FEBS Letters 585 (2011) 3689–3698

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Review Why is PTPN22 a good candidate susceptibility for autoimmune disease? ⇑ Garth L. Burn , Lena Svensson, Cristina Sanchez-Blanco, Manoj Saini, Andrew P. Cope

Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, Division of Immunology, Infection and Inflammatory Disease, King’s College School of Medicine, King’s College London, UK article info abstract

Article history: The PTPN22 locus is one of the strongest risk factors outside of the major histocompatability complex Received 23 March 2011 that associates with autoimmune diseases. PTPN22 encodes lymphoid tyrosine phosphatase Revised 13 April 2011 (Lyp) which is expressed exclusively in immune cells. A single base change in the coding region of this Accepted 13 April 2011 gene resulting in an arginine to tryptophan amino acid substitution within a polyproline binding Available online 20 April 2011 motif associates with type 1 diabetes, , systemic erythematosis, Hashimo- Edited by Richard Williams, Alexander tos thyroiditis, Graves disease, Addison’s disease, Myasthenia Gravis, , systemic sclerosis juve- Flügel and Wilhelm Just nile idiopathic arthritis and psoriatic arthritis. Here, we review the current understanding of the PTPN22 locus from a genetic, geographical, biochemical and functional perspective. Keywords: Crown Copyright Ó 2011 Published by Elsevier B.V. on behalf of Federation of European Biochemical PTPN22 society. All rights reserved. Lyp Phosphatase Genetics

1. Introduction 2. Protein tyrosine phophatases and protein tyrosine kinases are immune cell gatekeepers Lymphoid protein tyrosine phosphatase (Lyp) is encoded by the PTPN22 locus on 1p13.3–13.1. A single nucleotide Protein Tyrosine Phosphatases (PTPs) and Protein Tyrosine Ki- polymorphism in PTPN22 has been reported as an autoimmune nases (PTKs) are enzymes that specifically catalyse the reversible susceptibility locus that associates with type 1 diabetes [1–8], addition or release of phosphate groups from tyrosine residues rheumatoid arthritis [8–15], systemic lupus erythematosis on signalling intermediates. Broadly speaking, PTKs amplify signals [10,16–18], Hashimotos thyroiditis [19], Graves disease [20], Add- whilst the mode, tempo and duration of the signal are governed by isons disease [21,22], Myasthenia Gravis [23], vitiligo [24–26], sys- PTPs. PTPs and PTKs are divided into two groups: receptor (mem- temic sclerosis [27], and juvenile idiopathic arthritis [14,28] brane bound-RPTP or RPTK) or non-receptor (cytoplasmic-NRPTP suggesting that allelic variants of PTPN22 could predispose to a or NRPTK). These signalling intermediates are chemical messen- more general autoimmune diathesis. Genetic studies indicate that gers that are recruited when transducing messages from the extra- the association is highly reproducible in populations that carry the cellular environment into the cell and lead ultimately to changes in mutant allele. In spite of these validation studies, and the contin- cell behaviour. Phosphorylation/dephosphorylation by PTKs and ued growth in genetic association studies with disease in man, PTPs on inhibitory or activatory tyrosine on signalling intermedi- the functional consequences of a single base change at the DNA le- ates are the equivalent of molecular on/off switches. Post-transla- vel of PTPN22 remain unresolved. Here, we examine the current tional modifications of by the addition or release of understanding of Lyp function and explore the evidence providing phosphate groups on tyrosine alters enzymatic activity or binding insights into why the PTPN22 locus is a biologically plausible can- affinity of functional domains which act to propagate or inhibit a didate autoimmune susceptibility gene. series of molecular interactions and reactions. Protein phosphory- lation status can therefore be viewed as a fundamental, coordi- nated process allowing cells to adapt to gradual or sudden changes in the environment and must be tightly controlled. This is particularly true in the context of an immune response, where cells must be activated under appropriate circumstances to fight ⇑ Corresponding author. Fax: +44 20 7848 8632. infections and tumours, but avoid self directed responses that cul- E-mail address: [email protected] (G.L. Burn). minate in autoimmunity [29].

0014-5793/$36.00 Crown Copyright Ó 2011 Published by Elsevier B.V. on behalf of Federation of European Biochemical society. All rights reserved. doi:10.1016/j.febslet.2011.04.032 3690 G.L. Burn et al. / FEBS Letters 585 (2011) 3689–3698

There are around 110 PTPs that have been identified in the hu- cases the actual causative genetic variants cannot be precisely man phosphoproteome and at least 57 of these are expressed in identified because of linkage disequilibrium between causal vari- lymphocytes. PTPs and PTKs control the highly complex and dy- ants and the haplotype tagging single nucleotide polymorphisms namic nature of signal transduction pathways associated with (SNPs) employed to detect variants across the genome. Haplotype many cellular processes as diverse as differentiation, migration, defining SNPs in people who have been diagnosed with autoim- apoptosis, proliferation and activation of immune cells [30]. The mune disorders provide clues that, after fine genetic mapping at tonic inhibition of basal PTK activation requires PTPase activity selected loci, must then be investigated in a biological framework to be several orders of magnitude higher than that of PTK activity. to understand its contribution to disease development. This in turn PTPs were once thought largely to play housekeeping functions by can pave the way for exploring potential therapeutic interventions non-specifically binding and dephosphorylating signalling inter- by understanding pathological alterations. Common disease vari- mediates. It has, however, become increasingly evident that PTPs ants may point to pathways that might become plausible targets bind with exquisite specificity to certain signalling intermediates for therapeutic intervention. However, it should be noted that a re- through protein–protein interaction domains or lipid binding mod- cent study that sequenced two T1D associated in 13 715 ules, and in many cases play non-redundant roles. This notion is individuals revealed that European populations, probably as a re- exemplified in the context of the immune system by immunopa- sult of recent accelerating population growth, harbour many rare, thology exhibited by mouse models in which certain phosphatases deleterious variants that may contribute significantly to disease are defective in function or expression, and implicates signalling burden in these populations, but which are not detected through intermediates as important messengers that are responsible for conventional GWAS [36]. Hypothesis driven investigations of po- setting thresholds of immune activation. These mutations can be tential gene candidates in the diseased state are the only way rare naturally occurring or engineered and point to the importance of variants can currently be discovered. phosphatase function in maintaining immune homeostasis. For example, a loss-of-function of PTP SHP-1 mutation, which 4. PTPN22 associates with autoimmunity negatively regulates TCR signalling leads to the motheaten pheno- type characterised by immunodeficiency, lymphoproliferation In 2004 a seminal paper by Bottini et al. reported an association and autoimmunity [31,32]. A gain in function of CD45, which has between a SNP in PTPN22 and T1D in two distinct populations, been shown to be a positive regulator of TCR and BCR signalling, using a candidate gene approach [4]. A transitional mutation predisposes mice to an autoimmune-like syndrome [33]. While changing a cytosine to a thymine at position 1858 in the coding re- mouse models provide insight into the importance of phosphatase gion of the PTPN22 gene resulted in a single amino acid change of function, little is known about phosphatases that contribute to dis- an arginine to a tryptophan at codon 620 in exon 14. This R620W ease in humans. In recent years, genome wide association studies amino acid substitution is located in a polyproline motif, PLPXR, have repeatedly identified candidate PTPs that associate with auto- within the Lyp phosphatase protein thought to be involved in bind- immune disease. These include PTPN2, PTPRC, UBASH3A, PTPN11, ing to SH3 domains during protein–protein interactions [37]. PTPRT and PTPN22, pointing to dysregulation of protein tyrosine Within 1 year SLE and RA were reported to also associate with phosphorylation as a crucial facet of immunopathology. This re- R620W and following a multitude of studies it has now become view focuses on PTPN22, since genetic variants of this phosphatase evident that this polymorphism is an allelic variant associated with remain one of the strongest genetic risk factors for autoimmune susceptibility to a growing number of autoimmune diseases (Ta- disease outside of the major histocompatibility complex (MHC). ble 1). Strikingly, the association is highly reproducible, found in It follows that a better understanding of the biology of PTPN22/ many different populations and is restricted to disorders that usu- Lyp may unravel new and possibly common mechanisms that pro- ally have a strong autoantibody component. A perplexing gradient mote autoimmune disease. frequency of the allele has been reported to exist.

5. The geographical distribution of R620W in European 3. Genetic associations between allelic variants and populations autoimmunity The highest frequency of the allele is found in the most north- In recent years, human genetic studies have identified a grow- erly and easterly reaches of the continent in Finland (15%) and ing number of gene candidates that associate with autoimmune the Ukraine (14.1%) and their neighbouring lands. Moving south- disease. There are three approaches that can be utilised when west, the frequency of the allele gradually decreases to around investigating genetic variants that might contribute to the devel- 7% in France and Spain and is found in only 2–3% of Italian and Sar- opment of autoimmune disease states. These include candidate dinian populations (Fig. 1 [1–5,9,10,12,14,20,21,28,38,39]) [40]. gene association studies, linkage analysis in multiplex families and genome wide association studies (GWAS). Statistical tools have been developed to complement each approach, but a lack of Table 1 appreciation for careful case controls when conducting candidate PTPN22 associates with autoimmune diseases. The number of studies pooled is gene studies and low statistical power of genetic linkage analysis indicated in brackets. Based on references [1–34]. has compromised the validity of some of these studies [34].A Autoimmune disease Odds ratio strong publication bias of candidate gene studies that report posi- Rheumatoid arthritis (15) 1.3–2.13 tive findings has also been documented [35]. Results from these TID (7) 1.5–2.39 studies should therefore be treated with caution until they have SLE (5) 1.42–2.56 been validated unambiguously in replication studies. Hashimotos thyroiditis 1.62–1.77 GWAS are currently considered the most effective way of min- Addisons 1.69 Juvenile idiopathic arthritis 1.17–1.7 ing for new disease associated gene variants since this approach is Wegeners granulomatosis 1.76 purely discovery driven, conducted by scanning whole genomes Graves disease 1.7 without any particular hypothesis in mind. GWAS can be limited Systemic sclerosis 1.09 by the fact that only common gene variants are identified because Vitiligo 1.82 Psoriatic arthritis 1.49 of the reference genomes used and small sample sizes. In many G.L. Burn et al. / FEBS Letters 585 (2011) 3689–3698 3691

Fig. 1. The frequency distribution of the minor T allele across the European continent. An East to West gradient is demonstrated. Adapted from www.eduplace.com/ss/maps/.

American and Australian white European descendants carry the al- Structurally Lyp and PEP consist of an N-terminal catalytic do- lele at a frequency of 6–9%. In Asian and African populations the al- main, an interdomain and a C-terminal binding domain that con- lele is virtually absent [11]. This observation indicates that the tains four polyproline-rich motifs (P1–P4) that act as potential polymorphism in PTPN22 probably arose late in human evolution binding sites for Src Homology 3 (SH3) protein domains. Lyp and in a European population(s). It may confer a selective advantage. PEP are the most divergent example of phosphatase orthologues In support of this notion, extended haplotype homozygosity stud- between human and murine species (89% and 61% homology in ies have indicated that the PTPN22 locus may be under positive their catalytic and non-catalytic domains respectively) although selection, at least in populations of European ancestry [41]. most of these differences lie within the interdomain that may be There has been interest as to whether R620W may protect car- important for regulating phosphatase activity [52,53]. There have riers from infectious disease because there have been reports of been some hints in the literature of subtle biochemical differences potential autoimmune susceptibility loci protecting individuals between PEP and Lyp and so extrapolation from murine models to from environmental pathogens [42,43]. Studies have indicated that human biology should be undertaken with some caution. carriers of R620W may be protected from tuberculosis [44,45]. R620W is a risk factor in the development of invasive pneumococ- 7. Lyp isoforms cal disease, indicating that PTPN22 is a susceptibility gene for auto- immunity and certain infections [46]. Other studies investigating In humans, Lyp exists in three splice forms designated Lyp1–3 the involvement of R620W in the protection from or susceptibility [50,54] (Fig. 2). The full length protein of 807aa, Lyp1, is the most to Hepatitis C infection [47], Trypanosoma Cruzi infection [48] and abundant of the 3 isoforms and has been the subject of all func- brucellosis [49] have demonstrated no differences between control tional studies to date. Lyp2 is the shortest splice variant and con- groups and R620W carriers. Further association studies of R620W tains only the first P1 motif within the N-terminal whilst Lyp3 with infection might provide useful information about the fre- has a 28 amino acid deletion between P1 and P2. The functions quency distribution of the allele and its possible relationship with of Lyp2 and Lyp3 are not known. Lyp can be membrane bound an infectious agent. through interactions with other proteins, or cytosolic, but the sub- cellular location of the different isoforms has not yet been re- 6. The discovery of Lyp and PEP ported. The expression of Lyp is confined exclusively to haematopoietic cells and a clear hierarchy exists in the expression Catalytic domains of PTPs are highly conserved and can thus profile across immune cell lineages with respect to Lyp abundance be targeted using degenerate primers in PCR reactions that will [30]. Natural Killer cells and neutrophils express the highest levels amplify non-conserved flanking regions. The resultant PCR prod- of Lyp, followed by CD8+ T cells while CD4+ T cells and monocytes uct can then be compared to known PTP sequences and vali- express the lowest levels of Lyp [11]. Lyp expression in activated dated or investigated further if a novel stretch of DNA is versus non-activated cells requires further investigation. identified. Using this method, Lyp was identified in 1999 as a 105kDa Class 1 NRPTP, belonging to the proline-, glutamic 8. The function of Lyp in immune cells acid-, serine- and threonine-rich (PEST) family of phosphatases. Its mouse orthologue, proline enriched phosphatase (PEP), was Immune cells must cooperate to mount and maintain an appro- discovered in 1992 [50,51]. priate response and dysregulation of any cell type could lead to 3692 G.L. Burn et al. / FEBS Letters 585 (2011) 3689–3698

Fig. 2. Lyp isoforms 1–3. A schematic demonstrating the exons (vertical bars) and introns (horizontal lines) of Lyp1–3. Approximate sizes of exons and introns are shown. Lyp 1 is 807aa. Lyp2 is a truncated form of Lyp1 with a bigger exon 16 totalling 691aa. Lyp3 is 779aa and has a 28bp deletion in exon 15. Red denotes the changes in exon composition. Adapted from Zang et al 2010. immunopathology or immunodeficiency. Over the past 20 years have been shown to be essential for subcellular location as well much effort has been invested in understanding the various path- as protein binding [59,60]. In additional, immune cells can display ways that trigger immune cell activation, and the relative contribu- many different levels of activation that may also contribute to sub- tion of the different lineages to an immune response. T and B cell strate localisation and recognition [30]. receptors represent some of the best studied signalling machinery Substrate trapping experiments in the human Jurkat T cell line and even though they recognise peptides through completely dif- indicate that Lyp interacts with Lck, f-associated protein 70 ferent antigen receptor structures, they share many PTKs, PTPs (ZAP70), Vav, CD3e, TCRf, valosine containing protein (VCP), and adaptor molecules [55–57]. The Src and Syk kinase families Grb2and c-Cbl (Fig. 3) [61–63]. In addition to the substrates of are crucial positive regulators of immune cell activation through Lyp, PEP targets FynT [62]. These signalling intermediates are B and T cell receptors as well as other immunoreceptors like Fc known to participate in proximal signalling events following T cell receptors (FcR) and Natural Killer Cell receptors termed killer receptor (TCR) engagement with MHC-peptide complexes. One immunoglobulin receptors. At least some of the Src and Syk kinase exception to this is VCP, which has many apparently unrelated family are targeted and inactivated by Lyp as demonstrated by functions including retrotranslocation of unfolded proteins into substrate trapping experiments. Lyp may therefore represent a the endoplasmic reticulum, the homotypic fusion of smooth endo- key regulator of immunoreceptor dependent activation of many plasmic reticulum membranes, NF-jB activation, reformation of leukocyte functions. Despite its expression in all immune cells, golgi cisternae and nuclear envelope reassembly [64]. The signifi- only T cell substrates of Lyp have been reported. cance of this finding is currently unknown.

8.1. Substrate trapping in T cells 8.2. T cell receptor signalling

Although our understanding of Lyp function in T cells has T cells are educated in the thymus and emerge with a functional broadened since the association of R620W with autoimmune dis- and distinct TCR that recognises peptide in complex with MHC. The ease there is still a lack of data describing signalling pathways selection processes that occur in the thymus aim to generate TCR’s other than that of the TCR. Further study of Lyp is required in the that have a high affinity for foreign peptide in complex with MHC lymphoid compartment but is also merited in the context of other but T cells with a high affinity for self peptide are deleted. In the immune cells to generate ideas about fundamental biological pro- periphery, T cells, each of which bear a specific TCR, can undergo cesses that could lead to new hypotheses with respect to autoim- clonal expansion following an encounter with a foreign peptide mune diseases. in complex with MHC displayed by a professional antigen present- In order to delineate signalling pathways, it is important to ing cell. This paradigm is the cornerstone of adaptive immunity, identify target substrates of signalling intermediates. To this end, where T cells can perform a variety of effector functions following Flint et al. have developed a method whereby important PTP cata- their activation and proliferation. lytic residues in the N-terminus are mutated and the resultant Following the engagement of the TCR and co-receptors, signal- non-functional catalytic domain is then fused to a glutathione-S- ling intermediates proximal to the cell membrane are sequentially transferase protein allowing substrates to interact with but not activated. Lck is a critical positive regulator of TCR signalling and is disassociate from a PTP due to the lack of catalytic activity that either activated near or recruited to immunoreceptor tyrosine acti- leads to post-translational protein modifications and the release vation motifs (ITAMS) CD3 and TCR-f that are then phosphory- of modified substrate [58]. This method, or a variation thereof, lated. The doubly phosphorylated ITAM motifs in turn recruit has been widely employed as the substrate trap is sufficiently sta- ZAP-70 via its two tandem SH2 domains. The interaction between ble to allow subsequent isolation of PTP-substrate complexes by the SH2 domain and the phosphorylated ITAM motif releases ZAP- co-immunoprecipitation. Substrate trapping should always be 70 from its auto inhibited state, exposing activatory tyrosine resi- interpreted with care, especially when using candidate substrates dues that can be phosphorylated by Lck or in trans by ZAP-70 itself purified from whole cell lysates or PTP catalytic domain-GST fusion leading to increased catalytic activity. ZAP-70 then phosphorylates proteins as they do not respect binding domain interactions or sub- a number of downstream adaptors, scaffolds and signalling mole- cellular localisation of their native counterparts, both of which may cules including linker of activated T cells (LAT) and SH2 domain be intimately linked. This is because protein interaction domains containing lymphocyte protein (SLP-76). The activation of T cells G.L. Burn et al. / FEBS Letters 585 (2011) 3689–3698 3693

Fig. 3. Lyp acts on multiple proximal T cell receptor signalling intermediates. Lyp is thought to be targeted to the proximal TCR signalling machinery by an interaction of the first polyproline domain in Lyp and an SH3 domain on Csk. Csk, in turn binds phosphorylated PAG/CBP via an SH3 domain. Substrate trapping experiments indicate that Lyp can interact with Lck, ZAP-70, TCR f and CD3e ITAMS, Grb2 and Vav. Lck is thought to regulate Lyp activity but the nature and context of this interaction is not yet clear. through the TCR initiates these proximal signalling events that lead Following the activation of RPTP CD45, a positive regulator of eventually to fully functional, differentiated T cells. TCR signalling, Lck is activated through dephosphorylation of the inhibitory Y505 residue. Lck then auto phosphorylates activatory 8.3. The role of Lyp in T cell receptor signalling tyrosyl residue Y394 within the activation loop of the kinase do- main. Lyp is thought to dephosphorylate Lck at this activatory site Lyp targets various signalling intermediates involved in TCR sig- [61]. Recent studies indicate that activated Lck may also regulate nalling and may act at several points in the signalling cascade. Src Lyp function by phosphorylating an inhibitory tyrosine residue, and Syk family kinases are key activators of T cell receptor signal- Y536, thereby inactivating Lyp [71]. The precise stoichiometry of ling and are negatively regulated by Lyp (Fig. 3) [61,65]. The Src the interactions that promote Lyp phosphorylation by Lck is com- family kinase Lck is phosphorylated on an inhibitory tyrosyl resi- plex and remains to be determined, but Csk micro clusters may due Y505 under steady state by C-terminal Src kinase (Csk) creat- facilitate this interaction through SH2 binding sites that allows ing an internal SH2 binding domain which facilitates the so called the docking of Lck and subsequent phosphorylation and inactiva- ‘‘tail bite’’ inactive conformation of Lck [66,67]. Csk is located in the tion of Csk associated Lyp. Clarification of the sequence of events cytoplasm but can target the plasma membrane through direct that govern inhibition of Lck by Csk and Lyp is also required, since interactions with a high affinity SH2 binding site on a transmem- Csk preferentially targets activated Lck that is phosphorylated at brane protein found in lipid rafts termed Csk binding protein Y394, as demonstrated by in vitro kinase assays and phosphopep- (CBP) or protein associated with GEMS (PAG). It has been proposed tide mapping [72]. The regulation of Lck has also been brought into that Lyp may be targeted to the plasma membrane through inter- question in recent years since it is becoming evident that acute actions with Csk which results in a Csk/Lyp complex anchored to dephosphorylation of Y505 may not be necessary for Lck activation the cell membrane by PAG (Fig. 4). The positioning of Lyp and and TCR signalling. Instead, at steady state, Lck exists in differen- Csk near the cell membrane allows for access to their target sub- tially activated pools with substrate targeting occurring as a result strates which are localised in the same region. An unusually high of spatial temporal regulation [73,74]. Clarification of why there affinity interaction between the P1 domain motif PLPXR on Lyp are primed pools of Lck at steady state and the implications of and an SH3 binding domain on Csk promotes the formation of these findings with respect to TCR signalling is important to further the Csk/Lyp complex that may act to synergistically inhibit proxi- our understanding of the role Lyp plays during these events. mal TCR signalling [62,68]. Following TCR engagement, PAG is Once activated, Lck phosphorylates immunoreceptor tyrosine- dephosphorylated by an unknown phosphatase releasing the Csk/ based activation motifs (ITAMS) on TCRf, CD3e, CD3c and CD3d Lyp complex which may then form homodimers through SH3 do- creating high affinity binding sites for the tandem SH2 domains of- mains of Csk releasing Lyp and inhibiting its own catalytic activity ZAP-70, a Syk family protein tyrosine kinase that contains multiple [69].Using a human derived T cell line (Jurkat cells), the Gjorloff– inhibitory and activatory tyrosyl residues located at Y292, Y492 Wingren group showed that mouse PEP localises at the cell mem- and Y319, Y493 respectively [75,76]. ZAP-70 is phosphorylated brane but recent studies using mouse cells failed to identify the by Lck on residues Y319, Y493 and is activated following its phosphatase in lipid rafts [65,70]. The context in which the Lyp/ recruitment through tandemly arranged SH2 domains to phos- Csk complex is targeted to the plasma membrane requires further phorylated ITAM motifs located on TCR subunits. ZAP-70 then investigation. targets multiple substrates and is itself a substrate of Lyp through 3694 G.L. Burn et al. / FEBS Letters 585 (2011) 3689–3698

Fig. 4. Lyp and Csk inactivate Lck. The Lyp/Csk interaction is mediated through the first polyproline motif on Lyp and the SH3 domain of Csk. PAG, when phosphorylated by Fyn, acts as a scaffold and recruits Csk through an SH2 domain that binds phosphorylated proteins. Csk phosphorylates Y505 creating an SH2 binding site that results in an internal fold and partial deactivation of Lck. For Lck to be fully inactivated, Lyp must dephosphorylate the activatory Y394 residue within the kinase domain for Lck to inactivate its catalytic activity.

direct interactions. There is some speculation that Lyp might also of this phosphatase. Characterisation of the PEP deficient mouse target ZAP-70 through the E3 ubiquitin ligase c-Cbl which docks revealed an increase in the proportion of effector and memory pop- onto the phosphorylated inhibitory residue of ZAP-70 promoting ulations of T cells which would be entirely in keeping with the inactivation through dephosphorylation of activatory residues by putative functions of Lyp. To date, there have been no reports that Lyp. In support of this notion, Lyp has been reported to target c- PEP deficient mice are more susceptible to autoimmune or lym- Cbl, but this relationship remains unclear [77]. phoproliferative disease. One group have shown, however, that ZAP-70 phosphorylates proximal TCR signalling intermediates mice deficient of PEP which also harbour a CD45 SNP encoding a and adaptors that are targeted and perhaps negatively regulated constitutively active phosphatase that positively regulates antigen by Lyp including Grb2 and Vav. Grb2 positively regulates TCR acti- receptor signalling cooperate to break tolerance [83]. vation in cooperation with CD28 by activating Ras. It has been pos- tulated that Grb2 might play a dual role by delivering Lyp to CTLA- 8.4. The role of Lyp in B cell signalling 4 (a relative of CD28 and a negative regulator of co-stimulation) which has SH3 and SH2 domains capable of binding adaptor mol- Because B cells share much of their signalling machinery with T ecules thereby participating in downregulation of TCR signalling cells it is likely that Lyp may regulate proximal B cell receptor sig- mediated by CTLA-4 [30,78]. Studies have shown that Lyp and nalling in a similar fashion, but substrate trapping experiments CTLA-4 upregulate and co-localise following TCR activation [50]. have not been undertaken in B cells. There is some evidence that Vav, an atypical guanine exchange factor involved in cytoskele- there may be impairment of signalling in human individuals carry- tal rearrangements and immune synapse formation is targeted and ing the R620W variant (as discussed below). The PEP deficient activated by ZAP-70 and inactivated by Lyp [79]. Studies have yet mouse, on the other hand, shows no alteration in signalling to address the contribution of Lyp in regulating Vav function. Given through the BCR as evidenced by normal calcium mobilisation fol- the importance of the immune synapse in governing the outcome lowing BCR engagement. The impact of PEP null mutants on other of APC-lymphocyte and lymphocyte-target cell interactions, stud- haematopoietic lineages has not yet been reported [101]. ies exploring the role of Lyp in regulating such processes are war- ranted. Interestingly, a family member of Lyp, PTP-PEST, has been 9. Functional studies of mutant Lyp in T cells shown to dephosphorylate cytoskeletal and focal adhesion pro- teins CAS, PYK2, FAK and paxillin that mediate integrin activation The R620W mutation functions in an autosomal dominant fash- [80,81]. PTP-HCSF is also implicated in regulating cytoskeletal ion with increased clinical penetrance in carriers who are homozy- dynamics [82]. Taken together, the PTP-PEST family function as gous. Understanding precisely how and why this allelic variant important regulators of the cytoskeleton and adhesion molecules contributes to disease development is of considerable interest from in immune cells. both a biological and therapeutic point of view. These observations imply that Lyp plays an important function The arginine-tryptophan substitution within the P1 domain of in the tonic inhibition of TCR function as well the shutting down of Lyp at position 620 and the resulting autoimmune risk is difficult T cell activation after engagement of the TCR complex. Since Lyp is to study because our knowledge about the biochemistry of native upregulated following TCR engagement, it is quite feasible that Lyp and its functional importance remain limited. The expression modulation of signalling in effector T cells is an important function profile of Lyp across all immune cells and immune cell precursors G.L. Burn et al. / FEBS Letters 585 (2011) 3689–3698 3695

(like thymocytes) also makes it difficult to predict which cell types Reduced TCR signalling has also been reported by two other to prioritise when investigating functional differences between groups in healthy individuals (to exclude the effects of disease or R620 and W620. In addition to R620W, other PTPN22 variants have drug treatment) as well as patients with T1D, autoimmune thy- been reported to associate with or confer resistance to the develop- roiditis and RA [88,89].Both of these studies demonstrate blunted ment of autoimmune disease. For example, an R263Q mutant has calcium flux following TCR stimulation in healthy individuals and been shown to be protective against SLE and RA and is reportedly patients that were heterozygous for R620W with an even bigger a loss-of-function variant as demonstrated by in vitro phosphatase negative effect on calcium mobilisation seen in homozygous indi- assays [84]. There are also reports of a SNP found within the pro- viduals. Interestingly, T cell proliferation, a less sensitive and more moter region ofPTPN22 that associates with RA and T1D in an Asian distal reading of TCR signalling was shown to be profoundly de- cohort, leading to increased Lyp mRNA transcripts [85,86].Whether creased in one study [88], but not in another [89]. Rieck also re- the increase in mRNA correlates with higher expression levels of ported decreased levels of IL-4 and IL-10 expression and Lyp is not known. expansion of the memory T cell pool in R620W healthy donors, The mutation in the P1 domain of Lyp has been proposed to dis- as determined by the frequency of CD4+, CD45RO+ cells. rupt the binding of R620W to Csk [4] as compared to R620. Despite In another study using primary T cells derived from Myasthenia these two molecules having opposing kinase/phosphatase activity, Gravis sufferers, an increase in the number of cells producing IL-2 they both serve to inhibit T cell function, possibly synergistically (using ELISpot) was shown in R620W carriers compared to non- [62]. Disruption of the Csk/Lyp complex might then lead to altered carriers, but the measurement of secreted IL-2 was not undertaken, thresholds of antigen receptor signalling affecting the education of making these data difficult to interpret [90]. One way to reconcile T cells in the thymus or B cells in the bone marrow, and/or activation these studies, when taken together, would be that increased num- of naive T and B cells and effector/memory cell populations. bers of T cells in R620W carriers might respond to stimulation, accounting for the increase% of IL-2+ T cells, but the magnitude 9.1. Studies of R620W in immortalised cell lines of IL-2 response by individual cells is decreased. Together these studies flag up two important questions. First, Two groups have addressed the functional consequences of how does a mutation outside the catalytic domain lead to in- R620W in Jurkat cell transfectants [83,87]. Vang et al. used a lucif- creased enzyme activity? Second, how could an apparent attenua- erase NFAT/AP1 reporter to determine the effects of R620 versus tion of TCR signalling lead to autoimmune disease? The complex W620 following T cell stimulation with anti-CD3 and anti-CD28. regulation of PTPs through oxidation events that favour intramo- By transfecting cells with different concentrations of R620 or lecular formation of inhibitory disulphide bridges, inhibitory/acti- W620, it was shown that the mutant phosphatase inhibits NFAT/ vatory tyrosine, serine and threonine phosphorylation sites, AP1 much more efficiently in comparison to its non-disease asso- internal binding sites and pseudophophatases makes it difficult ciated counterpart. A phosphatase assay using a peptide modelled to delineate the mechanism by which R620W might function as on Lck, a physiological substrate of Lyp, that directly monitored the a gain-of-function mutant [91–93]. Recent studies have shown that enzyme kinetics of R620 and W620 demonstrated that the W620 Lck dependent phosphorylation of Lyp on an inhibitory tyrosine variant was catalytically more active, leading to a gain of phospha- residue might be an important regulatory mechanism which posi- tase activity and an associated attenuation of TCR signalling. tively influences TCR signalling [71].The interaction between Lyp Zikherman et al., on the other hand, used as a readout phos- and Csk is proposed to facilitate the interactions between Lck phorylation of the MAPK Erk after transfecting Jurkat cells with and Lyp. R620W cannot bind Csk efficiently and so this mutant R620 or W620 either alone or in conjunction with Csk and then may be a gain-of-function by virtue of its failure to be appropri- stimulated T cells through the TCR/CD3 complex. Phospho-Erk ately negatively regulated by Lck in vivo. Nonetheless, this model was significantly increased in cells co-expressing W620 and Csk. would not explain why, in vitro, R620W has increased phosphatase In addition, it was shown that calcium mobilisation in cells co- activity [4].Others have reported a negative regulatory S35 site lo- expressing W620 and Csk was more efficient in comparison to cated near the catalytic domain that is phosphorylated by PKC [94]. R620 and Csk. These results suggest that the mutant phosphatase, Direct interaction between the interdomain and the catalytic do- when co-expressed with Csk, is a loss-of-function variant leading main of Lyp and PEP has also been postulated as an important reg- to increased TCR signalling following stimulation. ulatory mechanism leading to decreased phosphatase activity but The differences in these two studies could be in part due to the the trigger and context of this interaction remains unknown different readouts used to measure TCR responses or to the use of [52,53]. different stimuli to initiate signalling. The significance of co- The second question pertaining to how hyporesponsive T cells expressing Csk and Lyp, which reveals a previously masked hypo- might lead to autoimmunity may be more straightforward. Studies morphic effect of the disease associated variant, could be crucial in SKG mice have demonstrated that attenuation of TCR signalling and should be investigated further. Most studies using unmanipu- due to a loss-of-function ZAP-70 mutation leads to impaired ZAP- lated primary human cells indicate that R620W is gain-of-function. 70 and dysregulated thymic selection. Our laboratory has also demonstrated the importance of reduced IL-2 production in this 9.2. Studies of R620W in primary human cells model, in the context of skewed Th cell differentiation (Somenzi and Cope, unpublished data). Thus, immunodeficiency manifested In addition to using human T cell lines, Vang et al. studied T cell by defective TCR signalling can, rather counter-intuitively, lead to activation thresholds in the context of R620W using primary T cells autoimmunity. The allelic series of ZAP-70 mutants described by and demonstrated reduced IL-2 production, decreased activity of Goodnow and co-workers highlights how this defective state can transcription factors NFAT/AP1 and reduced calcium mobilisation have subtle effects on distinct thymic and peripheral T cell subsets in response to anti-CD3 and anti-CD28 stimulation as compared within both effector and regulatory compartments [95]. to cells harbouring wild type R620 [87].These results should be interpreted with caution because the authors used primary cells 9.3. Functional studies using B cells from patients with T1D and it is possible that other autoimmune susceptibility genes encoding functionally and/or immunologically In considering B cells, signalling is apparently unperturbed in related proteins may be acting independently or in cooperation PEP null mice (there are however altered B cell pools and hyper- with R620W leading to the aforementioned cellular effects. gammaglobulinaemia)but signalling in human B cells harbouring 3696 G.L. Burn et al. / FEBS Letters 585 (2011) 3689–3698

R620W is altered [89,96]. The association of autoantibody produc- surface [97]. These naturally occurring self reactive T cells with tion with R620W might point to intrinsic B cell anomalies or al- immunoregulatory properties are proposed to be a main player tered help/activation by T cells or both. Studies using B cells in preventing the emergence of autoimmunity in the periphery. If derived from R620W carriers demonstrate smaller memory pools the gain-of-function hypothesis is correct, Treg function may be that are hyporesponsive to stimulation through the BCR in terms compromised leading to deficient regulation of autoreactive T cells of proliferation, display reduced basal level phosphorylation of sig- in the periphery. nalling intermediates and tyrosine phosphorylation following Finally, more work is required in the context of other immune stimulation, indicating a more active phosphatase. What is intrigu- cells. Immunoreceptor signalling is often governed by Src and Syk ki- ing about B cell studies is that naive cells seem unaffected by nases which are both substrates of Lyp. Lyp potentially influences R620W. These results imply that Lyp may have an important role any given immunoreceptor that utilises the Src and Syk kinases in modulating B cell signalling after activation and its role there- and some of these receptors have been shown to be important con- fore in tonic inhibition, at least in B cells, may be a minor one. This tributors to autoimmune disease. Recognition of immune deposits may have as much to do with differential expression of Lyp during through Fc receptors in lupus by immune cells is thought to be a ma- activation and differentiation than with Lyp phosphatase function. jor driving force of disease [98]. One study has already highlighted In fact, studies using the PEPÀ/À mouse indicate that signalling in the importance of Lyp in NK cell proliferation in vitro [99]. NK cells naïve T cells is also normal. Taken together these results suggest have been demonstrated to contribute to autoimmunity in various that in terms of signalling, Lyp appears to play a more important ways, and an imbalance of these cells in vivo might tip the scale in role in the memory and effector cell pools when compared to naïve favour of an autoimmune phenotype [100]. lymphocytes. The observation that expression of both Lyp and PEP are upregulated in activated T cells may be relevant [30]. 11. Concluding remarks

10. How does R620W contribute to disease development? In this review we have summarised the current understanding of the biology of an allelic variant of the PTPN22 gene resulting in an Studies indicate that there are biological differences between arginine to tryptophan substitution within an important polyproline the common Lyp R620 and the disease associated Lyp R620W var- binding motif that mediates SH3 domain interactions. Much work iant. How these differences might manifest in autoimmune disease remains to be done in order to understand R620W and the non-dis- remains to be uncovered. There is still no clear consensus as to ease associated R620. Its absence in African and Asian populations whether R620W is a gain- or loss-of-function variant and there is begs the question of its importance in human immunity and why a possibility that in the future there may be newly discovered func- it has been selected for in some populations and not others. Protec- tions of the mutant phosphatase. It is not inconceivable that tion from infection and susceptibility to autoimmune disease has R620W may exert both gain- and loss-of-function activity in differ- been proposed to have a reciprocal relationship. ent pathways but within the same cell. Another possibility is that Despite a wealth of genetic studies implicating R620W as risk the mutant Lyp may have different (or opposing) effects in cells factor for multiple autoimmune diseases, functional studies of different lineages, or that the mutant has opposing activities in exploring how the mutant phosphatase contributes to disease different pathways in the same cell. development have lagged behind. While T and B cell receptor sig- The most obvious subset of immune cells that are inextricably nalling have been the main focus of most investigations to date, linked to autoimmunity are lymphocytes but the coordination of other immune cell types must also be investigated so as to gain a an immune response requires the participation of all immune cells more holistic understanding of PTPN22 function both in sickness and the origin of an adaptive autoimmune response may not nec- and in health. essarily be due to intrinsic defects in the lymphoid compartment. New insights from the PEP deficient mouse may help to define For example, dendritic cells express high levels of Lyp and are the functions of Lyp in different cell lineages. Indeed, mouse stud- responsible for presenting antigen to and polarising Th cells. Inap- ies have contributed to our understanding of Lyp function and fur- propriate activation of T cells may therefore arise as a consequence ther studies may provide valuable information that can then be of aberrant antigen presenting cell function leading to the develop- used to direct future functional studies in humans. It is also imper- ment of autoimmune disease. ative to understand any functional discrepancies between PEP and One possibility by which R620W might contribute to disease is Lyp that may set these two phosphatases apart, as well as common through changes in thresholds of thymic selection. The involve- functions that bring them together. ment of Lyp during positive and negative selection has not been re- A gain-of-function mutation may present a situation in which ported but its high expression in thymocytes might point to an small molecule inhibitors might be of value. In order for the ther- important function during T cell education. Interestingly, the thy- apeutic potential of R620W to be realised, a clear consensus must mus is one of the only tissue in which all three Lyp isoforms can be reached as to the nature of the mutation ie gain- or loss-of-func- be detected [54]. If the disease associated variant turns out to be tion. Understanding perturbations caused by R620W in signalling gain-of-function, high affinity self-reactive TCR expressing T cells cascades that predisposes individuals to pathogenic autoimmunity might escape negative selection resulting in an expanded reper- remains a formidable challenge. toire of autoreactive T cells and an augmented predisposition to PTPN22 remains a fascinating gene candidate for further study autoimmune disease. Conversely, a loss-of-function R620W mu- and its promiscuous association with multiple autoimmune dis- tant may act on the very few cells that, per chance, escape negative eases might indicate that a common, hitherto unappreciated mech- selection, leading to enhanced and/or sustained T cell activation anism underlying the development of autoimmune disease. Such a and driving T cell differentiation of effector T cells in the periphery, finding would have huge implications in our current understand- leading ultimately to an autoimmune phenotype. ing of autoimmunity and may be of therapeutic benefit. An alternative explanation for autoimmune disease develop- ment in individuals carrying R620W is its effect on T regulatory References cells (Tregs). Experiments in the early 1990s by Sagakuchi and co-workers reignited the interest in these self reactive T cells that [1] Douroudis, K. et al. 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