Abbreviations BIM, Bcl2 like 11; DAG, diacylglycerol; Egr, early growth response protein; ERK, extracellular signal-regulated kinase; ITAM, immunoreceptor tyrosine-based activation motif
Trang 1ERK and cell death: ERK location and T cell selection
Emma Teixeiro and Mark A Daniels
Department of Molecular Microbiology and Immunology, Center for Cellular and Molecular Immunology, School of Medicine, University of Missouri, Columbia, MO, USA
Introduction
The development of a healthy immune system depends
on the generation of a diverse pool of T cells capable
of providing protection against a broad range of
pathogens, while avoiding an autoimmune attack on
healthy tissue T cells recognize specific peptide
anti-gens presented in the context of self-major
histocom-patibility complex (MHC) molecules through clonally
distributed T cell antigen receptors (TCRs) expressed
early during T cell ontogeny The TCR is formed by
the random association of variable and constant
genetic elements Although this process leads to the
production of a diverse pool of pathogen-specific T
cells, it also leads to the generation of T cells that are
either useless, due to an inability to recognize MHC,
or extremely dangerous, due to the potential for an overt reaction against self Therefore, it is essential that T cell development includes a selection process by which only the useful cells are instructed to mature and the nonfunctional and potentially harmful T cells are eliminated before they can fully develop
The shaping of the T cell repertoire begins when immature T cell precursors, called thymocytes, are selected by the ability of their TCR to recognize self-peptides presented by MHC (pMHC) on the various populations of antigen-presenting cells present in the thymus [1] A ‘Goldilocks’ affinity model of selection has been proposed to describe this process T cells that are unable to recognize self-peptide MHC undergo
Keywords
ERK; MAPK; T cell selection; TCR signaling;
thymocyte
Correspondence
M A Daniels, Department of Molecular
Microbiology and Immunology, Center for
Cellular and Molecular Immunology, M616
Medical Sciences Bldg, One Hospital Drive,
Columbia, MO 65212, USA
Fax: +573 882 4287
Tel: +573 884 1659
E-mail: danielsma@missouri.edu
(Received 19 June 2009, revised 14 August
2009, accepted 26 August 2009)
doi:10.1111/j.1742-4658.2009.07368.x
The selection of functional T cells is mediated by interactions between the
T cell antigen receptor and self-peptide major histocompatibility complex expressed on thymic epithelium These interactions either lead to survival and development or death The T cell antigen receptor is an unusual recep-tor able to signal multiple cell fates The precise mechanism by which this
is achieved has been an area of intense research effort over the years One model proposes that the differential activation of mitogen-activated protein kinase pathways contributes to this decision Here, the role of extracellular signal-regulated kinase in promoting or preventing apoptosis during thymic selection is discussed
Abbreviations
BIM, Bcl2 like 11; DAG, diacylglycerol; Egr, early growth response protein; ERK, extracellular signal-regulated kinase; ITAM, immunoreceptor tyrosine-based activation motif; JNK, c-JunNH2-terminal kinase; LAT, linker for activation of T cells; MAPK, mitogen-activated protein kinase; MHC, major histocompatibility complex; pERK, phospho-extracellular signal-regulated kinase; pJNK, phospho-c-JunNH2-terminal kinase; PLC, phospholipase C; SLP-76, SH2 domain containing leukocyte protein of 76 kDa; SAP-1, SRF accessory protein 1 (ELK4); SOS, son of sevenless; TCR, T cell antigen receptor.
Trang 2apoptosis (death by neglect) Weak or intermediate
TCR⁄ pMHC interactions induce positive selection and
lead to the survival, development and maturation of a
self-restricted, yet self-tolerant, T cell repertoire
(reviewed in [2]) TCRs that bind self-peptide MHC
with high affinity induce either anergy [3], receptor
editing [4], deviation into regulatory T cell lineage [5]
or clonal deletion (apoptosis), which collectively are
considered to be negative selection [6] Clonal deletion
is thought to be the dominant form of negative
selec-tion Although this model of selection is generally
accepted, the question remains as to how the TCR can
translate subtle changes in ligand binding parameters
to signal such distinct cell fates as survival⁄
differentia-tion and death The goal then is to establish the point
where the TCR signals diverge, leading to the ultimate
fate of either life or death for the developing
thymocyte A differential signaling model, where
different mitogen-activated protein kinase (MAPK)
signals lead to either positive or negative selection, has
begun to emerge (reviewed in [2,7,8]) MAPK signaling
is important for determining cell fate decisions in a
diverse number of organisms and cell types (reviewed
in [7,8]) In thymocytes, c-JunNH2-terminal kinase
(JNK) [9], p38 [10] and extracellular signal-regulated
kinase 5 (ERK5) [11] are MAPKs essential for
negative selection, but do not influence positive
selec-tion The small GTPase Ras initiates the MAPK
cas-cade that leads to Raf1–MEK1⁄ 2–ERK1 ⁄ 2 activation
The phosphorylation of ERK1⁄ 2 is important for
positive selection and dispensable for negative selection
[12–15] Interestingly, positive and negative selecting
ligands activate all four of these pathways The
conun-drum is how does a T cell integrate these signals to
distinguish positive from negative selection? One
possi-ble explanation may be the location of the active form
of these signaling molecules within a cell It was
recently shown, that in thymocytes, positive and
nega-tive selecting ligands induce the localization of the
components of the Ras⁄ ERK cascade and active ERK
into distinct subcellular compartments (Figs 1 and 2)
[16] Several groups have demonstrated that the
biolog-ical outcome of Ras⁄ MAPK activation is determined
by its subcellular localization [8,17] The role of ERK
in promoting or preventing clonal deletion (apoptosis)
during the thymic selection decision-making process is
the subject of this review
Is LAT the fork in the TCR signaling
road?
A long-time goal of immunologists is to establish
where signals emanating from the TCR diverge and
lead to such distinct cell fates as survival and death Much is known about the events that occur immedi-ately upon TCR engagement of pMHC Lck is acti-vated by CD45 and recruited to the TCR⁄ CD3 complex by the coreceptors CD4 or CD8 Lck then
Fig 2 Localization of pattern Ras ⁄ MAPK signaling intermediates during negative selection The figure depicts the location of mole-cules described in the text in the case of TCR engagement by a negative selecting ligand Note the separate location of pJNK and pERK, and Ras-GRP1 ⁄ Grb2 ⁄ SOS ⁄ Ras ⁄ Raf at the plasma membrane.
Fig 1 Localization of pattern Ras ⁄ MAPK signaling intermediates during positive selection The figure depicts the location of mole-cules described in the text in the case of TCR engagement by a positive selecting ligand Note the similar location of pJNK and pERK, and Ras-GRP1 ⁄ Ras ⁄ Raf at the Golgi.
Trang 3phosphorylates the immunoreceptor tyrosine-based
activation motifs (ITAMs) of the CD3 subunits and
TCRf This allows for the recruitment of the kinase
Zap-70 to the TCR and induces its activation
(reviewed in [18]) The TCRf chain contains six
ITAMs Initially, studies comparing variants of agonist
peptides suggested that qualitative differences in TCRf
phosphorylation of individual ITAMs contributed to
the selection decision In these studies, weak ligands,
capable of inducing positive selection, generated the
p21 form of phopho-f High-affinity ligands, capable
of inducing negative selection, generated p23-f [19,20]
More recently, the mutation of selected ITAMs and
studies on f-chain phosphorylation have provided
more support for a quantitative than a qualitative
TCRf phosphorylation model [21–23] One study has
suggested that phosphorylation of a defined number of
CD3 ITAMs is required for each developmental step
in selection [23] Another study showed that a gradual
decrease in TCR affinity correlates with a gradual
decrease in total TCRf phosphorylation Interestingly,
in spite of the small change in total TCRf
phosphory-lation between ligands that lie on either side of the
boundary of selection, the recruitment of active
Zap-70 to the membrane is markedly enhanced for negative
selecting ligands [16]
The TCRf chain does not have the capacity to
recruit a wide variety of signaling molecules [24]
How-ever, one of the downstream targets of active Zap-70,
the linker for activation of T cells (LAT), contains
nine phosphorylatable tyrosines that are able to
specifi-cally recruit several signaling molecules essential for
thymocyte selection (reviewed in [25]) This suggests
that LAT could be a more suitable candidate as a
branch point in TCR signal transduction Mutational
analyses specifically determined that the four distal
LAT tyrosines are critically important for T cell
devel-opment [26] Phosphorylation of these tyrosines
facili-tates the recruitment of phospholipase C (PLC)c1,
Gads, SLP-76 and Grb-2 PLCc1 is essential for the
mobilization of calcium The role of calcium, upstream
of calcineurin, has long been appreciated as being
important for thymocyte selection [27] Activation of
PLCc1 also induces the generation of diacylglycerol
(DAG), which is essential for the activation of protein
kinase C-h and the guanine exchange factor
Ras-GRP1 Protein kinase C-h and Ras-GRP1 are
impor-tant for the activation of Ras⁄ Raf1 ⁄ ERK and have
also been linked to positive selection in thymocytes
[28,29] Ras-GRP1, on the other hand, appears to be
much less important for negative selection [30] Gads
mediates the association of SLP-76, another Zap-70
substrate, to LAT Mice deficient in Gads or SLP-76
are largely defective for positive and negative selection [31,32] Members of the Tec family of kinases associate with LAT⁄ Gads ⁄ SLP-76 and contribute to the stability
of the complex and enhance the activation of PLCc1 Tec kinase deficiency alters both positive and negative selection [33,34]
Grb2 is the only adaptor molecule recruited to LAT that is uniquely required for negative selection Its ability to associate with the guanine exchange factor son of sevenless (SOS) in T cells makes it an important activator of the Ras⁄ Raf ⁄ ERK pathway Grb2 haploid insufficient mice demonstrate decreased induction of active JNK and p38 with a concomitant reduction in negative selection Interestingly, these mice do not have a defect in the generation of phospho-ERK (pERK) [35] Together with the role of Ras-GRP1 in ERK activation and positive selection, these data sug-gest that negative selecting ligands would exclusively activate Grb2⁄ SOS and positive selectors would acti-vate Ras-GRP1 In support of this, positive selectors
do not recruit Grb2⁄ SOS to LAT They only activate Ras-GRP1 and induce its recruitment to the Golgi [16] However, the fact that negative selecting ligands activate and recruit both Ras-GRP1 and Grb2⁄ SOS to the plasma membrane argues against this Mathemati-cal modeling of LAT phosphorylation [36] and studies
on the phosphorylation kinetics of individual LAT ty-rosines [37], indicate that the lack of Grb2–LAT inter-action during positive selection may be due to partial phosphorylation of LAT In line with this, positive and negative selecting ligands show quantitative differ-ences in total phosphorylation of LAT [16], although the phosphorylation state of individual tyrosines in thymocytes has not been assessed Taken together these data favor a model where the signal emanating from the TCR begins to diverge at LAT
outcome
The differential recruitment of signaling intermediates
to LAT appears to have a direct consequence on the regulation of downstream signaling pathways that are important for determining the selection decision One
of these is the Ras⁄ ERK pathway The role of the Ras⁄ ERK cascade in thymic selection has been well studied (reviewed in [7]) Phosphorylation of ERK1⁄ 2
is essential for positive selection, but their role in nega-tive selection is dispensable [12–15] The activation of ERK is linked to LAT through PLCc1⁄ DAG ⁄ Ras-GRP1⁄ Ras and Grb2 ⁄ SOS ⁄ Ras (reviewed in [8]) When thymocytes are stimulated by negative selecting ligands, they induce a rapid, robust, yet transient
Trang 4induction of pERK that is localized to the plasma
membrane (Fig 2) On the other hand, positive
select-ing ligands induce a slow and sustained activation of
ERK originating from the Golgi and leading to pERK
being distributed throughout the cell (Fig 1)
[16,38,39] The differences in the kinetics of pERK
induction may be explained by both the location and
the identity of the upstream activators of Ras Ras
activation by only Ras-GRP1 follows a graded
response that correlates with the stimulus, whereas
SOS, on the other hand, contains a positive feedback
loop that dramatically increases the rate of Ras
activa-tion [40] Furthermore, there is a marked increase in
negative regulation of Ras at the plasma membrane
versus the Golgi [8] Therefore, the differential
recruit-ment of Grb2⁄ SOS and Ras compartmentalization
describe a potential mechanism for the differences in
pERK kinetics observed between positive and negative
selecting ligands [16,38,39]
The method that thymocytes utilize for the
compart-mentalization of the components of the Ras⁄ ERK
pathway is not completely understood The regulation
of activation and membrane recruitment of Ras-GRP1
is dependent on calcium and DAG One could imagine
a scenario where the slow generation of calcium (and
DAG) induced by positive selectors activates
Ras-GRP1, which then binds to the DAG-rich Golgi [8,16]
Conversely, the robust calcium flux induced by
nega-tive selectors could correlate with the generation of
large quantities of DAG at the plasma membrane and
lead to Ras-GRP1 recruitment to that location Recent
work has demonstrated that in T cells, PLCc1
activa-tion leads to activaactiva-tion of Ras-GRP1 and its
recruit-ment to the Golgi and lymphocyte function-associated
antigen (LFA-1)-mediated activation of phopholipase
D2 leads to activation of Ras-GRP1 on both the Golgi
and the plasma membrane [41] In addition, DAG
kin-ases have also been shown to play an important role
in the regulation of Ras-GRP1 localization [42,43]
The location of Ras-GRP1 and SOS in turn lead to
the recruitment, activation and compartmentalization
of Ras to the different membranes within a cell
Downstream of Ras, various MAPK scaffolds that are
restricted to different membrane compartments are
probably responsible for the localization of pERK [8]
One such scaffold, kinase suppressor of Ras (KSR)1,
has been shown to be important for the membrane
localization of ERK and to somehow play a role in T
cell development [44,45] How these processes combine
to determine the localization pattern of Ras⁄ ERK in
thymocytes remains to be seen The classic paradigm
of ERK activation would predict that once
phosphory-lated, ERK dissociates from the scaffold where it can
either act on cytosolic targets or move to the nucleus
to activate transcription factors (reviewed in [7,8]) In light of this, the localization of pERK at the plasma membrane by negative selecting ligands is most curious (Fig 2) Whether it is somehow sequestered at the plasma membrane or subject to rapid dephosphoryla-tion is a quesdephosphoryla-tion that remains to be answered When all of this is considered together, differences in the location of pERK may provide the developing thymo-cyte with the ability to distinguish positive and nega-tive selecting ligands Although the precise mechanism
of how differential MAPK compartmentalization con-tributes to this decision is open to debate, possible implications of pERK localization are discussed below Several transcription factors that are downstream targets of ERK1⁄ 2 play an important role in mediating positive selection SRF accessory protein 1 (ELK4) (SAP-1) is a ternary complex factor subfamily member
of Ets transcription factors that is activated by pERK1⁄ 2 Deficiency of SAP-1 leads to a block in positive selection [46] Activation of SAP-1 leads to the expression of early growth response protein (Egr)1 Overexpression of Egr1 results in positive selection on
a nonselecting background [47], and Egr1-deficient mice are impaired for positive selection [48] ERK1⁄ 2 activation also leads to a reduction in DNA binding
by the basic helix–loop–helix protein E2A through the increased expression of the inhibitor of basic helix– loop–helix protein Id3 [49] E2A-deficient mice have enhanced positive selection [50]; Id3-deficient mice demonstrate a profound block in positive selection [51] Therefore, the sequestration of active ERK at the plasma membrane by negative selectors may effectively block the activation of these nucleus-resident transcrip-tion factors tipping the balance in favor of negative selection
Active ERK1⁄ 2 may also contribute to thymic selec-tion by regulating the balance of proapoptotic and prosurvival proteins in the cytosol (reviewed in [52]) Apoptosis induced by negative selection does not involve classical death receptor pathways, rather it depends (at least in part) on the nuclear orphan steroid receptor Nur77 (discussed later) and the proapoptotic BH3-only Bcl-2 family member Bcl2 like 11 (BIM) The prosurvival molecules Bcl-2 and Bcl-xLare able to bind and sequester the proapoptotic molecules Bax and Bak to prevent them from inducing apoptosis Once active, BIM is able to bind Bcl-2 or Bcl-xL, lead-ing to the release and activation of Bax⁄ Bak and apop-tosis [53] Thymocytes from male mice lacking Bim are severely impaired for negative selection of the auto-reactive male antigen specific HY-TCR [54] In addi-tion, the defect in negative selection in the nonobese
Trang 5diabetic mouse strain has been linked to defective
induction of BIM, among other proapototic molecules,
enhancing its importance for negative selection [55,56]
Post-translational modifications can affect both the
level of expression and the proapoptotic activity of
BIM For example, ERK-mediated phosphorylation of
BIM can target it for ubiquitination and degradation
(reviewed in [52,57]) or inhibit its proapoptotic activity
by reducing its binding to the prosurvival molecules
Mcl-1 and Bcl-xL [58,59] Interestingly, JNK
phospho-rylates BIM on the same residue as ERK However,
JNK also recruits the prolyl-isomerase Pin1 and
induces a conformational change in BIM that enhances
its proapoptotic potency in neuronal cells [60] JNK
has also been implicated in the upregulation of BIM
expression [52] and JNK-mediated phosphorylation of
BIM facilitates its release from sequestration by dynein
motor complex [61] Whether these findings hold true
in developing thymocytes is not known During
nega-tive selection, whether BIM is regulated through
tran-scription, post-translational modification or both
remains to be determined In summary, although BIM
is recognized to be critically important for negative
selection, the mechanism of its regulation is still
unclear
Negative selecting ligands induce a rapid and robust
induction of phopho-ERK1⁄ 2, whereas positive
select-ing ligands induce slow and sustained activation of
pERK1⁄ 2 [38,39] These data suggest the possibility of
a kinetic discrimination model for thymic selection
However, this cannot explain how strong induction of
pERK1⁄ 2 by negative selectors does not rescue the
thymocyte from apoptosis, especially in the light of the
roles of pERK1⁄ 2 just described Consider then, active
JNK is distributed throughout the cell and has the
same kinetics regardless of ligand strength [16,39]
Fur-thermore, positive selecting ligands induce pERK1⁄ 2
throughout the cell similar to phospho-JNK (pJNK)
By contrast, negative selecting ligands lead to the
acti-vation and retention of pERK at the plasma
mem-brane The net result is that negative selectors induce
segregation of pERK1⁄ 2 and pJNK [16] This suggests
that the localization of pERK1⁄ 2 determines the
selec-tion outcome Along this line, targeting of the Raf⁄
MEK⁄ ERK MAPK module to either the cytoplasm or
the plasma membrane in a neuronal cell line leads to
switch-like differences in biological outcome [62]
Additionally, studies from several groups have
demon-strated that the subcellular localization pattern of
Ras⁄ MAPK determines the signaling output in a
vari-ety of cell types (reviewed in [8]) Given the competing
roles of ERK1⁄ 2 and JNK in determining selection,
these data suggest that retention of pERK1⁄ 2 at the
plasma membrane mimics the effect of an ERK knock-out and gives pJNK an unopposed opportunity or at least a head start in activating the proapoptotic effec-tor molecules necessary for negative selection Alterna-tively, a model where a unique signal is provided by membrane-bound pERK cannot be ruled out Regard-less, it is attractive to hypothesize that the differential compartmentalization of Ras⁄ ERK pathways provides the thymocyte with the ability to distinguish between positive and negative selecting ligands [16] Future studies are needed to establish whether the localization pattern is sufficient to determine selection outcome
ERK5, Nur77 and negative selection
The orphan steroid receptor Nur77 is part of a small family of transcription factors (which also includes Nurr1 and Nor1) that is thought to play an important role in mediating TCR-induced apoptosis in immature thymocytes It acts in a pathway that is not redundant, but rather parallel to BIM (reviewed in [52]) The importance of Nur77 as a proapoptotic molecule in T cells was first described in hybridomas [63,64] Using various models of negative selection, dominant nega-tive Nur77 resulted in a decrease in neganega-tive selection, whereas constitutively active mutants led to an increase
in negative selection [65,66] However, Nur77-deficient mice do not have a defect in negative selection This apparent discrepancy can be explained by considering the redundant role of the related family member Nor1
in mediating clonal deletion and the fact that the dom-inant negative form of Nur77 is able to inhibit the function of the other family members and block dele-tion of autoreactive thymocytes [67]
Upon TCR stimulation, Nur77 transcription is upregulated through the ERK5⁄ MEF2 pathway [11,68,69] The activation of Nur77 occurs by a cal-cium-dependent pathway that ultimately leads to its phosphorylation by ERK5 [70,71] On the other hand, Akt-mediated phosphorylation of Nur77 inhib-its inhib-its DNA-binding activity [72], and there is specula-tion that ERK2 phophorylaspecula-tion can also inhibit Nur77 function by phosphorylation on a site distinct from the ERK5 target site [73] Given these and other roles of Akt and ERK2 [15], these data suggest
an additional mechanism by which thymocytes could distinguish positive from negative selecting ligands The mechanism by which Nur77 mediates the induc-tion of apoptosis is less clear Transcripinduc-tional activity correlates with apoptosis in Nur77 transgenic thymo-cytes and Nur77-deficient mice [70,74] In fact, Nur77 induces the proapoptotic gene Nur77 downstream gene 1 (NDG1), FasL and TNF-related
Trang 6apoptosis-inducing ligand (TRAIL) [75], but the physiological
relevance of some of these molecules in clonal
dele-tion of thymocytes has not been tested Other studies
have shown that Nur77 can translocate from the
nucleus to the mitochondria, where it binds to Bcl-2,
converting it from a prosurvival factor into a
proapo-totic molecule [76–78] A conflicting study reported
that efficient export of Nur77 was only observed in
mature T cells and immature thymocytes did not
translocate Nur77 to the cytosol [79] The observed
differences, which could be due to the type of cell
examined, experimental technique, model system or
maturation state of thymocytes tested, need to be
resolved to make an accurate conclusion
Further-more, although these two models of Nur77-induced
apoptosis are not necessarily mutually exclusive, it is
difficult to reconcile the mitochondrial data with the
correlation between transcriptional activity and
apop-tosis [52] Interestingly, the activation of ERK5,
dis-pensable for positive selection, has a kinetic level of
induction that is similar between positive and negative
selecting ligands [11] This resembles what has been
reported for JNK and p38 [39] and again suggests
that sequestration of pERK at the plasma membrane
by negative selecting ligands may be necessary for the
induction of signals necessary for negative selection
Conclusions
Understanding the mechanisms that determine central
tolerance is essential to the regulation of
autoimmuni-ty, infectious disease and cancer The mechanism by
which T cells translate the parameters of ligand
engagement into positive or negative selection has been
elusive The default for a preselection double-positive
thymocyte is death The time involved to complete the
selection process and the requirement for intact thymic
architecture have made studying the process of
nega-tive selection extremely difficult In spite of this,
MAP-Ks, ERK1⁄ 2, ERK5, JNK and p38, are known to be
involved in the induction of positive versus negative
selection in the thymus ERK5, JNK and p38 are
required for negative selection and dispensable for
positive selection On the other hand, ERK1⁄ 2 is only
involved in positive selection At first glance, this
appears to describe the mechanism of selection Yet,
the fact remains that these molecules are activated by
both positive and negative selecting ligands
Differen-tial subcellular localization of these, and possibly other
signaling intermediates, provides the developing
thymocyte with the tools to overcome this apparent
problem Interestingly, JNK activity and subcellular
location are the same regardless of the ligand strength,
whereas ERK activity and location change depending
of the nature of the selecting ligand In addition, the kinetics of the other MAPK important for negative selection are the same independent of the ligand Given their opposing function and downstream targets, this appears to give ERK a prominent role in determining the outcome of thymic selection Further studies are needed to demonstrate how ERK and JNK compete and if location is sufficient to determine selection out-come
Acknowledgements
We would like to thank Dr Bumsuk Hahm and Dr Mark McIntosh for critical reading of the manuscript and Dr Ed Palmer for his support Work from the laboratory of MAD and ET are supported by the University of Missouri Mission Enhancement Fund
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