Báo cáo khoa học: Regulation of STAT signalling by proteolytic processing Lisa Hendry and Susan John doc

MINIREVIEW
Regulation of STAT signalling by proteolytic processing
Lisa Hendry and Susan John
Peter Gorer Department of Immunobiology, Programme in Infection and Immunity, King’s College London, UK
Interaction of cytokines with their cognate receptors leads
to the activation of latent transcription factors, the signal
transducer and activator of transcription (STAT) proteins.
Numerous studies have identified the critical roles played
by STAT proteins in regulating cell proliferation, differ-
entiation and survival. Consequently, the activity of STAT
proteins is negatively regulated by a variety of different
mechanisms, which include alternative splicing, covalent
modifications, protein–protein interactions with negative
regulatory proteins and proteolytic processing by pro-
teases. Cleavage of STAT proteins by proteases results in
the generation of C-terminally truncated proteins, called
STATc, which lack the transactivation domain and behave
as functional dominant-negative proteins. Currently,
STATc isoforms have been identified for Stat3, Stat5a,
Stat5b and Stat6 in different cellular contexts and biolo-
gical processes. Evidence is mounting for the role of as yet
unidentified serine proteases in the proteolytic p rocessing
of S TAT proteins, although at least one cysteine protease,
calpain is a lso known to cleave these STATs in platelets
and mast cells. Recently, studies of acute myeloid leukae-
mia a nd cutaneous T cell lymphoma patients have revealed
important roles for the aberrant expression of Stat3c and
Stat5c proteins in the pathology of these diseases. To-
gether, these findings indicate that proteolytic processing is
an important mechanism in the regulation of STAT pro-
tein biological activity and provides a fertile area for future

studies.
Introduction
The Janus kinase-signal transducer and activator of tran-
scription ( JAK-STAT) s ignalling p athway, fi rst identified
for the interferon-a/b and c receptors, is now known to be
employed by many cytokine and growth factor receptors
and to be evolutionarily conserved [1,2]. STAT proteins
have a c ommon overall structure and are organized into
distinct functional modular domains (Fig. 1).
After a decade of intense investigation into the structure
and biological functions of STAT proteins, their essential
roles in cell proliferation, differentiation and survival have
been firmly established [2]. A number of studies have iden-
tified important negative regulatory mechanisms that exist to
curtail the activity of STAT proteins (Fig. 2). These include
the activities of phosphatases, suppressors of cytokine
signalling (SOCS), interaction of inhibitory proteins such
as protein i nhib itor of activated STATs ( PIAS), and targeted
proteasome-dependent degradation of active STATs [2,3].
In addition to these direct protein–protein interaction
methods o f n egative regulation, STATs a re also regulated a t
the level of alternative splicing. The STATb forms, gener-
ated by alternative splicing, possess an altered carboxy-
terminal (C-terminal) lacking the natural transactivation
domain and behave as functional dominant-negative pro-
teins when overexpressed in cells [4–6]. However, recent
evidence from transgenic mice indicates that STATb
proteins are not strict dominant-negatives, and actually
contribute to transcriptional activation of selective target
genes, despite the absence of the natural transactivation

domain [7–9]. The mechanism by which STATb isoforms
achieve transactivation remains to be elucidated, but
probably involves the differential interaction with other
transcription factors.
Another mechanism by which STAT signalling is regu-
lated occurs at the level of limited proteolytic processing in
cellular contexts where there is no evidence for alternative
splicing [10]. Proteolytic processing of STAT proteins also
results in the generation of C-terminally truncated STAT
proteins, referred to as STATc, but these proteins lack the
transactivation domain, without the addition of any extra
amino acid sequences at their C-termini. Thus, multiple
functional forms of STAT proteins, generated by distinct
mechanisms exist in different cell lineages. Here we review
the generation and function of STATc proteins and their
role in human diseases.
Processing of Stat5 in haematopoietic
progenitor cells
Stat5 is activated by a wide variety of haematological and
nonhaematological cytokines and growth factors including
those which regulate the proliferation and differentiation of
Correspondence to S. John, Peter G orer Department of Immunobiol-
ogy, Programme in Infection and Imm unity, K i ng’s College Lo ndon,
2nd floor New Guy’s House, St. Thomas Street, London SE1 9RT,
UK. E-mail:
Abbreviations: AML, acute myeloid leukaemia; BMMC, bone mar-
row-derived mast cells; CTCL, cuta neous T cell l ymphoma; G-CS F,
granulocyte colony stimulating factor; GM-CSF, granulocyte-
macrophage colony-stimulating factor; IL, interleukin; JAK, Janus
kinase; PBMC, peripheral blood mononuclear cell; PIAS, protein

inhibitor of activated STATs; PM SF, ph enylmethanesulfonyl fluoride;
SOCS, suppressors of cytokine signalling; SS , Sezary s yndrome;
STAT, signal transducer and activator of transcription.
(Received 17 August 2004, accepted 7 October 2004)
Eur. J. Biochem. 271, 4613–4620 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04424.x
myeloid [interleukin (IL)-3, IL-5, granulocyte-macrophage
colony-stimulating factor (GM-CSF) and thrombopoietin],
erythroid (erythropoietin) and lymphoid lineages (the
gamma-c family of cytokines, IL-2, IL-7 and IL-15)
[11,12]. Targeted deletions in mice of genes encoding Stat5
results in defects in myeloid ce ll differentiation through
effects o n early haematopoietic progenitor cells [13]. The
two Stat5 proteins, Stat5a and Stat5b, are encoded by
separate genes and are e xpressed as both full-length (Stat5a)
and shorter, C-termina lly truncated proteins [5,14].
Although alternative splicing generates Stat5b in certain
cellular contexts, the lack of abundance of the alternatively
Fig. 2. Negative r egulation o f S TAT s ignalling. Cyt okine-induced STAT activation can be in hibited by suppressors of c ytokine signalling (SOCS)
proteins, whose gene expression is regulated by STAT proteins, thus fulfilling a negative feedback loop. SOCS proteins inhibit STAT activation
either by inh ibition of the a ctivating JAKs or by c o mpetition with STATs for receptor bind ing. Activated STAT proteins can b e dephosphorylated
by cytoplasmic and/or nuclear phosphatases. C-terminally truncated STAT pro teins, STATb and STATc, behave as dominant-negative proteins to
functionally compete with their full-length coun terparts to alter or inhibit gene expre ssion, respectively. Protein inhibitor of activated STATs (PIAS)
proteins interact with STAT proteins to inhibit their DNA binding and/or potentially facilitate their covalent modification by sumoylation and
subsequent degradation. Ub, ubiquitin; SUMO, small ubiquitin-like modifier.
Fig. 1. Modular structure of STAT proteins. All STAT proteins share a common molecular topology and are organized into distinct functional
domains. The NH
2
-terminus (N-domain) is involved in protein–protein interactions between adjacent STAT dimers on DNA, facilitating the
formation of S TAT tetramers. It is a lso involved in the formation of d imers between nonphosphorylated STAT monomers, which is important for
receptor-mediated activation a nd nuclear translocation of certain STAT p roteins. Interactions with STAT cofactors, which positively o r negatively

modulate their t ranscriptional activity, occur via the N-domain, the a djacent coiled -coil domain and the carboxy-terminal transactivation domain
(TAD). The con served serine residue (p-S), which is ph osphorylated upon cytokine stimulation and is impo rtant for maximal transcriptional
activation, is located within the transactivation domain. The conserved tyrosine residue (p-Y), that becomes phospho rylated upon activation is
located immediately preceeding the transactivation domain.
4614 L. Hendry and S. John (Eur. J. Biochem. 271) Ó FEBS 2004
spliced message in haematopoetic progenitor cells led
investigators to evaluate other mechanisms for the genera-
tion of C-terminally truncated Stat5 proteins.
It was noted that distinct forms of Stat5 proteins were
activated upon IL-3 treatment of specific myeloid cell
lineages. Thus, in myeloid progenitor cell lineages s timula-
tion with IL-3, GM-CSF or e rythropoietin activates a
shorter, C-terminally truncated isoform of Stat5a (77 kDa)
and Stat5b (80 kDa), while full-length Stat5a (96 kDa) and
Stat5b (94 kDa) are only activated in differentiated mature
myeloid cells [10,15]. The S tat5c proteins in myeloid
progenitor cells are generated by a putative S tat5 protease,
which is primarily located in the nucleus and cleaves Stat5
proteins indepen dently of their tyrosine-phosphorylation
states [10,16]. The protease is an endopeptidase and is
inhibited by the broad-spectrum serine protease inhibitor,
phenylmethanesulfonyl fluoride (PMSF). Cellular fraction-
ation a nd chromatography studies indicate that the protease
has an approximate molecular mass of 25 kDa and cleaves
murine Stat5a between amino acids 719 (tyrosine; Y) and
720 (methionine; M) and Stat5b between Y724 and M725
[17]. M utant Stat5 proteins bearing amino acid substitutions
at these positions were resistant to cleavage by the protease.
Importantly, the Stat5-proteolytic activity was absent in
mature myeloid cells sugg esting that either the expression of

protease is down-regulated or a lternatively inactivated upon
myeloid cell differentiation [10,16,17].
Consistent with the distinct function of truncated Stat5
proteins in immature myeloid p rogenitors, they fail to
activate several known IL-3-induced target genes that are
activated by th e full-length proteins in differ entiated mature
myeloid cells [10]. The functional significance o f truncated
Stat5 p roteins in maintaining an undifferentiated immature
phenotype of myeloid cells was convincingly demon strated
by studies using stable enforced expression of mutant,
noncleavable forms of Stat5 in undifferentiated myeloid
cells [18]. The mutant cell lines developed a partially
differentiated phenotype and were resistant t o further
differentiation by cytokine treatment. Thus, proteolytic
cleavage of Stat5 is an important physiological mechanism
in regulating myeloid cell differentiation.
Proteolytic cleavage of Stat5 in peripheral
T cells
Despite the clear role of proteases in regulating myeloid cell
development, Schindler and colleagues were unable to
demonstrate an analogous situation for lymphoid cell
development i n m urine t hymic T cells [17]. However, studies
of human peripheral blood mononuclear cells (PBMCs)
indicate that naı
¨
ve T cells in the peripheral immune system
possess a similar mechanism f or regulating Stat5 a s myeloid
progenitor cells. Activation of naı
¨
ve T cells by antigen ic or

mitogenic stimulation leads to cell proliferation and differ-
entiation into effector T cells, m ediated b y the action of
immunologically important cytokines, which signal via
Type I and Type II cytokine receptors. Stat5 a ctivation,
mediated by IL-2 signalling upon T cell activation, is an
important regulator of cell proliferation a nd survival [19,20].
Recently, studies on Stat5 expression and activation in
normal human PBMC and peripheral T cells revealed that
Stat5 is expressed exclusively as a truncated protein in the
nucleus of naı
¨
ve PBMC/T cells [21]. Analysis of the
truncated Stat5 proteins using N- and C-terminal Stat5
antibodies revealed that the t runcation is at the C-terminus
of the Stat5 protein, as previously noted in myeloid cells.
The expression of the t runcated protein in the nucleus i s
independent of the phosphorylation state of Stat5a and
Stat5b. Unlike myeloid progenitor cells, the cytoplasmic
fraction expresses both the full-length and the truncated
Stat5 protein, although a t present we cannot exclude the
possibility t hat the truncated protein is exclusively generated
in the nucleus but is present in the cytoplasmic fraction due
to protein shuttling, which has been shown to occur in a
cytokine-dependent and independent manner for STAT
proteins [22,23].
Upon activation of naı
¨
ve T cells by mitogenic stimula-
tion, the expression of truncated Stat5a and Stat5b proteins
disappears and is replaced by the expression and activation

of the f ull-length Stat5 p roteins [21]. S ignificantly, the
normal regulation of truncated vs. full-length Stat5 is
dysregulated in cutaneous T cell lymphoma (CTCL)
patients and will be described in a later section. Ongoing
studies indicate th at the truncated Stat5 protein is generated
by the activity of a protease, which is down-regulated o r
inactivated upon mitogenic stimulation (Fig. 3). Future
biochemical characterization and purification of the prote-
ase(s) a nd the identification of t he exact c leavage site on
Stat5 will be important in enhancing our understanding of
the regulation of Stat5 function by proteolytic cleavage in
peripheral T cells.
Proteolytic regulation of Stat5 and Stat3
in mature human neutrophils
Stat3 and Stat5 isoforms have been identified in differen-
tiated human peripheral blood monocytes and polymor-
Fig. 3. Stat5a protein is cleaved to Stat5c by the activity of a protease
present in peripheral blood mononuclear cell (PBMC) extract. The
presence of Stat5-proteolytic activity was evaluated by coinc ubation
assay. Extracts prepared from either PBMC (lane 2) or PBMC mito-
genically stimulated with phytohaemagglutinin ( PHA-Blasts, lane 3),
or a buffer control containing no cell extrac t (lane 1), were incubated
with FLAG-tagged Stat5a protein at 37 °C for 15 min. Samples were
then analyzed by Western blot analysis using an anti-FLAG IgG.
Cleavage of the FLAG-Stat5a input protein was obtained specifically
with fresh PBMC extrcats and not with extracts made from PHA-
Blasts.
Ó FEBS 2004 STAT signalling by proteolytic processing (Eur. J. Biochem. 271) 4615
phonuclear neutrophils [24–27]. During t erminal differen-
tiation of n eutrophils, induced by granulocyte c olony

stimulating factor (G-CSF), the main STAT that is
activated is Stat3 and it is predominantly expressed as
Stat3c, generated by proteolytic cleavage of Stat3a [25,28].
Unlike the progenitor myeloid Stat5 protease, the Stat3
protease, activated by G-CSF can only cleave the a ctive,
phosphorylated form of Stat3a [25]. The exact specificity
of the Stat3 protease appears to be less clear, as the
proteolytic activity was shown to be inhibited by di-
isopropylfluorophosphate and not PMSF in living cells,
but neither was effective at inhibiting the protease in v it ro
[25]. The relationship between the S tat3 protease from
mature neutrophils and the Stat5 protease from immature
myeloid cells is also unknown at present, but the activation
of these proteases in different developmental contexts may
suggest that they are distinct proteases.
More recently, investigators have shown that Stat5 is
also similarly regulated by proteolytic processing in mature
human neutrophils [26]. Stat5 is activated in human
neutrophils by the cytokines IL-2 and GM-CSF, which
are both potent modulators of neutrophil activity [29]. In a
now familiar theme, these cytokines activate nuclear
expression of a C-terminally truncated form of Stat5 in
neutrophils, w hich results in a failure to induce expression of
known Stat5-regulated genes, such as osm and pim-1,
consistent with the inability of these cytokines t o induce
proliferation of these cells [26]. No evidence was found for
alternative splicing of S tat5 in the se cells and i nstead
truncated Stat5 proteins were generated by the activity of a
nuclear, PMSF-sensitive serine protease. The exact rela-
tionship between the various Stat5-serine proteases derived

from myeloid progenitors, human PBMC and mature
neutrophils awaits identification by future molecular c lo-
ning studies.
Regulation of Stat6 activity by proteolytic
cleavage in mast cells
Unlike Stat3 and Stat5, which are activated by a wide
variety of cytokines and growth factors, Stat6 is very
selectively activated by IL-4 and the related cytokine, IL-13
[30]. Stat6 deficient m ice reveal defects i n such crucial
aspects of normal immune function as Th2 cell differenti-
ation, B cell isotype switching and the loss of contact
hypersensitivity [30]. While IL-4 induced Stat6 signalling is
an activating signal in murine B a nd T cells, its role in bone
marrow-derived mast cells (BMMC) is less clear [31,32].
Analysis of Stat6 expression in murine BMMC provided a
clue to these apparent cellular differences in response to
IL-4. Brown and colleagues first observed that, whereas
Stat6 is expressed as a 100 kDa full-length protein in B and
T c ells, it is expressed as a 65 kDa protein i n murine B MMC
[33]. A similarly truncated Stat6 protein has not been
identified in human mast cells and it i s possible that this
mechanism of regulation of Stat6 has been lost during
evolution. Studies revealed that Stat6 is truncated at its C-
terminus and is lacking the transactivation domain in
murine BMMCs [33]. While no evidence for alternative
splicing of Stat6 was obtained in mast cells, several groups
have established that truncated Stat6 protein is generated
by proteolytic processing in these cells [33–35].
The activity of the murine Stat6 protease is exclusively
nuclear and can be inhibited b y t he serine protease

inhibitors, PMSF and 4-(2-aminoethyl)-benzenesulfonyl-
fluoride [35]. Moreover, the a ctivity of the Stat6 protease is
not dependent on the expression of Stat6, a s Stat6-deficient
BMMC also contained Stat6-specific proteolytic activity
[35,36]. More recently Iwamoto and c olleagues have further
characterized the serine protease to be inhibited by an
elastase inhibitor ONO-5046, suggesting that this protease
may belong to an elastase family [36,37]. The Stat6 p rotease
cleaves Stat6 between amino acids 685 (aspartic acid; D)
and 686 (methionine; M). The amino acid sequences
surrounding the cleavage site are not conserved in the
human Stat6 protein, providing an explanation for the lack
of observation of truncated Stat6 in human mast cells.
While cleavage-resistant point mutants of Stat6 (Stat6
D685A and M686A) have similar transcriptional activity as
their wild-type counterpart in cell transfection assays, the
stable expression of these Stat6 mutants in cell lines results
in prolonged nuclear accumulation of Stat6 and enhanced
IL-4-induced ap optosis and growth inhibition of the mutant
mast cell lines [35]. Furthermore, enforced coexpression of
truncated Stat6 with Stat6 D685A reverses the functional
effect of the l atter mutant indicating that the truncated Stat6
protein can potentially function as a dominan t-negative in
BMMC [35].
Despite the finding that both the Stat5 protease and the
Stat6 protease a re serine proteas es, the s imilarity apparently
does not extend any further. The Stat5 protease from
myeloid cells does not cleave Stat6 and is not inhibited by
ONO-5046, and the Stat6 protease from BMMC does not
cleave Stat5 [35–37]. Thus, t he serine proteases that r egulate

STAT activity show STAT and cell-type specificity.
Processing of Stat3, Stat5 and Stat6 by calpain
While the most common mechanism o f proteolytic process-
ing of STAT proteins is mediated by the action of serine
proteases, at least one other cellular protease is known to
specifically cleave certain STAT proteins. The calcium-
dependent cysteine protease calpain w as demonstrated to
cleave Stat3 and Stat5 in platelets and Stat6 in mast cells to
generate C-terminally truncated proteins [37,38]. Activation
of intracellular calpain by thrombin treatment of platelets
resulted in a significant i ncrease in the levels of C-terminally
truncated Stat3 and Stat5 [38]. Similarly, Stat6 was cleaved
upon activation of calpain by dibucaine treatment of
BMMC [37]. However, the truncated Stat6 protein that is
generated as a result of cleavage by calpain is a 70 kDa
protein as c ompared to t he 65 kDa protein generated by the
Stat6 protease. Furthermore, the generation of the 70 kDa
but not the 65 kDa Stat6 protein is inhibited by the calpain
inhibitor calpeptin [37]. Thus, multiple different STATc
isoforms can be generated by the activation of different
cellular proteases in BMMCs. It is unclear whether the
calpain cleaved Stat5 in platelets is identical in size and
function to the Stat5c proteins generated by proteolytic
processing by the Stat5 proteases from myeloid progenitor
or mature neutrophil cells. The physiological importance of
STATc isoforms generated by calpain is unknown at
present but, as calpain is potently a ctivated by increased
intracellular calcium concentrations following cellular
4616 L. Hendry and S. John (Eur. J. Biochem. 271) Ó FEBS 2004
activation, it is plausible t hat c alpain mediated processing of

STAT proteins may b e an i mportant mechanism f or
regulating STAT-dependent gene expression [39].
Dysregulated expression of proteolytically
processed STAT proteins in human diseases
The constitutive activation of full-length Stat3 and Stat5 is a
common f eature of many primary human tumours of
haematopoietic and nonhaematopoeitic origins and is
extensively reviewed elsewhere [40,41]. Recent studies of
acute m yeloid leukaem ia (AML) and CTCL patients
indicate that C-terminally truncated STAT proteins also
contribute to the pathology of these diseases.
Acute myeloid leukaemia
AML is characterized by the clonal expansion of myeloid
cells that have been arrested in their maturation. Like their
normal counterparts, AML b lasts can proliferate in
response to haematopoietic cytokines such as GM-CSF,
G-CSF, thrombopoietin and IL-3, which signal via the
JAK-STAT pathway [42]. H owever, unlike no rmal myeloid
cells, which undergo differentiation in response to specific
cytokine treatment, the leukaemic cells proliferate but do
not differentiate, sugge sting th at crucial s ignalling pathways
that regulate cell proliferation and differentiation may be
dysregulated in this disease. Analysis of a number of bone
marrow samples from pretreatment AML patients revealed
that  20–30% of AML b lasts expressed constitutively
activated full-length Stat3 and Stat5 proteins but a much
higher proportion ( 80%) expressed C-terminally trun-
cated Stat3 and Stat5 proteins [43]. Moreover, 94% of
patients in relapse expressed truncated STAT proteins
compared to 35% of patie nts with constitutively active full-

length STAT proteins, suggesting that the expression of
truncated Stat3 and Stat5 proteins may contribute adversely
to disease p rogression [44]. Nevertheless, the shortest
disease-free survival rate and overall survival was seen in
patients that had both constitutive activation of full-length
Stat3 and concurrent aberrant expression of truncated Stat3
[45]. These studies suggest that the relative ratio o f full-
length : truncated STAT protein may influence the out-
come of disease progression. Constitutive expression of
C-terminally truncated Stat5 proteins have also been
described previously in CD4 T cells from HIV patients
undergoing antiretroviral monotherapy or IL-2 treatment
and was associated with good response to therapy [46].
However, it is not known whether the truncated Stat5
protein in patient cells is generated by proteolytic activity or
by alternative splicing.
Biochemical characterization of the AML samples that
contained truncated STAT proteins, revealed t hat a pro-
teolytic activity was e xpressed in these samples, which could
selectively cleave Stat3 and Stat5, but not Stat6 [47]. The
serine protease inhibito r PMSF w as able to inhibit the
activity of the Stat3/5 prote ase from AML blasts, as
previously observed for progenitor myeloid cells. However,
this serine protease differed from that present in immature
myeloid cells in that it was present in both cytoplasmic and
nuclear fractions and chromatographic analysis of the
protease from AML blasts yielded a protein of approximate
molecular mass of 40 kDa. Thus, the active protease in
AML b lasts m ay either represent yet another member o f t he
STAT-serine protease family or alternatively may be

aberrantly post-translationally modified. Given the clearly
established dominant-negative functions of C-terminally
truncated STAT proteins, the aberrant constitutive expres-
sion of truncated Stat3 and Stat5 proteins in AML blasts
has important physiological implications for the pathology
of the disease. As cleavage-resistant mutant Stat5 proteins
induce differentiation and apoptosis of myeloid cells when
artificially expressed, it is plausible to speculate that the
selective expression of truncated Stat5 and Stat3 proteins
may enhance survival of leukaemic blasts cells in AML,
while at the same time preventing cellular differentiation
[18].
Cutaneous T cell Lymphoma
Primary CTCLs are one of the most frequent extranodal
lymphomas affecting the skin, and include mycosis fun-
goides and its leukaemic variant Sezary syndrome (SS) [48].
Tumour cells are typically CD4 T cells, which display a
memory activated phenot ype, and express Th2-like c yto-
kines (IL-4, IL-5 and IL-10) [49]. While the J ak3-Stat3
pathway is constitutively activated in SS, Stat5 activation is
inducible [21,50]. R ecently, a s tudy of SS patients with
advanced stage disease identified a different form of
dysregulation of Stat5 [21]. As mentioned earlier, Stat5 is
regulated by proteolytic processing in normal PBMC.
Analysis of PBMC from SS patients showed that, unlike
in healthy controls, there was elevated or exclusive expres-
sion of the C-terminally truncated Stat5 protein even in
potently activated cells. DNA binding studies revealed that
in SS patients, truncated Stat5 proteins are activated upon
IL-2 stimulation and preferentially bind to known S tat5

binding sites, even in patients where a mixture of full-length
and truncated Stat5 proteins are expressed. Consistent with
these findings, there was a loss of I L-2-induced Stat5-
dependent gene expression of target genes such a s pim-1, cis ,
and bcl-2 in patient samples. However, the Stat5-regulated
gene CD25 was still inducible by IL-2, consistent with
findings from other studies, which indicated that constitu-
tively activated Stat3 aberrantly regulates CD25 expression
in SS [50]. Thus, it seems likely t hat the regulation of other
important target genes, which a re s hared by Stat5 a nd Stat3
may be similarly dysregulated in SS. Future studies
investigating t he repertoire of target genes activated by
full-length vs. truncated Stat5 proteins in T cells will enable
us to better understand the functional differences between
the d ifferent forms o f S tat5 and t heir potential d ysregulation
in SS. Nevertheless, the preferential DNA binding of
truncated Stat5 proteins and the concomitant loss of
Stat5-dependent gene expression in SS patients demon-
strates that t runcated Stat5 proteins can behave as physio-
logical dominant-negatives.
Ongoing studies indicate that the dysregulated activity of
a Stat5 protease may be responsible for the elevated
expression of C-terminally truncated Stat5 proteins in SS
(L. Hendry and S. John, unpublished observations). Given
the critical role of IL-2 i nduced Stat5 signalling in normal
immune homeostasis and the maintenance of peripheral
tolerance, the loss of this pathway has important
Ó FEBS 2004 STAT signalling by proteolytic processing (Eur. J. Biochem. 271) 4617
implications for the pathogenesis of SS [20,51]. Thus,
sustained expression of C-terminally truncated Stat5 pro-

teins may be one m echanism adopted by indolent malignant
T cells in SS to escape apoptosis.
Conclusions and perspectives
Evidence is accumulating to suggest that proteolytic
processing is a general mechanism fo r the negative regula-
tion of STAT protein function (Fig. 4). Truncated forms of
Stat3, Stat5a, Stat5b and S tat6, g enerated by t he proteolytic
cleavage of t he C-termini, have been identified in progenitor
myeloid cells, mature neutrophils, mast cells and peripheral
T c ells. STATc proteins have different C -termini than
STATb proteins and behave as functional dominant-
negative proteins. Of the STAT proteases that have been
characterized most are serine proteases, whose activities are
regulated by the developmental or activation state of cells
depending on the cellular context. However, their expres-
sion and functional activities are not dependent on the
presence of the targe t S TAT protein itself and it i s likely that
other cellular targets exist f or these proteases. The exact
identities and mechanism o f action of the individual
proteases are currently unknown but they show STAT
and cell-type specificity. Future cloning of the proteases
from the different cell sources will reveal whe ther they
belong to a family of related serine proteases. In addition,
the cysteine protease, calpain, has also been shown to
process Stat3 and Stat5 in platelets and Stat6 proteins in
mast cells, respectively, although the physiological import-
ance of these findings are unknown. Truncated Stat3c and
Stat5c proteins generated b y proteases have been shown to
contribute s ignificantly to the pathology of AML and
CTCL. Thus, future identification of the relevant serine

proteases a nd their natural inhibitors from myeloid c ells and
T cells will enhance our understanding of these diseases and
also provide potential targets for therapeutic intervention b y
the rational design of drugs based on these proteins.
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Fig. 4. Proteolytic processing of STAT proteins. STAT proteins may be cleaved at the C-terminus by the action of nuclear (A1,A2; progenitor
myeloid cells, mature neutrophils, murine BMMC) and/or cytoplasmic (B1,B2; AML blasts, human PBMC) proteases. The activities of the
proteases are generally not dependent on STAT-ph osphorylation and therefore the protease can cleave activated (A1,B1) or unactivated STAT
proteins (A2,B2). Unactivated, fu ll-length STAT and STATc proteins can also s huttle between the cytoplasm and the nucleus in the absence of
cytokine stimulation . Th e t ru ncated S TATc protein lacks th e transactivation d o main a nd behaves a s a dominant-negative protein to functionally
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