoporins (Wigington et al. 2019). Calcineurin inhibitor-induced hypertension is Overall, these computational methods generate characterized by vasoconstriction as well as so- a large “parts list,” and provide a starting point dium and fluid retention and is likely caused by for investigations into new functions for Ca2+ multiple effects on the nervous system, vascula- and calcineurin signaling. ture, and kidney (Hoorn et al. 2012). The clear- est connection is between calcineurin inhibitors and the activity of the Na/Cl cotransporter ADVERSE EFFECTS OF (NCC), product of the SLC12A3 gene, which IMMUNOSUPPRESSION WITH functions in the distal convoluted tubule portion CALCINEURIN INHIBITORS SUGGEST of the kidney. Ellison and colleagues leveraged UNDISCOVERED SUBSTRATES AND similarities between familial hyperkalemic hy- FUNCTIONS pertension (FHHt), a rare genetic disorder, The use of calcineurin inhibitors (i.e., cyclospor- and calcineurin inhibitor-induced hyperten- in A (CsA) and tacrolimus (FK506)) as immu- sion, as a rationale to investigate regulation of nosuppressants revolutionized the field of organ NCC by calcineurin (Hoorn et al. 2011). Indeed, transplant surgery. In 1983, when CsA was ap- FK506 increases NCC phosphorylation, fails to proved for clinical use, the 1-year survival of induce hypertension in SLC12A3 knockout kidney transplant patients immediately in- mice, and evokes an exacerbated hypertensive creased from 50% to 80% (Kahan 1999). In response in mice that overexpress this gene. 1994, the more potent FK506 was introduced, Thus, NCC regulation is calcineurin-dependent, and these natural products remain the most but whether calcineurin acts directly on the commonly prescribed immunosuppressants transporter or through other regulators such as today, including treatments for autoimmune WNK/SPAK kinases that are upstream regula- disorders such as acute psoriasis. Both act by tors of this transporter is as yet unclear (Hoorn forming a complex with an intracellular immu- et al. 2012). Calcineurin inhibitors also activate nophilin protein (cyclophilin or FKBP, respec- NKCC2, the Na-K-2Cl cotransporter in the tively), which subsequently inhibits calcineurin thick ascending limb of the kidney, but this ef- and blocks the adaptive immune response by fect is apparently mediated by arginine vaso- preventing dephosphorylation of NFAT tran- pressin (AVP) signaling (Blankenstein et al. scription factors in T cells (Liu et al. 1991). How- 2017), consistent with other findings that calci- ever, improved patient survival has also led to neurin inhibitors alter endocrine signaling (i.e., chronic treatment with calcineurin inhibitors, vasopressin and the renin-angiotensin-aldoste- which results in many adverse effects. Hyper- rone system) (Lassila 2002). Calcineurin inhib- tension, new-onset diabetes, and neurologic itors may also inhibit vasodilation induced by complications commonly occur, likely because nitric oxide (NO), as calcineurin has been of inhibition of calcineurin in nonimmune tis- shown to regulate NO synthase activity and ex- sues (although some differences between CysA pression in endothelial cells (Kou et al. 2002; and FK506 are attributed to noncalcineurin ef- Yuan et al. 2016). These drugs also induce en- fects). Although disruption of calcineurin/ dothelial damage and inflammation, an effect NFAT signaling may contribute to some of these that is mediated by signaling through TLR4, a effects, many of the processes affected have no key component of the innate immune response

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New Substrates and Functions of Calcineurin

(Rodrigues-Diez et al. 2016). Finally, acute in- for glucose uptake (Hinke et al. 2012). These duction of hypertension by CsA may be medi- effects are recapitulated by disruption of the ated by renal sensory nerves, which also regulate PxIxIT motif in AKAP79/150, showing that cal- arterial pressure and sodium levels (Kopp 2015). cineurin signaling regulates diverse aspects of Synapsin, a calcineurin substrate (Chi et al. glucose handling. Calcineurin also affects sens- 2003) that is present in microvesicles in renal ing of the physiological range of glucose concen- sensory nerve terminals may mediate this effect, trations in β cells. Glucose metabolism enhances as knockout of synapsin I and II signiﬁcantly intracellular Ca2+ levels leading to activation of reduce acute induction of hypertension by the ERK1/2 mitogen-activated protein kinase CsA in mice (Zhang et al. 2000). In summary, (MAPK) pathway and calcineurin. Calcineurin calcineurin inhibitors likely disrupt kidney acts on at least two proteins involved in this function through complex effects on the vascu- pathway, B-Raf and KSR2. Calcineurin dephos- lar and nervous systems as well as the kidney phorylates B-Raf, an upstream kinase in the itself, and by disrupting regulation of multiple ERK1/2 pathway, to relieve negative feedback targets by calcineurin. and promote ERK pathway function (Duan and Cobb 2010). The KSR2 scaffolding protein is also dephosphorylated by calcineurin in re- Diabetes sponse to Ca2+ signals to activate the ERK path- New-onset diabetes after transplantation (NO- way (Dougherty et al. 2009). In fact, deletion of DAT) is a frequent complication occurring in KSR2 in mice causes increased insulin levels and almost 30% of patients after kidney transplanta- obesity, and human mutations in this gene sim- tion, resulting in increased risk of organ rejec- ilarly cause decreased glucose and fatty acid ox- tion and detrimental quality of life. Numerous idation, severe insulin resistance and obesity studies have implicated calcineurin inhibitors as (Pearce et al. 2013). These mutations include a cause of NODAT (Chakkera and Mandarino R397H, the terminal residue of an LxVP motif, 2013). The underlying physiological problems 391NTLSVPR397 (Dougherty et al. 2009). Thus, underlying NODAT appear to be enhanced ap- calcineurin inhibitors may disrupt ERK1/2 sig- optosis of pancreatic β cells (the predominant naling, which could also alter glucose homeo- insulin producing cells in the islets of Langer- stasis. In summary, the multiple roles for calci- hans) resulting in reduced insulin secretion. neurin suggest that the underlying etiologies of Calcineurin-mediated signaling has been shown NODAT are likely complex and involve several to regulate pancreatic β-cell growth and func- sites of action. tion. In fact, deletion of the regulatory subunit CN-B from pancreatic β cells in adult mice re- Neurologic Toxicities sults in decreased cell mass, reduced insulin bio- synthesis, and age-dependent development of Use of calcineurin inhibition is associated with a diabetes, effects that are improved by expression wide range of neurotoxicities, which is consis- of active NFATc1 (Heit et al. 2006). Further, a tent with the high concentration of calcineurin similar deletion in neonatal islets resulted in se- in many regions of the brain and its well-docu- verely defective β-cell proliferation and lethal mented effects on synaptic transmission and diabetes (Goodyer et al. 2012). However, other plasticity that underlie demonstrated roles for studies suggest more complex effects of calci- calcineurin in learning and memory (reviewed neurin on insulin signaling and glucose homeo- in Tarasova et al. 2018). These include dephos- stasis. Insulin release from pancreatic β cells is phorylation of synaptic vesicle proteins to regulated by Ca2+ and cAMP signaling, and loss regulate endocytosis and recycling of synaptic of AKAP79/150, the plasma membrane-local- vesicles, as well as modulation of N-methyl-D- ized anchor that localizes PKA and calcineurin, aspartate (NMDA) and α-amino-3-hydroxy- decreases insulin secretion from β cells but in- 5-methyl-4-isoxazolepropionic acid (AMPA) creases glucose sensitivity of muscle, a key tissue receptor activity and cell-surface expression.

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Multiple ion channels with critical functions in ceptors (Tarasova et al. 2018), and rats treated the nervous system including TRPV1, the Cav- with calcineurin inhibitors showed increased 1.2 subunit of the L-type Ca2+ channel, and pre- and postsynaptic NMDA receptor activity TRESK, the inwardly rectifying two pore K+ in spinal cords, producing pain hypersensitivity channel, are also known substrates of calci- that was reduced with an NMDA receptor in- neurin (Mohapatra and Nau 2005; Czirják and hibitor (Chen et al. 2014). Finally, increased vas- Enyedi 2006; Oliveria et al. 2007). However, eti- cular tone caused by calcineurin inhibitors ologies of the common neurotoxic effects of cal- might also contribute to CNIPS. cineurin inhibitors are not well understood. Cal- A somewhat surprising benefit of long-term cineurin inhibitors frequently induce posterior treatment with calcineurin inhibitors may be reversible encephalopathy syndrome (PRES), decreased susceptibility to Alzheimer’s disease, which is marked by cortical and subcortical ede- as suggested by a retrospective patient study (Ta- ma and can cause headaches, confusion, visual glialatela et al. 2015). This finding, although impairment, and seizures in patients (Chen et al. somewhat preliminary, is consistent with ani- 2016). PRES induced by calcineurin inhibitors mal studies showing that calcineurin inhibitors may be a result of hypertension (see above). improve learning and memory in a mouse mod- However, calcineurin inhibitors also compro- el of Alzheimer’s and block the toxic effects of mise the blood–brain barrier, which is also introduced Aβ amyloid oligomers into normal thought to contribute to PRES and may be the mice (Dineley et al. 2007, 2010; Taglialatela et al. result of altered signaling by adrenomedullin, a 2015). All of these findings are consistent with peptide hormone that improves epithelial barri- an emerging model of Alzheimer’s implicating er function (Dohgu et al. 2010) and/or apoptosis dysregulation of Ca2+ signaling as a key, causa- of brain capillary endothelial cells (Kochi et al. tive factor in pathology of the disease (Popu- 2000). The seizures induced by calcineurin in- gaeva et al. 2017). hibitors may be related to those caused by in- In summary, the long-term use of calci- herited mutations in PPP3CA, which inactivate neurin inhibitors as immunosuppressants has the enzyme and cause an early onset form of provided a wealth of data about the conse- epilepsy termed West syndrome (Myers et al. quences of calcineurin inhibition in humans. 2017; Mizuguchi et al. 2018; Rydzanicz et al. However, molecular events underlying many 2019). How calcineurin deficiency causes epi- of these clinical effects have not been elucidated, lepsy is unknown, but mutations in several genes and only ∼75 human proteins have been clearly involved in synaptic vesicle endocytosis, includ- attributed as calcineurin substrates. New studies ing the calcineurin substrate Dynamin I, also of calcineurin structure, its mechanisms of acti- cause epilepsy, suggesting that this function of vation and inactivation, and investigations of its calcineurin may be critical. splice isoforms have revealed the critical role of Calcineurin inhibitors also cause a pain SLiMs in substrate recognition by this and other syndrome termed CNIPS (calcineurin inhibi- phosphatases (Brautigan and Shenolikar 2018). tor-induced pain syndrome) characterized by Novel SLiM-directed approaches are revolu- symmetric pain in the lower extremities includ- tionizing methods for calcineurin substrate ing foot, ankle, and knee bones (Prommer identification and will undoubtedly reveal novel 2012). Although the etiology of this syndrome functions for this enzyme and thus Ca2+ signal- is unknown, calcineurin does have documented ing. Calcineurin inhibitors are still in wide clini- roles in nociception. Calcineurin dephosphory- cal use, and remain some of the most effective lates and activates TRESK, promoting the return treatments available for suppression of the of neurons to baseline after signaling, and sug- adaptive immune response despite their sig- gesting a mechanism for calcineurin inhibi- nificant negative consequences. Advancing our tors to induce hyperexcitability that would result understanding of calcineurin signaling through- in pain. Calcineurin also regulates TRPV1, out the human body is a critical step toward NMDA, and γ-amino butyric acid (GABA) re- elucidating the effects of calcineurin inhibitors

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New Substrates and Functions of Calcineurin

in patients, and for developing therapeutic strat- calcium handling functions. eLife 6: e27192. doi:10.75 egies to combat their toxicities. 54/eLife.27192 Chakkera HA, Mandarino LJ. 2013. Calcineurin inhibition and new-onset diabetes mellitus after transplantation. Transplantation 95: 647–652. doi:10.1097/TP.0b013e ACKNOWLEDGMENTS 31826e592e Chen SR, Hu YM, Chen H, Pan HL. 2014. Calcineurin in- J.R. and M.S.C. are supported by National Ins- hibitor induces pain hypersensitivity by potentiating pre- titutes of Health Grant 1R01GM119336. We and postsynaptic NMDA receptor activity in spinal cords. J Physiol 592: 215–227. doi:10.1113/jphysiol.2013.263814 thank Devin Bradburn, Idil Ulengin-Talkish, Chen S, Hu J, Xu L, Brandon D, Yu J, Zhang J. 2016. Poste- and Callie Wigington for useful discussion and rior reversible encephalopathy syndrome after transplan- critical reading of this manuscript. tation: A review. 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Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

Jagoree Roy and Martha S. Cyert

Cold Spring Harb Perspect Biol published online July 15, 2019

Subject Collection Calcium Signaling

The Endoplasmic Reticulum−Plasma Membrane Primary Active Ca2+ Transport Systems in Health Junction: A Hub for Agonist Regulation of Ca 2+ and Disease Entry Jialin Chen, Aljona Sitsel, Veronick Benoy, et al. Hwei Ling Ong and Indu Suresh Ambudkar Calcium-Handling Defects and Neurodegenerative Signaling through Ca2+ Microdomains from Disease Store-Operated CRAC Channels Sean Schrank, Nikki Barrington and Grace E. Pradeep Barak and Anant B. Parekh Stutzmann Lysosomal Ca2+ Homeostasis and Signaling in Structural Insights into the Regulation of Ca2+ Health and Disease /Calmodulin-Dependent Protein Kinase II (CaMKII) Emyr Lloyd-Evans and Helen Waller-Evans Moitrayee Bhattacharyya, Deepti Karandur and John Kuriyan Ca2+ Signaling in Exocrine Cells Store-Operated Calcium Channels: From Function Malini Ahuja, Woo Young Chung, Wei-Yin Lin, et al. to Structure and Back Again Richard S. Lewis Functional Consequences of Calcium-Dependent Bcl-2-Protein Family as Modulators of IP3 Synapse-to-Nucleus Communication: Focus on Receptors and Other Organellar Ca 2+ Channels Transcription-Dependent Metabolic Plasticity Hristina Ivanova, Tim Vervliet, Giovanni Monaco, et Anna M. Hagenston, Hilmar Bading and Carlos al. Bas-Orth Identifying New Substrates and Functions for an Calcium Signaling in Cardiomyocyte Function Old Enzyme: Calcineurin Guillaume Gilbert, Kateryna Demydenko, Eef Dries, Jagoree Roy and Martha S. Cyert et al. Fundamentals of Cellular Calcium Signaling: A Cytosolic Ca2+ Buffers Are Inherently Ca2+ Signal Primer Modulators Martin D. Bootman and Geert Bultynck Beat Schwaller

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Role of Two-Pore Channels in Embryonic Organellar Calcium Handling in the Cellular Development and Cellular Differentiation Reticular Network Sarah E. Webb, Jeffrey J. Kelu and Andrew L. Wen-An Wang, Luis B. Agellon and Marek Michalak Miller

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