Protein tyrosine phosphatases and the immune response

© 2004 Nature Publishing Group

R E V I E W S

NATURE REVIEWS | IMMUNOLOGY VOLUME 5 | JANUARY 2005 | 43

Tyrosine phosphorylation1 is a key mechanism for signaltransduction and the regulation of a broad set of physio-logical processes that are characteristic of multicellularorganisms. Processes that are regulated include the fol-lowing: decisions to proliferate, differentiate or die; acti-vation of large gene-transcription programmes; cellmotility and morphology; and transportation of mol-ecules into or out of cells. Tyrosine phosphorylation isalso instrumental in determining the duration of theseprocesses and their coordination among neighbouringcells, through soluble mediators and direct cell–cellcontacts, for example, within the immune system2–5.The importance of tyrosine phosphorylation in normalcell physiology is also clear from the many inherited oracquired human diseases that stem from abnormalitiesin protein tyrosine phosphatases (PTPs)6,7, whichdephosphorylate tyrosine residues, and protein tyro-sine kinases (PTKs), which phosphorylate tyrosineresidues (FIG. 1). Severe phenotypes are also observed inmany PTP- or PTK-knock-out mice, and in manycases, the immune system is affected.

All cells of the immune system have high levels oftyrosine phosphorylation and express more genesencoding PTKs and PTPs than any other cell type, withthe possible exception of neurons. Acute changes intyrosine phosphorylation regulate antigen-receptor-mediated lymphocyte activation, cytokine-induced dif-ferentiation and responses to many other stimuli8–12.

Both PTKs and PTPs can have activating and inhibitoryeffects, and the actions of both types of enzyme arerequired for a physiological immune response.Althoughthe molecular mechanisms that initiate T-cell receptor(TCR)- and B-cell receptor (BCR)-driven lymphocyteactivation have been intensely studied during the pasttwo decades2,5,13,14, only the PTK side of the phosphory-lation ‘equation’ (FIG. 1) is reasonably well understood. Bycontrast, the PTPs that regulate lymphocyte physiol-ogy4,5,15 have received the attention of only a handful oflaboratories. It is still far from clear what most of thenumerous PTPs found in immune cells do and which ofthem dephosphorylate key signalling proteins, includingthe PTKs and their immediate substrates.

One of the first indications of the importance ofPTPs in T cells came from experiments that were carriedout in our laboratory in the late 1980s (REF. 16), whichshowed that, although POLYCLONALLY ACTIVATED human T cells placed in fresh medium cease to proliferate andthen revert to a resting phenotype overnight, cells cul-tured in the presence of a low concentration of the non-specific PTP inhibitor sodium orthovanadate retain aBLASTOID MORPHOLOGY and continue to express activationmarkers and to proliferate for many days. This effect cor-related with persistent tyrosine phosphorylation of sev-eral proteins. Similarly, addition of other PTP inhibitors,either pervanadate17,18 or phenylarsine oxide19, to restinghuman T cells was shown to cause spontaneous T-cell

PROTEIN TYROSINE PHOSPHATASESAND THE IMMUNE RESPONSETomas Mustelin, Torkel Vang and Nunzio Bottini

Abstract | Reversible tyrosine phosphorylation of proteins is a key regulatory mechanism fornumerous important aspects of eukaryotic physiology and is catalysed by kinases andphosphatases. Together, cells of the immune system express at least half of the 107 proteintyrosine phosphatase (PTP) genes in the human genome, most of which encode multidomainproteins that contain protein- and phospholipid-interaction domains. Here, we discuss the diversebut specific, and important, roles that PTPs have in immune cells, focusing mainly on T and B cells, and we highlight recent evidence that even subtle alterations in PTPs can cause immunedysfunction and human disease.

POLYCLONALLY ACTIVATED

Activation of all T cells bymitogens in an antigen-independent manner.

BLASTOID MORPHOLOGY

The larger size and appearanceof an activated T cell.

Program of Inflammation,Inflammatory andInfectious Disease Center,and Program of SignalTransduction, CancerCenter, The BurnhamInstitute, 10901 NorthTorrey Pines Road, La Jolla,California 92037, USA.Correspondence to T.M.e-mail:[email protected]:10.1038/nri1530

© 2004 Nature Publishing Group44 | JANUARY 2005 | VOLUME 5 www.nature.com/reviews/immunol

R E V I E W S

immune system describe data obtained using lympho-cytes. Finally, we suggest that new discoveries showingthat even subtle alterations in PTPs can cause humandisease indicate that PTPs could be valuable drug targetsfor the treatment of immunological diseases.

Lymphocyte representatives of the ‘PTPome’We refer to the entire set of PTP genes in a genome asthe PTPome. Contrary to the assumption of manyresearchers a decade ago (and of some still today) thatPTPs are non-specific, unregulated housekeepingenzymes, of which cells only require one or two, it turnsout that the human PTPome is larger than expected. Infact, in the human genome, there are more genes thatencode PTPs6 than PTKs21 — 107 versus 90. Althoughthese numbers include several catalytically inactiveenzymes (both PTPs and PTKs) and PTPs that alsodephosphorylate other phospho-amino acids (that is,serine and/or threonine) or non-protein substrates(such as inositol phospholipids or mRNAs), they concurwith the growing experimental evidence that PTPs tendto be very specific and to have non-redundant, uniquefunctions. It is also becoming apparent that PTPs have amore active role in cellular processes than originallyappreciated; rather than being the enzymes that simplyreverse what PTKs accomplish, PTPs often have anactive regulatory role. In addition, many PTKs, such asthe SRC-FAMILY and SYK (spleen tyrosine kinase)-familyPTKs found in T and B cells, are tightly controlled byPTPs (BOX 1).

Lymphocytes express a remarkably high proportionof the 107 PTP genes in the human genome; T cellsexpress at least 45 different PTPs (TABLE 1), and B cellsseem to express a similar set. We anticipate that, after all107 PTP genes have been examined for lymphocyteexpression, the number of PTPs expressed by either

activation and secretion of interleukin-2 (IL-2).Althoughall of these pharmacological experiments were non-specific and were fraught with pitfalls, they indicated thatPTPs have a crucial role in both the maintenance of aresting lymphocyte phenotype and the reversion of acti-vated lymphocytes to a resting state during the termina-tion of an immune response. (Before these papers, othershad used vanadate in biochemical studies: for example, tostimulate tyrosine phosphorylation of membrane frac-tions from T and B cells20; however, at that time, themechanism by which vanadate mediated this effect wasunknown).

In this review, we discuss the repertoire of PTPs thatis present in cells of the immune system, the structures,functions and common themes of these enzymes andthe most recent advances in our understanding of theirmany roles in the immune response. We mainly focuson the importance of these enzymes in lymphocytes, inpart because most of the publications about PTPs in the

SRC FAMILY

A group of structurally relatedcytoplasmic and/or membrane-associated enzymes that arenamed after the prototypicalmember, SRC. Inhaematopoietic cells, SRCkinases — such as LCK, FYNand LYN — are the first proteintyrosine kinases that areactivated after stimulationthrough the immunoreceptors.They phosphorylate ITAMs(immunoreceptor tyrosine-based activation motifs) that are present in the signal-transducing subunits of theimmunoreceptors, therebyproviding binding sites for SRChomology 2 (SH2)-domain-containing molecules, such asSYK (spleen tyrosine kinase).

PTP

ATPPhosphorylatedsubstrate

Substrate P

P

PTK

Figure 1 | The phosphorylation ‘equation’. Tyrosinephosphorylation is a key mechanism for signal transduction andfor the regulation of many physiological processes. Proteins arephosphorylated on tyrosine residues by protein tyrosine kinases(PTKs) and dephosphorylated by protein tyrosinephosphatases (PTPs).

Box 1 | Regulation of crucial immune-cell protein tyrosine kinases by phosphorylation

Protein tyrosine kinases (PTKs) of the SRC, SYK (spleen tyrosine kinase), TEC and CSK (carboxy (C)-terminal SRCkinase) families are crucial for antigen-receptor-induced lymphocyte activation. SRC-family PTKs — that is, LCK, FYNand YES in T cells, and LYN, FYN and BLK (B-lymphoid kinase) in B cells — phosphorylate antigen-receptor subunitsand are required for lymphocyte activation. The activity of all SRC-family PTKs is positively regulated byphosphorylation of a conserved tyrosine residue in the activation loop (the tyrosine residue at position 394 (Y394) inLCK) and negatively regulated by phosphorylation of a tyrosine residue in the C-terminus (Y505 in LCK). The former is thought to be a site of autophosphorylation, and its phosphorylation is required for activity. Normally, SRC-familyPTKs are only phosphorylated at very low levels at this site. The C-terminal tyrosine residue is phosphorylated by thePTK CSK and results in the adoption of a suppressed conformation, in which the phosphorylated tail binds to the SRChomology 2 (SH2) domain in the same kinase molecule, which stabilizes an interaction of the SH3 domain with a motifin a short linker between the SH2 domain and the kinase domain. This conformation is referred to as the ‘tail bite’ and isshown for LCK in FIG. 3.

The SYK-family kinases — SYK and ZAP70 (ζ-chain-associated protein kinase of 70 kDa) — function downstream ofthe SRC-family kinases to amplify the signal and are focal points for the assembly of signalling complexes. ZAP70 isactivated by LCK-mediated phosphorylation of Y493 in its activation loop, and it subsequently autophosphorylatesabout ten tyrosine residues, including Y292,Y315,Y319 and Y492. Although some of these sites attract SH2-domain-containing substrates,Y292 functions as a negative-regulatory site by attracting the E3 ubiquitin ligase CBL (Casitas B-lineage lymphoma), which results in degradation of the T-cell receptor and disengagement and dephosphorylation of ZAP70.

TEC family PTKs — ITK (interleukin-2-inducible T-cell kinase), TXK and BTK (Bruton’s tyrosine kinase) inlymphocytes — function downstream of SRC PTKs and are positively regulated by phosphorylation, whereas CSK isphosphorylated only on the serine residue at position 364.

© 2004 Nature Publishing GroupNATURE REVIEWS | IMMUNOLOGY VOLUME 5 | JANUARY 2005 | 45

R E V I E W S

Table 1 | PTPs expressed by T cells and their main functions

Class PTP* Role in lymphocyte Mechanism, function and substratesactivation

Receptor-like PTPs (Class I) CD45 Positive Positively regulates LCK and FYN

RPTP-ε ND Unknown, might regulate SRC-family PTKs

CD148 Negative Might dephosphorylate LAT, PLC-γ and other substrates

Non-receptor, classical PTPs (Class I) HEPTP Negative Regulates ERK and p38 in cytosol

PTP1B None Regulates insulin receptor and other receptor PTKs

TCPTP Negative Dephosphorylates STAT1 in nucleus

PTPH1 Negative Inhibitory, dephosphorylates ITAMs and VCP

PTP-MEG1 Negative ND

PTP-MEG2 None Regulates secretion, no effect on early TCR-signalling events

PEZ ND ND

PTP-BAS None Binds CD95 (FAS)

LYP (PEP)‡ Negative Dephosphorylates activation loops of LCK, FYNand ZAP70

PTP-PEST Negative Binds CSK, dephosphorylates CAS, SHC, PYK2 and FAK

PTP-HSCF ND Binds CSK and ITK

SHP1 Negative Mediates inhibition through ITIM-containing receptors,dephosphorylates ZAP70 and SYK

SHP2 Positive Augments ERK activation, crucial for lymphoid development

HDPTP ND ND

Other class I PTPs PAC1 Negative Dephosphorylates ERK in nucleus

MKP1 Negative Dephosphorylates MAPKs in nucleus

MKP3 Negative Dephosphorylates ERK in cytosol

MKP5 Negative Dephosphorylates JNK in nucleus

MKP6 Negative Associates with CD28

VHR Negative Activated by ZAP70, dephosphorylates ERK, JNK and STAT5

VHX Negative ND

VHZ ND ND

RNGTT None Processes mRNA

PRL1, -3 ND ND

CDC14 None Regulates exit from mitosis

PTEN Negative Counteracts PI3K, reduces survival

MTM1 ND Presumably regulates intracellular-vesicle traffic

MTMR1, -2, -3, -4, -14 ND Presumably regulate intracellular-vesicle traffic

MTMR5 ND Catalytically inactive, regulates MTM1 and MTMR2

MTMR10 ND Catalytically inactive, regulates MTMR2

MTMR11 ND Catalytically inactive

Class II PTPs LMPTP Positive Dephosphorylates ZAP70 on the tyrosine residue at position 292

Class III PTPs CDC25A, -B, -C None Activates CDKs

Aspartic-acid-based PTPs EYA3, -4 ND Regulate transcription

*PTP (protein tyrosine phosphatase) classification is reviewed in REF. 6. ‡PEP (PEST (proline-, glutamic-acid-, serine- and threonine-rich)-domain-enriched PTP) is the mouseorthologue of human LYP (lymphoid-specific PTP). CAS, CRK-associated substrate; CDC, cell-division cycle; CDK, cyclin-dependent kinase; CSK, carboxy-terminal SRC kinase;ERK, extracellular signal-regulated kinase; EYA, eyes absent; FAK, focal adhesion kinase; HDPTP, histidine-domain PTP; HEPTP, haematopoietic PTP; ITAM, immunoreceptortyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; ITK, interleukin-2-inducible T-cell kinase; JNK, JUN amino-terminal kinase; LAT, linkerfor activation of T cells; LMPTP, low-molecular-weight PTP; MAPK, mitogen-activated protein kinase; MKP, MAPK phosphatase; MTM1, myotubularin 1; MTMR, MTM-relatedprotein; ND, not determined; PAC1, phosphatase of activated cells 1; PEZ, phosphatase with ezrin domain; PI3K, phosphatidylinositol 3-kinase; PLC-γ, phospholipase C-γ;PRL, phosphatase of regenerating liver; PTEN, phosphatase and tensin homologue; PTK, protein tyrosine kinase; PTP-BAS, PTP basophil; PTP-HSCF, PTP haematopoieticstem-cell fraction; PTP-MEG, PTP megakaryocyte; PTP-PEST, PTP with PEST domains; PYK2, PTK 2; RNGTT, RNA guanylyltransferase and 5′-phosphatase; RPTP-ε, receptor-type PTP-ε; SHC, SRC homology 2 (SH2)-domain-containing transforming protein C; SHP, SRC homology 2 (SH2)-domain-containing PTP; STAT, signal transducer andactivator of transcription; SYK, spleen tyrosine kinase; TCPTP, T-cell PTP; TCR, T-cell receptor; VCP, valosin-containing protein; VHR, Vaccinia virus-VH1 related; VHX, Vacciniavirus-VH1-related MKP X; VHZ, Vaccinia virus-VH1-like member Z; ZAP70, ζ-chain-associated protein kinase of 70 kDa.


R E V I E W S

T cells or B cells will be between 50 and 60. There areseveral PTPs that are restricted to haematopoieticcells, such as HEPTP (haematopoietic PTP), LYP(lymphoid-specific PTP; the mouse orthologue ofwhich is PEP, proline-, glutamic-acid-, serine- andthreonine-rich (PEST)-domain-enriched PTP), SHP1(SRC HOMOLOGY 2 (SH2)-DOMAIN-containing PTP 1) andCD45. The lymphocyte complement of PTPs includesfew transmembrane PTPs, perhaps only 3 of the 21encoded by the human genome — CD45, CD148 andRPTP-ε (receptor-type PTP-ε) — but a high proportion,14 of 17, of the intracellular (non-receptor), classicalPTPs. (For a review of PTP classification, see REF. 6.)

As might be expected, many PTPs are found at, ornear, the plasma membrane of lymphocytes22, and theyparticipate in the regulation of transmembrane sig-nalling (TABLE 1). Other PTPs are diffusely cytoplasmic,nuclear or associated with intracellular organelles22

(FIG. 2), and the latter typically have entirely different,and sometimes surprising, functions in lymphocytephysiology. For example, the 48-kDa isoform of TCPTP(T-cell PTP) is found in the endoplasmic reticulumanchored to the cytoplasmic membrane, where itdephosphorylates and inactivates recycled or newly syn-thesized PTKs. As a result of this localization, even ahigh level of overexpression of this TCPTP isoformdoes not affect TCR-ligation-induced activation of theIL-2 gene22. In fact, many other PTPs can also be over-expressed without any impact on signalling through theTCR, supporting the idea that PTPs have highly specificfunctions that are determined by both intracellular loca-tion and substrate specificity. By contrast, even a smallchange in the expression levels of PTPs such as LYP,PTPH1 or LMPTP (low-molecular-weight PTP), whichare found at, or near, the plasma membrane, can havemarked effects on signalling through the TCR22–25.

Domain architecture and regulation of PTPsEach PTP has a catalytic domain with an intrinsic sub-strate preference, which is determined by the identity ofthe amino-acid residues that generate the topographyand charge distribution of the substrate-interacting sur-face that surrounds the catalytic pocket. As well as thisdeterminant of specificity, most PTPs are targeted to specific locations or protein complexes within a cell(FIG. 2). For this purpose, most PTPs have non-catalyticamino (N)- or carboxy (C)-terminal extensions thatcontain targeting motifs, protein–protein interactiondomains or lipid-binding modules. Accordingly, manyPTPs are enriched at the plasma membrane, whereasothers are confined either to the nucleus or by otherspecific internal membranes (for example, the endo-plasmic reticulum, mitochondrion or secretory vesicles)or shuttle from the cytosol to any of these locations. Theexpression of distinct non-catalytic domains that areimportant for PTP targeting means that PTPs resemblePTKs21; however, a closer inspection reveals that therepertoire of non-catalytic domains of PTPs only partlyoverlaps with that of PTKs. For example, only two PTPshave SH2 domains, and none has an SH3 DOMAIN.Instead, many PTPs have domains that are not found in

PTKs: for example, the Sec14 (Saccharomyces cerevisiaephosphatidylinositol-transfer protein)-homologydomain of PTP-MEG2 (PTP megakaryocyte 2), whichmediates the association of PTP-MEG2 with phos-phatidylinositols; the CDC25 (cell-division cycle 25)-homology domain of mitogen-activated protein kinase(MAPK) phosphatases (MKPs), which mediates the asso-ciation of MKPs with their MAPK substrates; the FYVE(Fab1, YOTB, Vac1 and EEA1)-homology domain inmany myotubularin-related proteins (MTMRs), whichmediates their association with phosphatidylinositol-3-phosphate; and the ERM (EZRIN, RADIXIN AND MOESIN) DOMAIN

of PTPH1, PTP-MEG1, PTP-BAS (PTP basophil) andother PTPs, which enables their association with thecytoskeleton and/or plasma membrane. Functionally,an ERM domain compensates for the lack of an SH3domain in PTPs.We think that the differences in domainrepertoire between PTPs and PTKs probably reflect therequirement for regulation of these two classes ofenzyme in temporally and spatially distinct, and oftenreciprocal, manners.

The non-catalytic regions also participate in the regu-lation of phosphatase activity by intramolecular-foldingmechanisms. This is best illustrated by SHP1 and SHP2,the crystal structures of which show that their N-terminalSH2 domain fits into the catalytic pocket of the phos-phatase domain and thereby prevents substrate access.This inhibited conformation is opened by ligand bind-ing to the SH2 domain and results in a 100-fold increasein enzymatic activity26–30. This type of interaction-dependent regulation seems to be common among PTPsand provides a mechanism for rapid spatial and tempo-ral regulation of PTPs within cells. In addition, mostPTPs are post-translationally modified to assist with sub-cellular targeting, interactions with other proteins, stabil-ity, and regulation of catalytic activity. Glycosylationseems to be restricted to the transmembrane PTPs,which have high levels of N-LINKED CARBOHYDRATES and O-LINKED CARBOHYDRATES in their extracellular regions. Atleast one PTP found in lymphocytes, VHX (Vacciniavirus-VH1-related MKP X), is N-terminally MYRISTOYLATED

(and thereby associates with the plasma membrane)31, ina manner reminiscent of SRC-family PTKs. Similarly,C-terminal FARNESYLATION of two other PTPs, PRL1 (phos-phatase of regenerating liver 1) and PRL3, mediates theirassociation with the plasma membrane.

By far the most common post-translational modifi-cation of PTPs is phosphorylation of serine, threonineand/or tyrosine residues. Phosphorylation can regulatethe catalytic activity, subcellular location, physical asso-ciation with substrates or regulators, or stability ofPTPs. The tyrosine phosphorylation of PTPs is particu-larly intriguing as it implies a physical interaction withPTKs, which themselves are often substrates forPTPs25,32–37. At least 15 PTPs have been reported to betyrosine phosphorylated, but the physiological signifi-cance of this remains unclear in most cases. Tyrosinephosphorylation also introduces the possibility of auto-dephosphorylation, an idea that is supported by theobservation that many PTPs become noticeably morephosphorylated on tyrosine residues when they are

SRC HOMOLOGY 2 DOMAIN

(SH2 domain). A proteindomain that is commonly foundin signal-transductionmolecules. It specificallyrecognizes phosphotyrosine-containing peptide sequences in proteins.

SH3 DOMAIN

(SRC homology 3 domain).A protein domain that iscommonly found in signal-transduction molecules.It specifically interacts withcertain proline-containingpeptides. Classically, it containseither (R/K)XXPXXP orPXXPXR motifs, where Xdenotes any amino acid.

ERM DOMAIN

A protein–protein interactiondomain found in ezrin, radixinand moesin.

N-LINKED CARBOHYDRATES

Sugars that are attached toasparagine residues of proteins.

O-LINKED CARBOHYDRATES

Sugars that are attached toserine and/or threonine residuesof proteins.

MYRISTOYLATION

The covalent attachment of ahydrophobic myristoyl group to the amino-terminal glycineresidue of a nascent polypeptide.

FARNESYLATION

The covalent addition of farnesyl(15 carbon) isoprenoids toproteins at cysteine residues byfarnesyl transferases, which leadsto their constitutive associationwith the plasma membrane.


R E V I E W S

LMPTP

ERM

PTPH1, PTP-MEG1 PTPD2, PEZ PTP-BAS

Sec14

PTP1B, TCPTP-48 PTP-MEG2

LYP, PTP-PEST, PTP-HSCFHEPTP

HDPTP

VHX

PRL1, PRL3

CD45

PH-G Coil

C2

PTEN

MTM1, MTMR1, MTMR2 MTMR3, MTMR4 MTMR11

MTMR10MTMR5

RPTP-ε

MTMR14

VHR, MKP6

CD148

MKP3

Cytosol

Endoplasmic reticulum Endosomes, secretory vesicles

VHZ

Plasmamembrane

PTPD1

D2

PDZKIND PBM

FN

FN

BRO

KIM

Pro-rich

CH

2

FYVE

PH

CDC25A, CDC25B, CDC25C

EYA3, EYA4KAP

mRC

RNGTT

MKP5

PAC1, MKP1

CDC14A, CDC14B

TCPTP-45

Nucleus

SHP1, SHP2

SH2

DNA

Figure 2 | Protein tyrosine phosphatases in lymphocytes. The domain architecture and the subcellular location of proteintyrosine phosphatases (PTPs) in lymphocytes are illustrated. Catalytic PTP domains are shown in green, and a green domain witha red cross denotes a catalytically inactive PTP domain. A star denotes a farnesylation motif. BRO, baculovirus Bro homology;C2, protein-kinase-C conserved region 2; CDC, cell-division cycle; CH2, CDC25-homology region 2; Coil, coiled coil; D1, domain 1of PTPs with two catalytic domains; D2, domain 2 of PTPs with two catalytic domains; DNA, DNA binding; ERM, ezrin, radixinand moesin; EYA, eyes absent; FN, fibronectin-like; FYVE, Fab1, YOTB, Vac1 and EEA1 homology; HDPTP, histidine-domainPTP; HEPTP, haematopoietic PTP; KAP, kinase-associated phosphatase; KIM, kinase-interaction motif; KIND, kinase amino-lobe-like domain; LMPTP, low-molecular-weight PTP; LYP, lymphoid-specific PTP; MKP, mitogen-activated protein kinase (MAPK)phosphatase; mRC, mRNA capping; MTM1, myotubularin 1; MTMR, MTM-related protein; PAC1, phosphatase of activatedcells 1; PBM, PDZ-binding motif; PDZ, PSD95, DLGA and ZO1 homology; PEZ, phosphatase with ezrin domain; PH, pleckstrinhomology; PH-G, pleckstrin homology-GRAM (glucosyltransferases, RAB-like GTPase activators and MTMs) domain; PRL, phosphatase of regenerating liver; Pro-rich, proline rich; PTEN, phosphatase and tensin homologue; PTP-BAS, PTP basophil;PTP-HSCF, PTP haematopoietic stem-cell fraction; PTP-MEG, PTP megakaryocyte; PTP-PEST, PTP with PEST (proline-,glutamic-acid-, serine- and threonine-rich) domains; RNGTT, RNA guanylyltransferase and 5′-phosphatase; RPTP-ε, receptor-type PTP-ε; Sec14, Saccharomyces cerevisiae phosphatidylinositol-transfer protein homology; SH2, SRC homology 2; SHP, SH2-domain-containing PTP; TCPTP-45, T-cell PTP of 45 kDa; TCPTP-48, T-cell PTP of 48 kDa; VHR, Vaccinia virus-VH1 related;VHX, Vaccinia virus-VH1-related MKP X; VHZ, Vaccinia virus-VH1-like member Z.


R E V I E W S

the first PTP found to be present in T cells was immedi-ately found to have an important physiological functionas a positive regulator of T-cell activation.

Since then, numerous studies have supported thefinding that CD45 mainly has a positive role. Forexample, humans and mice that are deficient in CD45develop a severe-combined immunodeficient pheno-type50. At the molecular level, CD45 was shown tofunction upstream of TCR-triggered tyrosine phos-phorylation of signalling molecules, activation ofphospholipase C (PLC)51 and mobilization of cal-cium52. Furthermore, loss of CD45 correlates withincreased phosphorylation of LCK at Y505 (REF. 53),and the abnormalities in thymic development that areobserved in mice that lack exon 6 of the Cd45 gene are rescued by a transgene that expresses a constitu-tively active form of LCK54. Together with similar find-ings for CD45 in B cells and other immune cells, thesefindings have led to the idea5,13,55 that CD45 functionsas a positive regulator of antigen- and immuno-globulin-receptor signalling, by dephosphorylatingthe C-terminal negative-regulatory tyrosine residue ofSRC-family PTKs (that is, LCK, FYN and YES in T cells, and LYN, FYN and BLK (B-lymphoid kinase)in B cells). Recently, CD45 was also suggested to dephos-phorylate the LIPID-RAFT-associated transmembrane-adaptor molecule PAG (protein associated with glycolipid-enriched membrane domains; also knownas CBP)56; PAG recruits the PTK CSK (C-terminalSRC kinase) to plasma-membrane lipid rafts throughan interaction between the SH2 domain of CSK and thephosphorylated Y317 in PAG57,58 (FIG. 3b). The interac-tion between PAG and CSK in lipid rafts of restingcells is important for keeping lipid-raft-associatedSRC-family PTKs in an inactive state because CSKmediates phosphorylation of their negative-regulatorytyrosine residues58–60. In response to stimulationthrough the TCR, Y317 in PAG is rapidly dephospho-rylated, perhaps by CD45 (REF. 56), thereby resulting inthe dissociation of CSK from lipid rafts58,59. This leadsto reduced CSK activity towards LCK and FYN, tip-ping the balance towards their activation61, therebyproviding another way in which CD45 positively reg-ulates lymphocyte activation. However, the role ofCD45 in dephosphorylating PAG is controversial. Arecent paper indicates that SHP2, rather than CD45,dephosphorylates PAG, at least in non-haematopoieticcells62, indicating that further studies are necessary toresolve this issue. In addition, there are also severalpapers indicating that CD45 itself can have a negativerole in signalling through the TCR63–71. Although weconsider that this is possible, most of the evidencecomes from studies of leukaemic cells or artificial systems in which CD45 is forced into either non-physiological subcellular locations or excessive contactwith the TCR. Therefore, better evidence that CD45has a negative role in immune cells under physiologicalconditions is needed to solve this question.

Similar to CD45, two other PTPs (SHP2 (REF. 42) andLMPTP22) also seem to have a mainly positive influenceon lymphocyte activation. LMPTP seems to preferentially

expressed as catalytically inactive mutants15. In mostcases, however, the phosphorylation sites are clearlyinaccessible for intramolecular dephosphorylation,indicating that dephosphorylation must occur in trans.So, regulatory networks of PTPs and other proteinphosphatases, perhaps in the form of ‘phosphatase cascades’, are likely to exist in cells.

An interesting additional layer of regulation of PTPsis provided by the high sensitivity of the PTP-domaincatalytic cysteine residue to reversible oxidation38,39,which was recently shown to occur in intact cells inresponse to stimulation with growth factors40. Suchregulation of enzymatic activity might be particularlyrelevant in cells of the immune system, which areimmersed in a highly oxidative environment at sites ofinflammation. Such conditions might therefore cause adecrease in the activity of many PTPs, thereby stronglyaugmenting tyrosine phosphorylation in response toreceptor ligation and subsequently increasing activationof the immune response.

PTPs involved in lymphocyte activationWe estimate that nearly 20 T-cell-expressed PTPs regu-late the signalling events that occur between ligation ofthe TCR and transactivation of the IL-2 gene. At leastthree of these PTPs (CD45, SHP2 and LMPTP) mainlyhave an activation-promoting role (a ‘positive’ role),whereas most of the other PTPs function to dampensignalling (a ‘negative’ role). Naturally, many of thesePTPs also have functions in other cellular processes: forexample, PTEN (phosphatase and tensin homologue)in survival41 and SHP2 in lymphoid-cell development42.Indeed, some of these PTPs are essential, and disrup-tion of the genes encoding these (for example, SHP2and PTP-PEST (PTP with PEST domains)) results inearly embryonic lethality, whereas mice that lack otherPTPs (such as CD45 (REF. 43), HEPTP44, PTP1B45,or TCPTP46) have milder and/or immune-restrictedphenotypes.

Positive regulation of lymphocyte activation. The firstPTP implicated in lymphocyte activation was CD45.CD45 is a 180–220-kDa transmembrane enzyme with along and slender, highly glycosylated extracellulardomain and two cytoplasmic PTP domains, the secondof which is catalytically inactive. The discovery thatCD45 was a PTP came as a surprise in 1988, when thefirst amino-acid sequence of a PTP — a fragment ofPTP1B purified from placental tissue — was found tohave homology to the cytoplasmic portion of CD45(REF. 47). Indeed, CD45 was then shown to have PTPactivity48. As this finding was published, we were search-ing for the identity of a membrane-associated PTP thatrapidly activated the PTK LCK in membrane prepara-tions of normal T cells32,33. This activity was found toreside with CD45, which activated LCK by dephosphory-lating its negative-regulatory site, the tyrosine residue atposition 505 (Y505) (FIG. 3a). At a similar time (andentirely independently), Matthew Thomas’s group49

showed that only T cells expressing CD45 could respondto stimulation by antigen or mitogenic antibodies. So,

LIPID RAFT

Area of the plasma membranethat is rich in cholesterol,glycosphingolipids, severalsignalling proteins (such as SRCkinases, RAS, LAT and PAG) andglycosylphosphatidylinositol-anchored proteins. Also knownas glycolipid-enrichedmembrane domains (GEMs)and detergent-insolubleglycolipid-enriched membranes(DIGs).


R E V I E W S

LYP). Therefore, the dephosphorylation of Y292 byLMPTP results in prolonged phosphorylation of thetyrosine residues of ZAP70 that are responsible fortransducing TCR-initiated signals and slower down-modulation of cell-surface TCR. By contrast, SHP2 aug-ments RAS and MAPK activation by dephosphorylatingan unknown substrate (possibly PAG62) and is requiredfor thymic development42.

dephosphorylate the negative-regulatory site (Y292) ofZAP70 (ζ-chain-associated protein kinase of 70 kDa)25

(FIG. 3b). When phosphorylated, this functions as abinding site for the CBL (Casitas B-lineage lymphoma)E3-ubiquitin-ligase complex, which negatively regu-lates TCR signalling by accelerating TCR internaliza-tion and degradation, and promotes the inactivation ofZAP70 through dephosphorylation (perhaps through

Plasma membrane

a

c

b

Lipid raft

CD45ROCD45RABC

f

LYP,SHP1, PTPH1,CD45?

CD45

LYP,SHP1

LMPTP

CD45,SHP2?

Y317

Y394

Y505

Y292

Y493

e

Y394

Y505

CD45?LYP, SHP1, PTPH1

ATP

Inactive Increased activity Fully active

P

P

SH3

LCK

P

SH2

SH3

SH2 SH2

CD45

CD45

SH3

SH2

P

PP

P

PTPH1

SH3

SH2

P

PAG

P

P

SH2

SH3

P

SH2P

SH3

SH2

d

More active Less active More active Less active

CD45RO

Transmembraneprotein

Transmembraneprotein

CD45RABC

Fodrin

CD45-AP

CD45

P

P

P

CK2, PKC, CSK

SH2

SH3

FN

FN

FN

FN

LCK

LCK

CSK

ZAP70PITAMs

FN

Figure 3 | CD45 in the dephosphorylation of SRC- and SYK-family PTKs, and modes of regulation of CD45.a | Dephosphorylation of LCK at the tyrosine residue at position 505 (Y505) by CD45 results in a relief of the ‘tail-bite’ suppressionof LCK. This is followed by autophosphorylation at Y394, which results in full activation of LCK. b | Although CD45 is the onlyprotein tyrosine phosphatase (PTP) in T cells that is known to dephosphorylate LCK at Y505, several other PTPs participate indephosphorylation at Y394 and of other positive- and negative-regulatory tyrosine residues in SRC- and SYK (spleen tyrosinekinase)-family protein tyrosine kinases (PTKs) (such as CSK (carboxy-terminal SRC kinase) and ZAP70 (ζ-chain-associatedprotein kinase of 70 kDa)). Arrows denote phosphorylation. Blocking arrows denote dephosphorylation. c | Given the preferentialpartitioning of LCK into lipid rafts and the exclusion of most CD45 molecules from lipid rafts, it remains controversial whetherCD45 dephosphorylates LCK at Y505 within lipid rafts or outside lipid rafts. d | Differential regulation of CD45 isoforms byhomodimerization is shown. e | CD45-isoform-specific associations with other transmembrane proteins might increase ordecrease the juxtapositioning of CD45 with LCK and/or other substrates. f | CD45 might be regulated by several other proteins:by binding the CD45-associated protein (CD45-AP) or the cytoskeletal protein fodrin, or by being phosphorylated by caseinkinase 2 (CK2), protein kinase C (PKC) or CSK. FN, fibronectin-like; ITAM, immunoreceptor tyrosine-based activation motif;LMPTP, low-molecular-weight PTP; LYP, lymphoid-specific PTP; PAG, protein associated with glycolipid-enriched membranedomains; SH, SRC homology; SHP, SH2-domain-containing PTP.


R E V I E W S

CD45 dimerization. In addition, several crystal structuresof CD45, with or without substrate peptides bound, showthat the wedge does not exist in CD45 (H. Saito, unpub-lished observations). So, the molecular mechanism ofdimerization-mediated inhibition remains to be deter-mined. CD45 also associates with other glycoproteins inthe plane of the T-cell plasma membrane79 (FIG. 3e). Theseinteractions differ between CD45 splice variants andmight account for the differences in CD45 activitybetween the distinct isoforms, although this remains tobe experimentally determined. Finally, CD45 is phospho-rylated at numerous sites (FIG. 3f), but the physiologicalrelevance of these sites also remains unclear.

Negative regulation of antigen-receptor-associatedPTKs by multiple PTPs. In contrast to CD45, LMPTPand SHP2, a number of PTPs dephosphorylate posi-tively acting tyrosine-phosphorylation sites in the TCRand/or BCR signal-transduction subunits (that is,CD3, Igα and Igβ) and the receptor-coupled PTKs,thereby suppressing lymphocyte activation (FIG. 3b). Atpresent, these ‘gate-keepers’ are known to includeSHP1, LYP (PEP80), PTP-PEST, PTPH1 and PTP-HSCF (PTP haematopoietic stem-cell fraction), butothers might also be involved. Of these, the SH2-domain-containing SHP1 is probably the best under-stood PTP in immune cells. SHP1 has a crucial role inboth natural killer cells and B cells as the main regula-tor of signalling for receptors that have an ITIM(immunoreceptor tyrosine-based inhibitory motif) intheir intracellular region81,82. After ligand binding,ITIMs are phosphorylated by SRC-family PTKs, creat-ing docking sites for the SH2 domains of SHP1, whichis activated 100-fold by the binding event and, at thesame time, is brought into proximity with its sub-strates, which include SRC- and SYK-family PTKs35,83

and their substrates84. This therefore downmodulatessignal-transduction pathways. Studies of the moth-eaten mouse, which lacks SHP1 (REF. 85), have revealedthe importance of SHP1 for the function of both theinnate and adaptive immune systems. The mice showsigns of systemic autoimmunity that increase with ageand severe inflammation of many organs, and theyalso die young 86. Thymic cells from these mice arehyper-responsive to TCR stimulation87, indicating thatSHP1 either directly affects TCR signalling or affectsT-cell development in the thymus. Indeed, SHP1 candephosphorylate ZAP70, resulting in dampening ofdownstream signalling35, but it seems that an undefinedITIM-containing receptor is required for this effect.One such receptor might be CD22, which was recentlyfound to be expressed by T cells and to inhibit T-cellactivation through SHP1 (REF. 88).

Our present knowledge of LYP is mainly derivedfrom studies of its mouse orthologue PEP. Although thehomology between the N-terminal PTP domains of LYPand PEP is high, LYP and PEP differ substantially in thecentral portion of the enzyme and to some extent at theC-terminal region. In both mice and humans, expressionis restricted to leukocytes, including T cells. PEP residesin the cytosol close to the plasma membrane22, in other

Regulation of CD45 activity. The regulation of CD45activity is not completely understood, but it seems toinvolve spatio-temporal mechanisms, autoinhibition bydimerization, and phosphorylation (FIG. 3c–f). Spatio-temporal regulation is indicated by biochemical studiesshowing that, in thymocytes and resting T cells, ∼5–10%of CD45 molecules partition into lipid rafts57,72,73, wherethey have access to TCR-associated signalling molecules.This fraction of CD45 seems to be of physiological rele-vance as it has been shown to support the initial steps ofTCR signalling74 (FIG. 3c). However, in response to TCRstimulation, there is a reduction in the amount of CD45that is associated with lipid rafts73. This removal mightprevent CD45 from dephosphorylating the ITAMs(immunoreceptor tyrosine-based activation motifs)of the CD3 ζ-chain67, the activating tyrosine residuesof SRC-family PTKs69,70 and perhaps other substrates.Indeed, artificial retention of CD45 in proximity tothe TCR can strongly inhibit T-cell activation63. Further-more, lipid-raft-targeted chimeric molecules consistingof the N-terminus of LCK and the cytoplasmic region ofCD45 inhibit TCR signalling, seemingly as a result ofreduced SRC-family PTK activity in lipid rafts but per-haps also as a result of dephosphorylation of other sig-nalling molecules71. The idea that CD45 is regulated ina spatio-temporal manner in activated T cells is alsosupported by confocal microscopy and FLUORESCENCE

RESONANCE ENERGY TRANSFER (FRET) analysis75, which hasshown that, after the initial contact between a T cell andan antigen-presenting cell, CD3 and CD45 co-localizein the early cSMAC (central supramolecular activationcluster), at which stage, no tyrosine phosphorylation ofsignalling proteins is seen. A few minutes later, CD45leaves the cSMAC and is observed to be outside thepSMAC (peripheral SMAC), while phosphotyrosineresidues (such as in ZAP70) appear in the cSMAC. Itshould be noted, however, that the movements ofCD45 remain unclear and are controversial.

CD45 activity is also controlled by homodimerization(FIG. 3d), which reportedly inhibits its PTP activity50. Themolecular mechanism for this inhibition has been pro-posed to entail a wedge-like structure in CD45, whichwould block the catalytic site of the other CD45 moleculein the dimer76. In support of this model, mutation of akey residue (from glutamic acid to arginine at position624) in the putative wedge disrupts its function andalso abolishes dimerization-induced inhibition of CD45(REF. 76). Furthermore, mice with a corresponding ‘knock-in’ mutation in CD45 develop a lymphoproliferativesyndrome and are prone to autoimmune diseases, indi-cating aberrant CD45 activation77. Perhaps also related tothe wedge model, it has been reported that CD45 splicevariants with a smaller extracellular domain (that is,CD45RO) tend to homodimerize more easily than thosewith a larger extracellular domain (that is, CD45RA,CD45RAB and CD45RABC)78 (FIG. 3d). This mechanismmight allow differential regulation of CD45 between T-cell subsets because naive and memory T cells expressdifferent repertoires of CD45 isoforms. However,although the wedge model is intriguing, there have beenno reports that directly show reduced PTP activity by

FLUORESCENCE RESONANCE

ENERGY TRANSFER

(FRET). This is used to measureprotein–protein interactionsmicroscopically or by a FACS(fluorescence-activated cellsorter)-based method. Proteinsfused to cyan, yellow or redfluorescent proteins areexpressed and assessed forinteraction by measuring the energy transfer betweenfluorophores, which can onlyoccur if proteins physicallyinteract. FRET can also be usedto examine the activation state of certain proteins if theiractivation results in specificprotein–protein interactions.

cSMAC

(Central supramolecularactivation cluster). The centralregion of the immune synapse.


R E V I E W S

The close relatives of LYP, PTP-PEST and PTP-HSCF, also associate with CSK98,99. PTP-PEST isexpressed broadly in numerous tissues and by variouscell types, including cells of the immune system,whereas PTP-HSCF is expressed only in the brain andby stem cells and haematopoietic cells, includingmature B cells. In contrast to Pep –/– mice, Ptp-pest –/–

mice die in utero100, indicating that PTP-PEST isessential for normal development. PTP-PEST dephos-phorylates several cytoskeletal and focal adhesionproteins (such as CAS (CRK-associated sub-strate)100,101, PYK2 (PTK 2), FAK (focal adhesionkinase) and paxillin), which mediate signal transduc-tion from integrins (such as lymphocyte function-associated antigen 1, LFA1) that are involved in theadhesion of lymphocytes and the formation of theimmune synapse (FIG. 4). In the B-cell line A20, PTP-PEST was shown to inhibit BCR signalling bydephosphorylating these and other signalling pro-teins that participate in BCR signalling upstream ofRAS102. On the basis of this limited information, wepropose that PTP-PEST might be crucial for the for-mation of the immune synapse of T cells, through itsregulation of signalling pathways that control the cyto-skeleton and the lymphocyte equivalent of the FOCAL

ADHESION PLAQUE. This might also be the case for PTP-HSCF, which seems to act on signalling molecules thatare involved in cytoskeletal dynamics103.

Another potent inhibitor of TCR signalling isPTPH1 (REFS 22,24). At least part of this inhibition resultsfrom its dephosphorylation of ITAM tyrosine residuesin CD3-ζ104 and LCK (T.M., unpublished observations)(FIGS 3b,5), which leads to the loss of TCR-inducedMAPK activation24. The ability of PTPH1 to inhibitTCR signalling depends on its targeting to the plasma-membrane actin network by its ERM domain24. PTPH1has also been reported to dephosphorylate the 100-kDavalosin-containing protein (VCP)105, a hexameric ATPasethat is heavily tyrosine phosphorylated in activated T cells106. However, there is no information that linksVCP to early TCR-signalling events. Instead, VCP hasbeen reported to have several diverse and seeminglyunrelated functions — including roles in S26 protea-some function, peroxisome assembly, transcriptionalregulation and vesicle-fusion events — involving VCPin different subcellular locations107. PTPH1 also has a PDZ (PSD95, DLGA AND ZO1 HOMOLOGY) DOMAIN, which bindsthe C-terminal tail of TACE (tumour-necrosis-factorconverting enzyme), a transmembrane disintegrin andmetalloproteinase (ADAM) that is involved in theectodomain shedding of several proteins108. Presumablythe PDZ domain also allows PTPH1 to interact withother PDZ-domain-containing, T-cell-relevant trans-membrane proteins. (Candidates include selectins,MHC class I molecules, CD26, CD95 (FAS) and CD45-AP.) Interactions with such T-cell proteins could‘steer’ PTPH1 to sites of physiological action for thisPTP. All of the defined aspects of PTPH1 are schemati-cally summarized in FIG. 5. Finally, the closely relatedPTP-MEG1 might overlap with PTPH1 in negativelyregulating T-cell activation24.

intracellular structures and in the nucleus89. PEP is amore potent negative regulator of TCR signalling thanSHP1 (REFS 22,23), and it acts on receptor-coupled PTKs(FIG. 3b), including the positive-regulatory Y394 of LCK23,Y417 of FYN82, and ZAP70 (REF. 82). The dephosphoryla-tion of LCK and FYN by PEP is regulated by its associa-tion with the SH3 domain of CSK90,91, whereas thedephosphorylation of ZAP70 by LYP might be related tothe association of LYP with CBL92. Surprisingly, PEP isnot detected in lipid rafts56, indicating that PEP–CSKcomplexes do not use PAG for targeting LCK and FYN.The mechanism for targeting LYP to these PTKs inhuman T cells also remains unclear. Nevertheless, theLYP–CSK interaction is of crucial importance for theadequate regulation of immune function in humans: asingle nucleotide polymorphism in LYP that changesamino acid 620 from arginine to tryptophan in theCSK-binding motif markedly reduces CSK binding toLYP93 and is associated with autoimmune diseases, suchas type 1 diabetes93, rheumatoid arthritis94, systemiclupus erythematosus (SLE)95 and Graves’ disease96.

A negative role for LYP and PEP in TCR signallinghas been confirmed by two recent studies. First, positiveselection is enhanced in the thymus of Pep–/– mice,resulting in clonal expansion of the memory and effec-tor T-cell pools97. Effector T cells generated in vitrofrom these mice show increased and sustained phos-phorylation of both ZAP70 and Y394 of LCK inresponse to TCR stimulation, as well as increased pro-liferative responses. Second, acute elimination of LYPfrom Jurkat T cells, using RNA INTERFERENCE, resulted inaugmented nuclear factor-κB activation after TCRtriggering94.

RNA INTERFERENCE

(RNAi). The use of double-stranded RNAs with sequencesthat precisely match a given geneto ‘knock-down’ the expressionof that gene by directing RNA-degrading enzymes to destroythe encoded mRNA transcript.

FOCAL ADHESION PLAQUE

The closest contact site of a cellwith its environment, which isformed by integrin clustering.The integrins link theextracellular environment to theactin cytoskeleton by a complexassembly of adaptor proteins.

PDZ DOMAIN

(PSD95, DLGA and ZO1homology domain). A proteindomain that is commonly foundin signal-transductionmolecules. It can interact withdifferent amino-acid motifs thatare present at the carboxylterminus of proteins. PDZdomains can also interact withother PDZ domains, withcertain internal peptidesequences and even with lipids.

Plasma membrane

PSTPIP

Talin

CAS

Vinculin

FAK Paxillin

CSK

LFA1

P

P

P

P

P

P

PTP-PEST

PTP-PEST FYN

ABLP

P

P

P P

P

P

F-actin

Figure 4 | Role of PTP-PEST in control of integrin and/or cytoskeleton interactions in theimmune synapse. The lymphocyte adhesion molecule LFA1 (lymphocyte function-associatedantigen 1) accumulates in the pSMAC (peripheral supramolecular activation cluster) region of theimmune synapse, where it signals the reorganization of the actin cytoskeleton. We propose thatPTP-PEST (protein tyrosine phosphatase (PTP) with PEST (proline-, glutamic-acid-, serine- andthreonine-rich) domains) has a regulatory role in this signalling process, by dephosphorylatingfocal adhesion proteins (such as CAS (CRK-associated substrate) FAK (focal adhesion kinase)and paxillin). We also propose that PTP-PEST, through its association with CSK (carboxy-terminalSRC kinase), dephosphorylates FYN and other SRC-family protein tyrosine kinases (PTKs) thatare involved in LFA1 signalling. Targeted by the cytoskeletal protein PSTPIP (proline-serine-threonine phosphatase-interacting protein), PTP-PEST also dephosphorylates the F-actin-associated PTK ABL. Blocking arrows denote dephosphorylation.


R E V I E W S

MAPKs and the marked differences that the durationof MAPK activation and the subcellular transport ofactive MAPKs can make. The various MAPK-specificphosphatases have different substrate preferenceswithin the MAPK family, use different mechanisms forassociation with MAPKs, are expressed in response todifferent stimuli and in lineage-specific manners, residein different subcellular locations and are subject to dif-ferent modes of post-translational regulation. So,although the kinase cascades that trigger MAPK activa-tion are stereotypical, the phosphatases provide theguidance and fine-tuning that are crucial for cell- andsituation-specific responses.

In resting lymphocytes, HEPTP associates with inac-tive forms of ERK and p38 through an N-terminal KIM(kinase-interaction motif)113–115. This complex can bedisrupted by phosphorylation of HEPTP at S23, whichis in the KIM, by cAMP-dependent kinase (PKA)115

or by phosphorylation of HEPTP on the threonineresidue at position 45 (T45) and at S72 by MAPKs114.Interestingly, TCR signalling also induces phosphoryla-tion of S23 and dissociation of ERK and p38 from theinhibitory HEPTP116. HEPTP remains in the cytosoland, seemingly, cannot reach activated ERK and p38 inthe nucleus, which therefore remain active. About 30–60minutes later, mRNA encoding PAC1 (phosphatase ofactivated cells 1) is produced, and PAC1 protein beginsto accumulate in the nucleus, where it dephosphorylatesthe two phospho-amino acids in the activation loop ofERK. This results in complete inactivation of ERK, whichshuttles back to the cytosol and reassociates with HEPTP.We refer to this as the ‘sequential phosphatase model’(FIG. 6) and use it as an example of how different PTPsare used in a spatially and temporally ordered mannerto control the extent, location and duration of MAPKactivation. Clearly, it is even more complex in live cells,which also express many other phosphatases that candephosphorylate MAPKs.

PTPs functioning downstream of membrane-receptor-coupled PTKs. PTPs not only function as regulatorsof membrane-receptor-associated PTKs, by dephos-phorylating their stimulatory or inhibitory tyrosine-phosphorylation sites, but also function as downstreameffectors of these PTKs. For example, LMPTP is acti-vated by LCK-mediated phosphorylation of Y131 ofLMPTP109, and the PTP VHR (Vaccinia virus-VH1related) — which dephosphorylates and inactivatesthe MAPKs ERK (extracellular signal-regulatedkinase) and JNK (JUN N-terminal kinase)110 — isphosphorylated at Y138 by ZAP70 (REF. 111). Only the phosphorylated form of VHR seems to have biolog-ical activity against ERK and JNK. So, ZAP70 controlsboth MAPK activation through the RAS–RAF–MEKpathway and MAPK inactivation through activationof VHR (FIG. 6). Because ZAP70 has differential roles inT-cell subsets and in response to different stimuli, thismechanism might be associated with the differenttime courses of MAPK activation that have beenobserved in T helper 1 (T

H1) and T

H2 cells and in

response to partial agonists.The MAPKs ERK, JNK and p38 function as integra-

tion points for numerous signalling pathways thatcontrol immune responses. These kinases are also true‘hot spots’ for phosphatases. The human genome encodes11 PTPs that specifically inactivate MAPKs by dephos-phorylating both the phosphotyrosine and phospho-threonine residues in their activation loop112. We referto these as the ‘classical’ MKPs6. In addition, severalrelated small PTPs (such as VHR) that lack the MAPK-targeting domain found in MKPs, as well as the strictlytyrosine-specific phosphatase HEPTP and several serine/threonine phosphatases, also dephosphorylate MAPKsin lymphocytes. Altogether, many more phosphatasesthan kinases act on the MAPKs112. The reasons for thisabundance of phosphatases that can dephosphorylateMAPKs are probably related to the numerous roles of

ERMPTP

PDZ

Peroxisome assembly

tER transport

Gene transcription

S26 proteasome function

F-actin

PTPH1

VCP

TCR

CD4

CD3 CD3TACE

Plasma membrane Lipid raft

SH2

SH3P

P

P

P

P P

SH2

SH2

ZAP70LCK

PP

P

P

Figure 5 | Proposed functions of PTPH1 in T cells. In T cells, most of the PTPH1 (protein tyrosine phosphatase H1) molecules aretargeted to the plasma membrane through their ERM (ezrin, radixin and moesin) domains, where they dephosphorylate T-cell receptor(TCR) subunits and valosin-containing protein (VCP). The specific ligand of the ERM domain is unknown, but it co-localizes with F-actin atthe plasma membrane. PTPH1 is also anchored to some transmembrane proteins that have a PDZ (PSD95, DLGA and ZO1 homology)-binding motif, such as TACE (tumour-necrosis-factor converting enzyme). VCP is phosphorylated by TCR-associated protein tyrosinekinases and has been ascribed many functions, some of which are regulated by its tyrosine phosphorylation. SH, SRC homology; tER, transitional endoplasmic reticulum; ZAP70, ζ-chain-associated protein kinase of 70 kDa.


R E V I E W S

PTPs with other functions in immune cellsBecause tyrosine phosphorylation is closely involved inso many different physiological processes — includingsignalling from receptors for immunoglobulins, comple-ment, extracellular-matrix components and cytokines— it seems clear that numerous PTPs found in immunecells participate in the regulation of these physiologicalprocesses.A few reports describe PTPs that participate insignalling though the JAK (Janus activated kinase)–STAT(signal transducer and activator of transcription) path-ways, which mediate signalling from receptors for inter-ferons, hormones, cytokines and interleukins. The JAKsthemselves seem to be regulated in a negative-feedbackloop by SHP1, which can be recruited to phosphorylatedtyrosine residues in the C-terminus of some cytokinereceptors that associate with JAK-family PTKs117. JAKsare also dephosphorylated by PTP1B118. Several PTPshave also been reported to dephosphorylate phosphory-lated STATs; these include PTP1B, SHP2 and VHR.STAT1 is dephosphorylated by the 45-kDa nuclear iso-form of TCPTP119 in a manner that is regulated by argi-nine methylation of STAT1 (REF. 120). Although most ofthese observations were made using transformed non-haematopoietic cell lines, it seems probable that, inimmune cells, JAK–STAT pathways, which influence somany aspects of the immune response, are also regulatedby these PTPs.

One PTP that is potentially important in the immuneresponse is PTP-MEG2, which resides on the cytoplas-mic surface of secretory vesicles in leukocytes, where itregulates the homeostasis and size of these vesicles121–123,as well as their exocytosis123 (FIG. 7). A key substrate forPTP-MEG2 is the vesicle-fusion protein N-ethyl-maleimide-sensitive factor, which is inhibited by phos-phorylation of Y83 and is activated to set up vesiclefusion after dephosphorylation by PTP-MEG2 (REF. 123).PTP-MEG2 is itself activated by inositol phospho-lipids121,122, thereby connecting the phosphorylation ofthese lipids to tyrosine dephosphorylation and trigger-ing of vesicle fusion: for example, during secretion ofimmune mediators by activated lymphocytes or duringdegranulation of mast cells. Further understanding of theimmune function of PTP-MEG2 is likely to be providedby Ptp-Meg2–/– mice, which have severe immune-systemabnormalities (G. Downey, unpublished observations)and are currently under investigation.

Finally, a typical immune response involves the rapidclonal expansion of activated lymphocytes, followed by acontraction phase, during which most effector cells die byapoptosis and only a few memory cells remain. We pre-dict that many PTPs will also be shown to participate inthese later phases of the immune response. For example,several PTPs are crucial for cell-cycle progression and celldivision, such as the three CDC25s, the two CDC14s andKAP (kinase-associated phosphatase), all of which regu-late (positively or negatively) the cyclin-dependentkinases15. Because MAPKs also regulate (positively andnegatively) the passage of cells through cell-cycle check-points, we anticipate that MAPK-specific phosphataseswill affect the clonal expansion and contraction oflymphocytes. Clearly, much work remains to be done.

We propose that several PTPs are involved in thedephosphorylation of many other downstream signallingproteins in lymphocytes, such as PLC-γ1, LAT (linker foractivation of T cells), SLP76 (SH2-domain-containingleukocyte protein of 76 kDa), SLAP (SLP76-associatedprotein) and VAV1. In fact, the transient and low stoi-chiometry of phosphorylation of most of these proteinsafter antigen-receptor triggering indicates that PTPs havea dominant role in the regulation of their phosphoryla-tion. However, the identities of these PTPs and whetherthey have an important role in regulating the activity ofthese signalling molecules are still largely unknown.

Plasma membraneTCR PGE2R

ZAP70

LAT

cAMP

SOS

RAS

VHRPKA

RAF

MEK

HEPTP

PP

P

PP

PP

PP

PP1P

P

PP

ERK2

PAC1, MKP1

Nucleus PAC1MKP1

Figure 6 | The circuitry of MAPK regulation by multiple PTPs. According to the ‘sequentialphosphatase’ model, multiple protein tyrosine phosphatases (PTPs) — including HEPTP(haematopoietic PTP), VHR (Vaccinia virus-VH1 related), PAC1 (phosphatase of activated cells 1)and MKPs (mitogen-activated protein kinase (MAPK) phosphatases) — act on MAPKs at differentpoints during the activation cycle, in response to different inputs and in different subcellularcompartments of the T cell. Together, these PTPs fine-tune the extent, duration and subcellularlocation of MAPK activation. Orange arrows denote phosphorylation; blocking arrows denotedephosphorylation. cAMP, cyclic AMP; ERK2, extracellular signal-regulated kinase 2; LAT, linkerfor activation of T cells; MEK, MAPK kinase; PGE2R, prostaglandin E2 receptor; PKA, cAMP-dependent protein kinase; PP1, protein phosphatase 1; SOS, son of sevenless homologue; TCR, T-cell receptor; ZAP70, ζ-chain-associated protein kinase of 70 kDa.


R E V I E W S

T cells from patients with SLE125, and loss of CD45 wasfound in some patients with severe combined immuno-deficiency126. A polymorphism that impairs alternativesplicing of CD45 was reported to be associated withmultiple sclerosis127, but this association was not repro-ducible in studies of different populations128, exempli-fying that the interpretation of genetic-associationstudies of polygenic diseases is complicated by linkagedisequilibrium and complex interactions with othergenetic and environmental factors129. A missensepolymorphism in PTPN22 (which encodes LYP) thatmarkedly affects the binding of LYP to CSK wasfound to be associated with type 1 diabetes in twopopulations93 and with rheumatoid arthritis94, SLE95

and Graves’ disease96. In addition, the allelic polymor-phism in the gene encoding LMPTP has been shownto correlate with increased IgE levels and atopy130,131.In all of these cases, human disease seems to be causedby small perturbations in early signalling events inimmune cells.

The power of PTPs to affect the immune responsehas also been exploited by several pathogenic bacteriaand viruses. A classic example is that of Yersinia pestis,the causative agent of bubonic plague; it injectsYopH, a highly active PTP, directly into the cytoplasmof immune cells (including macrophages, T cells andB cells) during the early stages of infection132–134. Theintracellular YopH then efficiently dephosphorylateskey signalling proteins and thereby inhibits the acti-vation of these cells and consequently the initiationof an immune response. This allows the bacteria tomultiply unopposed and to cause a rapidly fatal dis-ease. In T cells, YopH efficiently dephosphorylatesLCK at Y394 in the activation loop, resulting in thecomplete ‘paralysis’ of TCR signalling135.

Virulent Salmonella spp. use a similar strategy: theyinject host cells with the phosphatase SptP, which inter-feres with the activation of MAPKs136. Mycobacteriahave also been reported to secrete a tyrosine-specificPTP, which is involved in virulence137. Studies of themolecular mechanism of these and other bacterial PTPsmight help us to understand how microorganismsevade the immune system and will provide new insightsinto the function of PTPs in signalling pathways inhuman immune cells.

PTPs as drug targets. Although inhibitors of PTKs havebeen approved for clinical use, PTP inhibitors have onlyrecently become of interest to the pharmaceutical indus-try. The main impetus for this newly found interest wasthe discovery that PTP1B counteracts signalling throughthe insulin receptor45. So, small-molecule inhibitors ofPTP1B are considered to be promising for the treatmentof type 2 diabetes. Recent discoveries regarding the roleof other PTPs in human metabolic, neurological, mus-cular and immunological diseases6, as well as in cancer7,are increasing the interest in PTPs as drug targets138.Currently considered drug targets include CD45, PTP1B,MKP1 and CDC25. Bacterial PTPs that have a key role inimmune evasion and virulence are also promising drugtargets for treating infectious disease139.

Disease and clinical applicationPTPs in human immunological and infectious disease.The discovery that the motheaten mouse had a muta-tion in SHP1 (REF. 85) provided the first example of anautoimmune disease caused by a defect in a PTP. Thesemice have overly active phagocytic cells and hyper-responsive T and B cells, and they develop inflamma-tory pathology of several tissues. Autoimmune diseasealso develops in mice that are transgenic for a gain-of-function mutant CD45 (in which arginine replacesglutamic acid at position 613)77 and in mice that lackPEP97 (the mouse orthologue of LYP). The latter animalsare characterized by exaggerated activation of memoryT cells with age, as well as expansion of secondarylymphoid organs with age.

Two PTPs have also been found to be associatedwith human disease: namely, CD45 and LYP. A fewgenetic polymorphisms have also been described in thehuman PTPN6 gene (which encodes SHP1)124, but noassociations have yet been reported with humanautoimmunity. CD45 abnormalities were detected in

P

P

t-SNARE

Inactive NSF

NSF

Fusion

Fusion-competent secretory vesicle Resting secretory vesicle

Phosphatidylinositolkinases

Plasma membrane

PTKs

v-SNARE

Granzyme

PtdIns(4,5)P2

PtdIns(3,4,5)P3

PtdIns(3,5)P2

P

Sec14 PTP

Inactive PTP-MEG2Active PTP-MEG2

Figure 7 | Schematic model for the function of PTP-MEG2 in secretory-vesicle fusionwith the plasma membrane. The substrate for PTP-MEG2 (protein tyrosine phosphatasemegakaryocyte 2) is the hexameric ATPase and vesicle-fusion regulator NSF (N-ethylmaleimide-sensitive factor), which is activated to set up the fusion machinery after dephosphorylation byPTP-MEG2. PTP-MEG2 is itself activated by phosphatidylinositols, which interact with the Sec14(Saccharomyces cerevisiae phosphatidylinositol-transfer protein)-homology domain of the PTP.PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; PTK, protein tyrosinekinase; t-SNARE, target soluble-NSF-accessory-protein receptor; v-SNARE, vesicle soluble-NSF-accessory-protein receptor.


R E V I E W S

oxidation, nitrosylation, acetylation and methylation)of PTPs and their substrates. As such, global phospho-proteomics studies are likely to become important.Perhaps most importantly, we predict that more PTPswill be found to be involved in human disease and willbe used as drug targets to treat these diseases. Giventhe crucial role that PTPs have in immune cells andgiven our growing understanding of these enzymes,we expect that PTP inhibitors will be developed forthe treatment of allergic and autoimmune diseaseswithin the next few years.

Note added in proofH. Saito’s unpublished observations have now beenaccepted for publication. Nam, H.-J., Poy, F., Saito, H. &Frederick, C. A. Structural basis for the function andregulation of the receptor protein tyrosine phosphataseCD45. J. Exp. Med. (in the press).

Future perspectives for PTPs in immune cellsThe PTP field is heading into increasingly excitingtimes. An important recent milestone was the clarifi-cation of the entire repertoire of PTP genes inhumans and mice6. It is now possible to venture intomore global studies of the PTPome and to addressquestions of specificity, redundancy and tissue expres-sion of all PTPs within the immune system. A princi-pal challenge is now to elucidate the physiologicalfunctions of individual PTPs, their substrates andtheir importance in physiological processes. Genedeletions in mice will undoubtedly continue to be animportant avenue for this work, but newer and fastertechniques, such as RNA interference, are also gainingground. We also predict that improved proteomicstechnologies will be developed for faster and moresensitive identification of PTP substrates and post-translational modifications (such as phosphorylation,

1. Hunter, T. & Sefton, B. M. Transforming gene product ofRous sarcoma virus phosphorylates tyrosine. Proc. NatlAcad. Sci. USA 77, 1311–1315 (1980).

2. Mustelin, T., Abraham, R. T., Rudd, C. E., Alonso, A. &Merlo, J. J. Protein tyrosine phosphorylation in T cellsignaling. Front. Biosci. 7, 918–969 (2001).

3. Mustelin, T. Src Family Tyrosine Kinases in Leukocytes1–155 (Landes, Austin, 1994).

4. Mustelin, T., Rahmouni, S., Bottini, N. & Alonso, A. Role ofprotein tyrosine phosphatases in T cell activation. Immunol.Rev. 192, 139–147 (2003).

5. Mustelin, T. & Taskén, K. Positive and negative regulation ofT cell activation through kinases and phosphatases.Biochem. J. 371, 15–27 (2003).

6. Alonso, A. et al. Protein tyrosine phosphatases in the humangenome. Cell 117, 699–711 (2004).This paper reviews the entire complement of 107 PTPgenes in the human genome.

7. Andersen, J. N. et al. A genomic perspective on proteintyrosine phosphatases: gene structure, pseudogenes, and genetic disease linkage. FASEB J. 18, 8–13 (2004).

8. Samelson, L. E., Patel, M. D., Weissman, A. M., Harford, J. B. & Klausner, R. D. Antigen activation of murine T cells induces tyrosine phosphorylation of apolypeptide associated with the T cell antigen receptor. Cell 46, 1083–1090 (1986).

9. Hsi, E. D. et al. T cell activation induces rapid tyrosinephosphorylation of a limited number of cellular substrates. J. Biol. Chem. 264, 10836–10842 (1989).

10. Mustelin, T., Coggeshall, K. M., Isakov, N. & Altman, A.Tyrosine phosphorylation is required for T cell antigenreceptor-mediated activation of phospholipase C. Science247, 1584–1587 (1990).

11. Saltzman, E. M., Thom, R. R. & Casnellie, J. E. Activation ofa tyrosine protein kinase is an early event in the stimulationof T lymphocytes by interleukin-2. J. Biol. Chem. 263,6956–6959 (1988).

12. Gold, M. R., Matsuuchi, L., Kelly, R. B. & DeFranco, A. L.Tyrosine phosphorylation of components of the B cellantigen receptors following receptor crosslinking. Proc. NatlAcad. Sci. USA 88, 3436–3440 (1991).

13. Mustelin, T. T cell antigen receptor signaling: three families oftyrosine kinases and a phosphatase. Immunity 1, 351–356(1994).

14. Weiss, A. T cell antigen receptor signal transduction: a taleof tails and cytoplasmic protein-tyrosine kinases. Cell 73,209–212 (1993).

15. Mustelin, T. et al. Protein tyrosine phosphatases. Front.Biosci. 7, 85–142 (2002).

16. Iivanainen, A. V., Lindqvist, C., Mustelin, T. & Andersson, L. C. Phosphotyrosine phosphatases are involved in reversion of T lymphoblastic proliferation.Eur. J. Immunol. 20, 2509–2512 (1990).

17. O’Shea, J. J., McVicar, D. W., Bailey, T. L., Burns, C. &Smyth, M. J. Activation of human peripheral blood Tlymphocytes by pharmacological induction of protein-tyrosine phosphorylation. Proc. Natl Acad. Sci. USA 89,10306–10310 (1992).

18. Secrist, J. P., Burns, L. A., Karnitz, L., Koretzky, G. A. &Abraham, R. T. Stimulatory effects of the protein tyrosine

phosphatase inhibitor, pervanadate, on T-cell activationevents. J. Biol. Chem. 268, 5886–5893 (1993).References 17 and 18 were the first papers to showthat acute inhibition of PTPs is sufficient to cause T-cell activation.

19. Oetken., C. et al. Phenylarsine oxide augments tyrosinephosphorylation in hematopoietic cells. Eur. J. Haematol.49, 208–214 (1992).

20. Earp, H. S., Austin, K. S., Buessow, S. C. & Gillespie, G. Y.Membranes from T and B lymphocytes have differentpatterns of tyrosine phosphorylation. Proc. Natl Acad. Sci.USA 81, 2347–2351 (1984).

21. Manning, G., Whyte, D. B., Martinez, R., Hunter, T. &Sudarsanam, S. The protein kinase complement of thehuman genome. Science 298, 1912–1934 (2002).

22. Gjörloff-Wingren, A. et al. Subcellular localization ofintracellular protein tyrosine phosphatases in T cells. Eur. J. Immunol. 30, 2412–2421 (2000).

23. Gjörloff-Wingren, A., Saxena, M., Williams, S., Hammi, D. &Mustelin, T. Characterization of TCR-induced receptor-proximal signaling events negatively regulated by the proteintyrosine phosphatase PEP. Eur. J. Immunol. 29, 3845–3854(1999).

24. Han, S., Williams, S. & Mustelin, T. Cytoskeletal proteintyrosine phosphatase PTPH1 reduces T cell antigenreceptor signaling. Eur. J. Immunol. 30, 1318–1325 (2000).

25. Bottini, N. et al. Activation of ZAP-70 through specificdephosphorylation at the inhibitory Tyr-292 by the lowmolecular weight phosphotyrosine phosphatase (LMPTP).J. Biol. Chem. 277, 24220–24224 (2002).

26. Townley, R., Shen, S.-H., Banville, D. & Ramachandran, C.Inhibition of the activity of protein tyrosine phosphatase 1C by its SH2 domains. Biochemistry 32, 13414–13418(1993).

27. Pei, D., Lorenz, U., Klingmüller, U., Neel, B. G. & Walsh, C. T.Intramolecular regulation of protein tyrosine phosphataseSH-PTP1: a new function for Src homology 2 domains.Biochemistry 33, 15483–15493 (1994).

28. Zhao, Z., Larocque, R., Ho, W.-T., Fischer, E. H. & Shen, S.-H. Purification and characterization of PTP2C, a widely distributed protein tyrosine phosphatase containing two SH2 domains. J. Biol. Chem. 269,8780–8785 (1994).

29. Pei, D., Wang, J. & Walsh, C. T. Differential functions of thetwo Src homology 2 domains in protein tyrosinephosphatase SH-PTP1. Proc. Natl Acad. Sci. USA 93,1141–1145 (1996).

30. Hof, P., Pluskey, S., Dhe-Paganon, S., Eck, M. J. &Shoelson, S. E. Crystal structure of the tyrosinephosphatase SHP-2. Cell 92, 441–450 (1998).

31. Alonso, A. et al. VHY, a novel, myristoylated testis-restricteddual specificity protein phosphatase related to VHX. J. Biol.Chem. 279, 32586–32591 (2004).

32. Mustelin, T., Coggeshall, K. M. & Altman, A. Rapid activationof the T cell tyrosine protein kinase pp56lck by the CD45phosphotyrosine phosphatase. Proc. Natl Acad. Sci. USA86, 6302–6306 (1989).This paper was the first to show that the PTP CD45can activate the PTK LCK by direct dephosphorylation.

33. Mustelin, T. & Altman, A. Dephosphorylation and activationof the T cell tyrosine kinase pp56lck by the leukocytecommon antigen (CD45). Oncogene 5, 809–813 (1990).

34. Mustelin, T. et al. Regulation of the p59fyn protein tyrosinekinase by the CD45 phosphotyrosine phosphatase. Eur. J. Immunol. 22, 1173–1178 (1992).

35. Brockdorff, J., Williams, S., Couture, C. & Mustelin, T.Dephosphorylation of ZAP-70 and inhibition of T cellactivation by activated SHP1. Eur. J. Immunol. 29,2539–2550 (1999).

36. Zheng, X. M., Wang, Y. & Pallen, C. J. Cell transformationand activation of pp60c-src by overexpression of a proteintyrosine phosphatase. Nature 359, 336–339 (1992).

37. Mustelin, T. & Hunter, T. Meeting at mitosis: cell cycle-specific regulation of c-Src by RPTPα. Sci. STKE [online]2002, PE3 (2002).

38. Salmeen, A. et al. Redox regulation of protein tyrosinephosphatase 1B involves a sulphenyl-amide intermediate.Nature 423, 769–773 (2003).

39. van Montfort, R. L. M., Congreve, M., Tisi, D., Carr, R. &Jhoti, H. Oxidation state of the active-site cysteine in proteintyrosine phosphatase 1B. Nature 423, 773–777 (2003).References 38 and 39 report the crystallization ofoxidized PTP1B and reveal that oxidation of thecatalytic cysteine residue results in the formation of a sulphenyl amide moiety, which is resistant tofurther oxidation (which would be irreversible) andcan be reduced back to the catalytically active freecysteinyl.

40. Meng, T.-C., Buckley, D. A., Galic, S., Tiganis, T. & Tonks, N. K. Regulation of insulin signaling throughreversible oxidation of the protein-tyrosine phosphatasesTC45 and PTP1B. J. Biol. Chem. 279, 37716–37725(2004).

41. Wang, X. et al. The tumor suppressor PTEN regulates T cell survival and antigen receptor signaling by acting as a phosphatidylinositol 3-phosphatase. J. Immunol. 164, 1934–1939 (2000).

42. Qu, C.-K., Nguyen, S., Chen, J. & Feng, G.-S. Requirement of Shp-2 tyrosine phosphatase in lymphoidand hematopoietic cell development. Blood 97, 911–914(2001).

43. Kishihara, K. et al. Normal B lymphocyte development butimpaired T cell maturation in CD45-exon6 protein tyrosinephosphatase-deficient mice. Cell 74, 143–156 (1993).

44. Gronda, M., Arab, S., Iafrate, B., Suzuki, H. & Zanke, B.Hematopoietic protein tyrosine phosphatase suppressesextracellular stimulus-regulated kinase activation. Mol. Cell.Biol. 21, 6851–6858 (2001).

45. Elchebly, M. et al. Increased insulin sensitivity and obesityresistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283, 1544–1548 (1999).

46. You-Ten, K. E. et al. Impaired bone marrowmicroenvironment and immune function in T cell proteintyrosine phosphatase-deficient mice. J. Exp. Med. 186,683–693 (1997).

47. Charbonneau, H., Tonks, N. K., Walsh, K. A. & Fischer, E. H.The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase. Proc. Natl Acad. Sci.USA 85, 7182–7186 (1988).


R E V I E W S

48. Tonks, N. K., Charbonneau, H., Diltz, C. D., Fischer, E. H. &Walsh, K. A. Demonstration that the leukocyte commonantigen CD45 is a protein tyrosine phosphatase.Biochemistry 27, 8695–8701 (1988).Although reference 47 was the first to report thatCD45 has a high degree of homology with the firstidentified PTP, PTP1B, and therefore was likely to be a PTP itself, reference 48 directly showed this by providing evidence that CD45 has tyrosine-phosphatase activity.

49. Pingel, J. T. & Thomas, M. L. Evidence that the leukocyte-common antigen is required for antigen-inducedT lymphocyte proliferation. Cell 58, 1055–1065 (1989).This paper was the first to show that expression of CD45 is important for T-cell activation.

50. Hermiston, M. L., Xu, Z. & Weiss, A. CD45: a criticalregulator of signaling thresholds in immune cells. Annu. Rev. Immunol. 21, 107–137 (2003).

51. Koretzky, G. A., Picus, J., Thomas, M. L. & Weiss, A.Tyrosine phosphatase CD45 is essential for coupling T-cellantigen receptor to the phosphatidyl inositol pathway.Nature 346, 66–68 (1990).

52. Volarevic, S. et al. The CD45 tyrosine phosphatase regulatesphosphotyrosine homeostasis and its loss reveals a novelpattern of late T cell receptor-induced Ca2+ oscillations. J. Exp. Med. 176, 835–844 (1992).

53. Ostergaard, H. L. et al. Expression of CD45 altersphosphorylation of the Lck-encoded tyrosine protein kinasein murine lymphoma T-cell lines. Proc. Natl Acad. Sci. USA86, 8959–8963 (1989).

54. Seavitt, J. et al. Expression of the p56lck Y505F mutation inCD45-deficient mice rescues thymocyte development. Mol.Cell. Biol. 19, 4200–4208 (1999).

55. Mustelin, T. & Burn, P. Regulation of src family tyrosinekinases in lymphocytes. Trends Biochem. Sci. 18, 215–220(1993).

56. Davidson, D., Bakinowski, M., Thomas, M. L., Horejsi, V. &Veillette, A. Phosphorylation-dependent regulation of T cellactivation by PAG/Cbp, a lipid raft-associatedtransmembrane adaptor. Mol. Cell. Biol. 23, 2017–2025(2003).

57. Kawabuchi, M. et al. Transmembrane phosphoprotein Cbpregulates the activities of Src-family tyrosine kinases. Nature404, 999–1003 (2000).

58. Brdicka, T. et al. Phosphoprotein associated withglycosphingolipid-enriched microdomains (PAG), a novelubiquitously expressed transmembrane adapter protein,binds the protein tyrosine kinase Csk and is involved inregulation of T cell activation. J. Exp. Med. 191, 1591–1604(2000).

59. Torgersen, K. M. et al. Release from tonic inhibition of T cellactivation through transient displacement of C-terminal Srckinase (Csk) from lipid rafts. J. Biol. Chem. 276,29313–29318 (2001).

60. Vang, T., Abrahamsen, H., Myklebust, S., Horejsi, V. &Taskén, K. Combined spatial and enzymatic regulation ofCsk by cAMP and protein kinase A inhibits T cell receptorsignaling. J. Biol. Chem. 278, 17597–17604 (2003).

61. Vang, T. et al. Knockdown of C-terminal Src kinase bysiRNA-mediated RNA interference augments T cell receptorsignaling in mature T cells. Eur. J. Immunol. 34, 2191–2199(2004).

62. Zhang, S. Q. et al. Shp2 regulates Src family kinase activityand Ras/Erk activation by controlling Csk recruitment. Mol.Cell 13, 341–351 (2004).

63. Ledbetter, J. A., Tonks, N. K., Fischer, E. H. & Clark, E. A.CD45 regulates signal transduction and lymphocyte activationby specific association with receptor molecules on T or B cells. Proc. Natl Acad. Sci. USA 85, 8628–8633 (1988).

64. Kiener, P. A. & Mittler, R. S. CD45-protein tyrosinephosphatase cross-linking inhibits T cell receptor CD3-mediated activation in human T cells. J. Immunol. 143,23–28 (1989).

65. Ostergaard, H. L. & Trowbridge, I. S. Coclustering of CD45with CD4 or CD8 alters the phosphorylation and kinaseactivity of p56lck. J. Exp. Med. 172, 347–350 (1990).

66. Volarevic, S. et al. Regulation of TCR signaling by CD45lacking transmembrane and extracellular domains. Science260, 541–544 (1993).

67. Furukawa, T., Itoh, M., Krueger, N. X., Streuli, M. & Saito, H.Specific interaction of the CD45 protein-tyrosinephosphatase with tyrosine phosphorylated CD3 ζ chain.Proc. Natl Acad. Sci. USA 91, 10928–10932 (1994).

68. Mustelin, T. et al. Regulation of the p70zap tyrosine proteinkinase by the CD45 phosphotyrosine phosphatase in Tcells. Eur. J. Immunol. 25, 942–946 (1995).

69. D’Oro, U., Sakaguchi, K., Appella, E. & Ashwell, J. D.Mutational analysis of Lck in CD45-negative T cells:dominant role of tyrosine 394 phosphorylation in kinaseactivity. Mol. Cell. Biol. 16, 4996–5003 (1996).

70. Katagiri, T. et al. CD45 negatively regulates Lyn activity bydephosphorylating both positive and negative regulatorytyrosine residues in immature B cells. J. Immunol. 163,1321–1330 (1999).

71. He, X., Woodford-Thomas, T. A., Johnson, K. G., Shah, D. D.& Thomas, M. L. Targeting of CD45 protein tyrosinephosphatase activity to lipid microdomains on the T cellsurface inhibits TCR signaling. Eur. J. Immunol. 32,2578–2584 (2002).

72. Montixi, C. et al. Engagement of T cell receptor triggers itsrecruitment to low-density detergent-insoluble membranedomains. EMBO J. 17, 5334–5340 (1998).

73. Edmonds, S. D. & Ostergaard, H. L. Dynamic association of CD45 with detergent-insoluble microdomains in T lymphocytes. J. Immunol. 169, 5036–5046 (2002).

74. Irles, C. et al. CD45 ectodomain controls interaction withGEMs and Lck activity for optimal TCR signaling. NatureImmunol. 4, 189–192 (2003).

75. Freiberg, B. A. et al. Staging and resetting T cell activation inSMACs. Nature Immunol. 3, 911–916 (2002).

76. Majeti, R., Bilwes, A. M., Noel, J. P., Hunter, T. & Weiss, A.Dimerization-induced inhibition of receptor protein tyrosinephosphatase function through an inhibitory wedge. Science279, 88–91 (1998).

77. Majeti, R. et al. An inactivating point mutation in the inhibitorywedge of CD45 causes lymphoproliferation andautoimmunity. Cell 103, 1059–1069 (2000).

78. Xu, Z. & Weiss, A. Negative regulation of CD45 by differentialhomodimerization of the alternatively spliced isoforms.Nature Immunol. 3, 764–770 (2002).

79. Dianzani, U. et al. Isoform-specific associations of CD45 with accessory molecules in human T lymphocytes.Eur. J. Immunol. 22, 365–371 (1992).

80. Matthews, R. J., Bowne, D. B., Flores, E. & Thomas, M. L.Characterization of hematopoietic intracellular proteintyrosine phosphatases: description of a phosphatasecontaining an SH2 domain and another enriched in proline-,glutamic acid-, serine-, and threonine-rich sequences. Mol.Cell. Biol. 12, 2396–2405 (1992).

81. Vivier, E. & Daëron, M. Immunoreceptor tyrosine-basedinhibition motifs. Immunol. Today 18, 286–291 (1997).

82. Mustelin, T. et al. T cell activation: the coming of the phosphatases. Front. Biosci. 3, D1060–D1096 (1998).

83. Chiang, G. G. & Sefton, B. M. Specific dephosphorylation ofthe Lck tyrosine protein kinase at Tyr-394 by the SHP-1protein-tyrosine phosphatase. J. Biol. Chem. 276,23173–23179 (2001).

84. Raab, M. & Rudd, C. E. Hematopoietic cell phosphatase(HCP) regulates p56LCK phosphorylation and ZAP-70binding to T cell receptor ζ chain. Biochem. Biophys. Res.Commun. 222, 50–57 (1996).

85. Tsui, H. W., Siminovitch, K. A., de Souza, L. & Tsui, F. W. L.Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene. Nature Genet.4, 124–129 (1993).

86. Shultz, L. D. & Green, M. C. Motheaten, an immunodeficientmutant of the mouse. II. Depressed immune competenceand elevated serum immunoglobulins. J. Immunol. 116,936–943 (1976).

87. Pani, G., Fischer, K.-D., Mlinaric-Rascan, I. & Siminovitch, K. A. Signaling capacity of the T cell antigenreceptor is negatively regulated by the PTP1C tyrosinephosphatase. J. Exp. Med. 184, 839–852 (1996).

88. Sathish, J. G. et al. CD22 is a functional ligand for SHP1 inprimary T cells. J. Biol. Chem. 279, 47783–47791 (2004).

89. Roy, G., Matthews, J., Woodford-Thomas, T. & Thomas, M. L. The function of protein tyrosinephosphatases in immune regulation. Adv. ProteinPhosphatases 9, 121–138 (1995).

90. Cloutier, J.-F. & Veillette, A. Association of inhibitory tyrosineprotein kinase p50csk with protein tyrosine phosphatase PEPin T cells and other hemopoietic cells. EMBO J. 15,4909–4918 (1996).

91. Cloutier, J.-F. & Veillette, A. Cooperative inhibition of T-cell antigen receptor signaling by a complex between a kinase and a phosphatase. J. Exp. Med. 189, 111–121(1999).References 90 and 91 document the high-stoichiometry association between the PEPphosphatase and the inhibitory kinase CSK. Thepapers conclude that the interaction between the twoenzymes helps PEP reach its SRC-family PTK targets.

92. Cohen, S., Dadi, H., Shaoul, E., Sharfe, N. & Roifman, C. M.Cloning and characterization of a lymphoid-specific,inducible human protein tyrosine phosphatase, Lyp. Blood93, 2013–2024 (1999).

93. Bottini, N. et al. A functional variant of lymphoid tyrosinephosphatase is associated with type 1 diabetes. NatureGenet. 36, 337–338 (2004).

This paper reports that a single amino-acidsubstitution at position 620 of the human PTP LYP issufficient to cause a marked increase in the incidenceof type 1 diabetes. This increase correlates with a lossof association with CSK.

94. Begovich, A. B. et al. A missense single-nucleotidepolymorphism in a gene encoding a protein tyrosinephosphatase (PTPN22) is associated with rheumatoidarthritis. Am. J. Hum. Genet. 75, 330–337 (2004).

95. Kyogoku, C. et al. Genetic association of the R620Wpolymorphism of protein tyrosine phosphatase PTPN22with human SLE. Am. J. Hum. Genet. 75, 504–507 (2004).

96. Smyth, D. et al. Replication of an association between thelymphoid tyrosine phosphatase locus (LYP/PTPN22) withtype 1 diabetes, and evidence for its role as a generalautoimmunity locus. Diabetes 53, 3020–3023 (2004).

97. Hasegawa, K. et al. PEST domain-enriched tyrosinephosphatase (PEP) regulation of effector/memory T cells.Science 303, 685–689 (2004).This paper describes the defects in T-cell immunitythat are found in Pep –/– mice.

98. Davidson, D., Cloutier, J. F., Gregorieff, A. & Veillette, A.Inhibitory tyrosine protein kinase p50csk is associated withprotein-tyrosine phosphatase PTP-PEST in hemopoieticand non-hemopoietic cells. J. Biol. Chem. 272,23455–23462 (1997).

99. Wang, B., Lemay, S., Tsai, S. & Veillette, A. SH2 domain-mediated interaction of inhibitory protein tyrosine kinase Cskwith protein tyrosine phosphatase-HSCF. Mol. Cell. Biol. 21,1077–1088 (2001).

100. Cote, J. F., Charest, A., Wagner, J. & Tremblay, M. L.Combination of gene targeting and substrate trapping toidentify substrates of protein tyrosine phosphatases usingPTP-PEST as a model. Biochemistry 37, 13128–13137(1998).

101. Garton, A. J., Flint, A. J. & Tonks, N. K. Identification ofp130cas as a substrate for the cytosolic protein tyrosinephosphatase PTP-PEST. Mol. Cell. Biol. 16, 6408–6418(1996).

102. Davidson, D. & Veillette, A. PTP-PEST, a scaffold proteintyrosine phosphatase, negatively regulates lymphocyteactivation by targeting a unique set of substrates. EMBO J.20, 3414–3426 (2001).

103. Spencer, S. et al. PSTPIP: a tyrosine phosphorylated cleavagefurrow-associated protein that is a substrate for a PESTtyrosine phosphatase. J. Cell Biol. 138, 845–860 (1997).

104. Sozio, M. S. et al. PTPH1 is a predominant protein-tyrosinephosphatase capable of interacting with anddephosphorylating the T cell receptor ζ subunit. J. Biol.Chem. 279, 7760–7768 (2004).

105. Zhang, S.-H., Liu, J., Kobayashi, R. & Tonks, N. K.Identification of the cell cycle regulator VCP (p97/CDC48)as a substrate of the band 4.1-related protein-tyrosinephosphatase PTPH1. J. Biol. Chem. 274, 17806–17812(1999).

106. Egerton, M. et al. VCP, the mammalian homolog of CDC48,is tyrosine phosphorylated in response to T cell antigenreceptor activation. EMBO J. 11, 3533–3540 (1992).

107. Lavoie, C. et al. Tyrosine phosphorylation of p97 regulatestransitional endoplasmic reticulum assembly in vitro. Proc.Natl Acad. Sci. USA 97, 13637–13642 (2000).

108. Zheng, Y., Schlondorff, J. & Blobel, C. P. Evidence forregulation of the tumor necrosis factor α-convertase (TACE)by protein-tyrosine phosphatase PTPH1. J. Biol. Chem.277, 42463–42470 (2002).

109. Tailor, P., Williams, S., Gilman, J., Couture, C. & Mustelin, T.Regulation of the low molecular weight phosphotyrosinephosphatase (LMPTP) by phosphorylation at tyrosines 131and 132. J. Biol. Chem. 272, 5371–5376 (1997).

110. Alonso, A., Saxena, M., Williams, S. & Mustelin, T. Inhibitoryrole for dual-specificity phosphatase VHR in T cell antigenreceptor and CD28-induced Erk and Jnk activation. J. Biol.Chem. 276, 4766–4771 (2001).

111. Alonso, A. et al. Tyrosine phosphorylation of VHRphosphatase by ZAP-70. Nature Immunol. 4, 44–48 (2003).

112. Saxena, M. & Mustelin, T. Extracellular signals and scores ofphosphatases: all roads lead to MAP kinase. Semin.Immunol. 12, 387–396 (2000).

113. Saxena, M., Williams, S., Gilman, J. & Mustelin, T. Negativeregulation of T cell antigen receptor signaling byhematopoietic tyrosine phosphatase (HePTP). J. Biol.Chem. 273, 15340–15344 (1998).

114. Saxena, M., Williams, S., Brockdorff, J., Gilman, J. &Mustelin, T. Inhibition of T cell signaling by MAP kinase-targeted hematopoietic tyrosine phosphatase (HePTP). J. Biol. Chem. 274, 11693–11700 (1999).

115. Saxena, M., Williams, S., Taskén, K. & Mustelin, T. Crosstalkbetween cAMP-dependent kinase and MAP kinase throughhematopoietic protein tyrosine phosphatase (HePTP).Nature Cell Biol. 1, 305–311 (1999).


R E V I E W S

116. Nika, K. et al. Hematopoietic protein tyrosine phosphatase(HePTP) phosphorylation by cAMP-dependent proteinkinase in T cells: dynamics and subcellular location.Biochem. J. 378, 335–342 (2004).

117. Klingmüller, U., Lorenz, U., Cantley, L. C., Neel, B. G. &Lodish, H. F. Specific recruitment of SH-PTP1 to theerythropoietin receptor causes inactivation of JAK2 andtermination of proliferative signals. Cell 80, 729–738 (1995).

118. Myers, M. P. et al. TYK2 and JAK2 are substrates of protein-tyrosine phosphatase 1B. J. Biol. Chem. 276,47771–47774 (2001).

119. ten Hoeve, J. et al. Identification of a nuclear Stat1 proteintyrosine phosphatase. Mol. Cell. Biol. 22, 5662–5668 (2002).

120. Zhu, W., Mustelin, T. & David, M. Arginine methylation ofSTAT1 regulates its dephosphorylation by TcPTP. J. Biol.Chem. 277, 35787–35790 (2002).

121. Wang, X. et al. Enlargement of secretory vesicles by proteintyrosine phosphatase PTP-MEG2 in RBL mast cells andJurkat T cells. J. Immunol. 168, 4612–4619 (2004).

122. Huynh, H. et al. Homotypic secretory vesicle fusion inducedby the protein tyrosine phosphatase MEG2 depends onpolyphosphoinositides in T cells. J. Immunol. 171,6661–6671 (2003).

123. Huynh, H. et al. Control of vesicle fusion by a tyrosinephosphatase. Nature Cell Biol. 6, 831–839 (2004).

124. Matsushita, M., Tsuchiya, N., Oka, T., Yamane, A. &Tokunaga, K. New variations of human SHP-1.Immunogenetics 49, 577–579 (1999).

125. Jury, E. C., Kabouridis, P. S., Flores-Borja, F., Mageed, R. A.& Isenberg, D. A. Altered lipid raft-associated signaling andganglioside expression in T lymphocytes from patients withsystemic lupus erythematosus. J. Clin. Invest. 113,1176–1187 (2004).

126. Tchilian, E. Z. et al. A deletion in the gene encoding theCD45 antigen in a patient with SCID. J. Immunol. 166,1308–1313 (2001).

127. Jacobsen, M. et al. A point mutation in PTPRC is associatedwith the development of multiple sclerosis. Nature Genet.26, 495–499 (2000).

128. Barcellos, L. F. et al. PTPRC (CD45) is not associated withthe development of multiple sclerosis in U.S. patients.Nature Genet. 29, 23–24 (2001).

129. Vorechovsky, I. et al. Does 77C→G in PTPRC modifyautoimmune disorders linked to the major histocompatibilitylocus? Nature Genet. 29, 22–23 (2001).

130. Bottini, N., Bottini, E., Gloria-Bottini, F. & Mustelin, T. Low-molecular-weight protein tyrosine phosphatase and humandisease: in search of biochemical mechanisms. Arch.Immunol. Ther. Exp. (Warsz.) 50, 95–104 (2002).

131. Bottini, N. et al. Genetic control of serum IgE levels: a studyof low molecular weight protein tyrosine phosphatase. Clin.Genet. 63, 228–231 (2003).

132. Cornelis, G. R. & Wolf-Watz, H. The Yersinia Yop virulon: abacterial system for subverting eukaryotic cells. Mol.Microbiol. 23, 861–867 (1997).

133. Andersson, K. et al. YopH of Yersinia pseudotuberculosisinterrupts early phosphotyrosine signalling associated withphagocytosis. Mol. Microbiol. 20, 1057–1069 (1996).

134. Yao, T., Mecsas, J., Healy, J. I., Falkow, S. & Chien, Y.Suppression of T and B lymphocyte activation by a Yersiniapseudotuberculosis virulence factor, YopH. J. Exp. Med.190, 1343–1350 (1999).

135. Alonso, A. et al. Lck dephosphorylation at Y394 andinhibition of T cell antigen receptor signaling by Yersiniaphosphatase YopH. J. Biol. Chem. 279, 4922–4928 (2004).References 134 and 135 show that the phosphataseYopH from Yersinia spp. is a potent inhibitor of T-cellactivation. Reference 135 shows that the molecularmechanism of inhibition involves thedephosphorylation of Y394 of LCK, resulting incomplete abrogation of signalling through the TCR.

136. Murli, S., Watson, R. O. & Galan, J. E. Role of tyrosinekinases and the tyrosine phosphatase SptP in theinteraction of Salmonella with host cells. Cell. Microbiol.3, 795–810 (2001).

137. Singh R. et al. Disruption of MptpB impairs the ability ofMycobacterium tuberculosis to survive in guinea pigs. Mol.Microbiol. 50, 751–762 (2003).

138. Lazo, J. S. & Wipf, P. Small molecule regulation ofphosphatase-dependent cell signaling pathways. Oncol.Res. 13, 347–352 (2003).

139. Liang, F. et al. Aurintricarboxylic acid blocks in vitro and invivo activity of YopH, an essential virulent factor of Yersiniapestis, the agent of the plague. J. Biol. Chem. 278,41734–41741 (2003).

AcknowledgementsWe apologize for being unable (owing to space limitations) to citemany important contributions made by our colleagues in the field.T.M. is supported by the National Institutes of Health, UnitedStates. T.V. is supported by The Norwegian Cancer Society. N.B.is supported by the American Italian Cancer Foundation, UnitedStates, and The Litta Foundation, Switzerland.

Competing interests statementThe authors declare no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Entrez Gene:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneCD45 | CSK | HEPTP | LCK | PTPH1 | PTP-MEG2 | PTP-PEST |SHP1 | ZAP70Access to this interactive links box is free online.

Protein tyrosine phosphatases and the immune response

Documents

Transcript of Protein tyrosine phosphatases and the immune response