Download Calcium-independent calcineurin regulation

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Calcium-independent calcineurin regulation
Richard V Parry & Carl H June
CaM
CHP
+
–
CnB
nB
–
FK506
CnA
–
Csp1
A238L
NFAT
CnA
— P
P
CABIN
MEF 2
No response
NFAT
Im
mmune response gene
es
CC
C.C.
–
Richard V. Parry and Carl H. June are at the
Abramson Family Cancer Research Institute and
Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Philadelphia,
Pennsylvania 19104, USA.
e-mail: [email protected]
2+
Ca
–
Although intracellular signal transduction
is often portrayed as a protein kinase
‘domino effect’, the counterbalancing function of phosphatases, and thus the control
of phosphatase activity, is equally relevant
to proper regulation of cellular function.
Indeed, a notable example of the importance of phosphatases is found in the
plague–causing agent Yersinia pestis, which
depends on a phosphatase to exert its
deadly effect1. In contrast to the many protein kinases that have been discovered and
studied in detail, relatively few phosphatases have received similar attention.
One prominent exception is found in calcineurin (PP2B), a calcium-responsive
enzyme that dephosphorylates nuclear factor of activated T cells (NFAT), thereby
promoting its nuclear translocation, and
thus occupies a unique niche linking calcium signaling to transcriptional regulation2. In this issue of Nature Immunology,
Ryeom and colleagues describe a new level
of calcineurin regulation mediated by a
group of proteins called calcipressins3.
Using calcipressin 1 (Csp1)-deficient mice,
they demonstrate Csp1 as an in vivo regulator of calcineurin, and explore its involvement in the acquired immune response.
Furthermore, these results have provided
more insights about the function of calcineurin itself.
Protein phosphatases are subdivided into
three classes: serine/threonine specific, tyrosine specific and dual specificity. Based on
biochemical parameters, substrate specificity and sensitivity to various inhibitors,
serine/threonine protein phosphatases are
divided into two main classes. Type I protein phosphatases can be inhibited by two
heat-stable proteins, called inhibitor 1 and
inhibitor 2. Type II protein phosphatases are
insensitive to heat-stable inhibitors and are
+
© 2003 Nature Publishing Group http://www.nature.com/natureimmunology
Calcineurin is negatively regulated by calcipressin 1. Analysis of calcipressin-deficient mice shows that survival of
T helper type 1 cells is dependent on calcipressin, demonstrating another function for the regulation of calcineurin
activity in T cells.
Figure 1 Multiple levels of regulation occur in the calcium-calcineurin-NFAT pathway. Calcineurin (Cn) is
activated by the binding of calcineurin A (CnA) to calcineurin B (CnB) and calmodulin (CaM), displacing
an auto-inhibitory domain. Dephosphorylation of NFAT promotes its nuclear import. FK506- and FKBPimmunophilin complexes bind CnB, calcipressin (Csp1) binds CnA, and CHP competes with CaM for CnA
binding. The viral inhibitor A238L blocks Cn-NFAT interaction, and cabin1 interacts with activated
calcineurin in the nucleus, preventing MEF2 activation, in this example.
further subdivided into spontaneously
active (PP2A), calcium-dependent (PP2B;
calcineurin) and magnesium-dependent
(PP2C) classes of phosphatases. Calcineurin
was first identified as a principal calmodulin-binding protein from brain, and is
abundantly expressed in areas of the brain
that are vulnerable to stroke, epilepsy and
neurodegenerative diseases. Calcineurin is
important in inflammation and, in the
clinic, immunosuppression can be achieved
by administration of cyclosporin A and
FK506, which target calcineurin phosphatase activity, blocking T cell activation.
Calcineurin is a heterodimer consisting
of a 60-kDa catalytic A subunit and a
19-kDa regulatory B subunit. The catalytic
subunit shares 40% sequence homology
with PP2A but contains a C-terminal region
that functions as a calmodulin-binding
domain. The catalytic activity of calcineurin
is positively regulated by the binding of cal-
NATURE IMMUNOLOGY VOLUME 4 NUMBER 9 SEPTEMBER 2003
cium both directly to calcineurin B and as a
calcium-calmodulin complex, which displaces an autoinhibitory domain, allowing
access of substrates to the catalytic domain.
Recent work, particularly in cardiac myocytes, has indicated that calcipressins may
negatively regulate calcineurin activity by
directly binding CnA4. Calcipressins are a
family of proteins derived from three genes.
Calcipressin 1 is also known as modulatory
calcineurin-interacting protein 1 (MCIP1),
Adapt78 and Down syndrome critical
region 1 (DSCR1). Calcipressin 2 is variously known as MCIP2, ZAKI-4 and
DSCR1-like 1. Calcipressin 3 is also called
MCIP3 and DSCR1-like 2. The Human
Nomenclature Committee has assigned
DSCR designations to these genes; however,
for clarity, the Csp1, Csp2 and Csp3 nomenclature will be used here. In a series of in
vitro experiments, Ryeom et al. demonstrate
that calcipressins bind activated calcineurin
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© 2003 Nature Publishing Group http://www.nature.com/natureimmunology
NEWS AND VIEWS
through their C termini and exert a potent
inhibitory effect on calcineurin phosphatase
activity and calcium-driven nuclear import
of NFATc, similar to that seen with
immunophilin-cyclosporin A or FK506
complexes.
Despite the profound influence of calcineurin on lymphocyte function, the
expression and function of calcipressins in T
lymphocytes has received scant attention so
far. Here, Ryeom et al. confirm the expression
of Csp1 in wild-type mouse lymph node CD4
T cells, and the generation of Csp1-deficient
mice, achieved by targeting DSCR1 exon 6.
As all four reported splice variants of Csp1
encode exon 6, no Csp1 protein is expressed.
Despite the absence of Csp1, deficient mice
show no apparent abnormalities in lymphocyte development or homeostasis. Catalytic
activity of endogenous calcineurin in
response to ionomycin, however, was found
to be greater in the absence of Csp1, consistent with the proposed inhibitory function of
Csp1 (ref. 5). Unexpectedly, the proliferation
of CD4 T cells in response to stimulation
with antibodies to CD3 and CD28 was
impaired in Csp1-deficient cells compared
with that of wild-type T cells. Csp1-deficient
cells also showed a notable deficit in production of interferon-γ, but not interleukin 4,
indicating that T helper type 1 (TH1) cells
were specifically affected. Furthermore,
analysis of cells after primary stimulation
showed low expression of T-bet but normal
expression of GATA-3 (TH1- and TH2-specific transcription factors, respectively).
These in vitro data were recapitulated in vivo
by immunization of mice with 2,4,6-trinitrophenol–keyhole limpet hemocyanin (TNPKLH), an antigen that can induce a mixed
TH1-TH2 response. Csp1-deficient mice
showed TNP-specific immunoglobulin M
(IgM) and IgG1 concentrations comparable
to those of wild-type mice, but decreased
concentrations of TNP-specific IgG2a. This is
taken as further evidence of defective TH1
cytokine production, as the TH1 cytokine
interferon-γ promotes switching to IgG2a
isotype, whereas the TH2 cytokine interleukin 4 promotes switching to IgG1 and IgE.
The defect in the TH1 immune response
noted in Csp1-deficient lymphocytes could
result from a defect in TH1 differentiation,
proliferation or survival. Investigating
the mechanism underlying the impaired
TH1 response, the authors demonstrated
increased apoptosis of Csp1-deficient TH1
cells but not Csp1-deficient TH2 cells. In
mice, homeostasis of TH1 cells is achieved
by apoptosis as a result of calcineurindependent Fas ligand expression during
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secondary stimulation6. Therefore, the
authors investigated whether aberrant
expression of Fas ligand mediated increased
cell death after primary stimulation of
Csp1-deficient TH1 cells. The authors were
able to restore TH1 function in Csp1-deficient cells by carefully titrating cyclosporin
A to mimic Csp1-mediated calcineurin
inhibition and by blocking cell death in
Csp1-deficient TH1 using Fas ligand–neutralizing antibodies, which increased proliferation
and
restored
interferon-γ
production in Csp1-deficient cells. Thus,
there is compelling evidence that aberrant
expression of Fas ligand precipitates the TH1
defect in Csp1-deficient lymphocytes.
Finally, the authors propose a hierarchical
model for Csp1 alteration of calcineurin
dependent transcription. The activation
thresholds of several genes were measured
in terms of T cell receptor (TCR) signal
strength; these thresholds were found to be
lower in Csp1-deficient cells. As expression
of Fas ligand required the greatest TCR
signal strength of the genes examined, the
consequence of losing Csp1-mediated calcineurin inhibition maybe to allow aberrant
Fas ligand expression after primary stimulation of TH1 cells. Thus the data indicate that
Csp1 has a key regulatory function in the
calcium-calcineurin-NFAT signaling pathway, altering the transcriptional profile
induced by calcium signaling.
The present work provides insight into
the involvement of calcipressins in T lymphocytes, and thus many questions remain
unanswered. As previously described, the
calcipressin family is derived from three
genes, and the expression of these genes
and their splice variants has not been fully
described in T lymphocytes. Although
Ryeom et al. demonstrate that Csp1, Csp2
and Csp3 are all capable of blocking calcineurin activity, these molecules are presumably not functionally redundant, and
the effect of Csp2 and Csp3 deficiency
remains to be described. The authors show
that the C termini of calcipressins are sufficient for calcineurin inhibition; however,
different calcipressins may have distinct
functions beyond calcineurin regulation,
and thus their deficiency may demonstrate
unforeseen phenotypes. The mechanisms
controlling tissue-specific expression probably differ for individual calcipressins;
indeed, work in other cell types has indicated that the expression of Csp1 and Csp2
is independently controlled by calcineurin
and thyroid hormone, respectively7. As
Csp1 expression is increased by calcineurin
activity, this may form the basis of a
feedback inhibition loop, which could serve
to protect T cells from high or sustained
intracellular calcium when antigen excess is
encountered, as has been suggested for neurons5. Precedence has been set for such a
system in T lymphocytes by the molecule
A20, which is induced by NF-κB, but promotes NF-κB nuclear export.
The upstream signaling mechanisms that
regulate calcipressin activity remain a chief
unexplored question. Csp1 encodes a highly
conserved central serine-proline repeat
sequence that is subject to phosphorylation
by glycogen synthase kinase (GSK) 3 and
mitogen-activated protein (MAP) kinase,
and is itself a substrate for calcineurin.
Phosphorylation status will likely modulate
calcipressin activity, and as both GSK3 and
MAP kinase are regulated at least in part by
costimulatory signals, the influence of costimulation on calcipressin activity may be
substantial in T lymphocytes. Positive costimulatory signals delivered by CD28 and
ICOS mediate increased intracellular calcium8. In contrast, negative costimulatory
molecules such as CTLA-4, PD-1 and the
recently described BTLA9 exert an inhibitory effect over interleukin 2 transcription, and it is possible that regulation of
calcipressin activity may help them achieve
this. Although work so far has focused on
the inhibitory function of calcipressins, it
has been suggested that Csp1 may suppress
or enhance the activity of calcineurin,
depending on the context of the cellular
stimulus10. The function of calcipressin may
therefore be more complex than its merely
acting as a calcineurin inhibitor. Furthermore, there are several classes of calcineurin
inhibitors in addition to calcipressins
(Fig. 1). The calcium-dependent activation
of calcineurin is prevented by calcineurin B
homologous protein, which competes with
calcineurin B for binding to calcineurin A11.
Cabin 1 (also call Cain) is a mainly nuclear,
noncompetitive inhibitor that interacts with
activated calcineurin12. Of particular interest to immunologists is the A238L protein
encoded by the African swine fever virus.
This protein shares sequence similarity with
the calcineurin interaction domain of NFAT
family members, and thus can mediate
immunosuppression and permit the virus to
evade the host immune response13. A further appreciation of the signals that regulate
these calcineurin inhibitors may yield clues
as to their functions, and their potential for
manipulation in lymphocytes.
The mechanisms by which diverse cell
types generate appropriate and specific
responses to changes in intracellular calcium
VOLUME 4 NUMBER 9 SEPTEMBER 2003 NATURE IMMUNOLOGY
© 2003 Nature Publishing Group http://www.nature.com/natureimmunology
NEWS AND VIEWS
concentration has been an enduring biological question. Answers probably lie in tissuespecific regulatory molecules, and in this
respect the emergence of calcipressins as regulators of calcineurin function is a promising development. This demonstration of the
regulatory function of calcipressins in T
lymphocytes advances our knowledge of the
immune system, adding a subtlety to calcineurin function. The phenotype of Csp1
deficiency in T cells shows the potential
involvement that calcineurin regulatory proteins have in the therapeutic manipulation
of the immune system.
1. Guan, K.L. & Dixon, J.E. Science 249, 553–556
(1990).
2. Clipstone, N.A. & Crabtree, G.R. Nature 357,
695–697 (1992).
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F. Nat. Immunol. 4, 874–881 (2003).
4. Rothermel, B.A., Vega, R.B. & Williams, R.S. Trends
Cardiovasc. Med. 13, 15–21 (2003).
5. Ermak, G., Harris, C.D. & Davies, K.J. FASEB J. 16,
814–824 (2002).
6. Zhang, X. et al. Unequal death in T helper cell (Th)1
and Th2 effectors: Th1, but not Th2, effectors
undergo rapid Fas/FasL-mediated apoptosis. J. Exp.
Med. 185, 1837–1849 (1997).
7. Yang, J. et al. Circ. Res. 87, E61–E68 (2000).
8. Parry, R.V., Rumbley, C.A., Vandenberghe, L.H., June,
C.H. & Riley, J.L. J. Immunol. 171, 166–174 (2003).
9. Watanabe, N. et al. Nat. Immunol. 4, 670–679 (2003).
10. Vega, R.B. et al. Proc. Natl. Acad. Sci. USA 100,
669–674 (2003).
11. Lin, X., Sikkink, R.A., Rusnak, F. & Barber, D.L.
J. Biol. Chem. 274, 36125–36131 (1999).
12. Sun, L. et al. Immunity 8, 703–711 (1998).
13. Miskin, J.E., Abrams, C.C., Goatley, L.C. & Dixon, L.K.
Science 281, 562–565 (1998).
SIGIRR puts the brakes on Toll-like receptors
Luke AJ O’Neill
Members of the Toll-like receptor–interleukin 1 receptor superfamily signal inflammatory responses. However, a
member of this family is now shown to modulate these responses by acting as a negative regulator.
The initial phase of host defense against
invading microbes involves a family of proteins called Toll-like receptors (TLRs). These
proteins are expressed on various cell types,
most notably dendritic cells, where they act
as primary sensors of microbial products
and activate signaling pathways that lead to
the induction of immune and inflammatory
genes. TLRs belong to a broader family of
proteins, which include receptors for the
pro-inflammatory cytokines interleukin 1
(IL-1) and IL-18 (ref. 1). Among the bestcharacterized TLRs are TLR4, TLR5 and
TLR9, which sense lipopolysaccharide
(LPS), flagellin and CpG motifs, respectively.
Although these receptors have important
functions in host defense, their unrestrained
stimulation may be detrimental to the host.
Thus, negative regulators of IL-1 receptor
(IL-1R), IL-18R and TLRs may be required
to modulate their responses. In this issue
of Nature Immunology, Wald et al.2 describe
an intriguing inhibitor of this receptor
superfamily.
All members of the TLR–IL-1R superfamily
signal inflammation in a very similar way. This
is because they all contain a conserved protein
sequence in their cytosolic regions, called the
Toll–IL-1R (TIR) domain, which activates
common signaling pathways, most notably
those leading to the activation of the tran-
Luke A.J. O’Neill is in the Cytokine Research
Group, Department of Biochemistry, Trinity College,
Dublin, Ireland.
e-mail: [email protected]
scription factor NF-κB and stress-activated
protein kinases. However, Wald et al. show
that an orphan receptor, which has the rather
cumbersome but accurate name, single
immunoglobulin IL-1R–related protein
(SIGIRR)3, is an inhibitory member of this
receptor superfamily. SIGIRR seems to temper
cellular activation by IL-1, LPS and probably
other activators of receptors in the TLR–IL-1R
superfamily, such that the biological outcome
will be the result of a balance between activation by a receptor and dampening by SIGIRR.
SIGIRR therefore acts as a ‘brake’ on the TLR
system, which may be essential for regulating
the detrimental effects of innate immunity, as
occurs in sepsis and chronic inflammation.
The TLR–IL-1R superfamily can be divided
into three subgroups1. The first contains
extracellular immunoglobulin (Ig) domains
and includes IL-1RI. The second is the TLRs,
which lack Ig domains, but have extracellular
leucine-rich repeats; recent years have seen
tremendous progress in determining their
function. The third subgroup consists of
upstream adapter molecules, including
MyD88, MyD88 adapter-like (Mal, also
known as TIRAP) and TIR domain–containing adaptor–inducing interferon-β (IFN-β;
TRIF, also known as TICAM-1). These
adapters are recruited to receptor TIR
domains and initiate signalling processes
through IL-1R–associated kinases (of which
there are four) and the adaptor molecule
TRAF-6, which leads to activation of four
protein kinase cascades, culminating in the
activation of NF-κB and kinases p38, JNK
and p42/p44 MAP kinase1. These molecules
in turn promote the production of many
NATURE IMMUNOLOGY VOLUME 4 NUMBER 9 SEPTEMBER 2003
proinflammatory proteins and enhance
immune reactivity. Recent evidence indicates
differences in adapter usage, such that
although almost all the receptors recruit
MyD88, only some receptors use Mal and
TRIF, leading to specificity in outcome4. The
best example is TRIF usage by TLR3 and
TLR4, which leads to the activation of
IFN-regulatory factor 3 and the induction of
IFN-β5,6. To some extent, because there has
been much more progress made in the understanding of the TLR and adapter subgroups,
the Ig subgroup has been neglected. Most
members in this subgroup remain as orphan
receptors of unknown function, the exceptions being IL-1R and IL-18R and their
respective accessory proteins, and IL-1Rrp2,
which may be a receptor for IL-1F9, a ‘paralog’ of IL-1 (ref. 7). Five other IL-1 paralogs
occur in humans and it seems likely that they
will be ligands or antagonists for the orphan
receptors8. Defining a function for SIGIRR
therefore assigns a function to one of the
orphans; it is of considerable interest that this
function is inhibitory for other members of
the protein superfamily.
Wald et al. show that SIGIRR is expressed
in various mouse tissues, including epithelial
cells in the kidney, and is highly expressed in
the colon but less so in spleen cells. Bone
marrow–derived macrophages did not express SIGIRR. Because LPS down-regulates
SIGIRR expression in epithelial cells, this
indicates that SIGIRR might be inhibitory,
consistent with the fact that SIGIRR has a TIR
domain that lacks two amino acids essential
for signaling by IL-1RI. To test this possibility,
the authors over-expressed SIGIRR in Jurkat
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