Báo cáo khoa học: Nucleocytoplasmic shuttling of STAT transcription factors Thomas Meyer and Uwe Vinkemeier doc

MINIREVIEW
Nucleocytoplasmic shuttling of STAT transcription factors
Thomas Meyer and Uwe Vinkemeier
Abteilung Zellula
¨
re Signalverarbeitung, Leibniz-Forschungsinstitut fu
¨
r Molekulare Pharmakologie, Freie Universita
¨
t Berlin, Germany
The s ignal transducer an d activator of transcription (STAT)
proteins have initially been described as cytoplasmic proteins
that enter the nucleus only after cytokine treatment of cells.
Contrary to this assumption, it was demonstrated that
STATs a re constantly shuttling between nucleus and cyto-
plasm i rrespective o f c ytokine stimulation. This happens
both via carrier-dependent as well as carrier-independent
transportation. Moreover, it was also recognized that cyto-
kine stimulation triggers nuclear retention of dimeric
STATs, rather than affecting the rate of nuclear import. In
summary, it is increasingly being appreciated that STAT
nucleocytoplasmic cycling determines the quality of cytokine
signaling and also constitutes an important area for micro-
bial intervention.
Introduction
Multicellular organisms utilize a n integrated n etwork of
cell–cell communications and humeral interactions to
coordinate complex cellular processes such as proliferation,
differentiation, and homeostasis. Cells recognize external
stimuli and transfo rm the signals i nto a cellular response,
which most often result in an alteration in the pattern of

expressed genes. Many s ignal transducers that f unction as
transcription factors have to traverse the barrier of the
nuclear envelope in order t o gain a ccess to specific target
genes within the nuclear compartment. The Janus kinase
(JAK)-signal tr ansducer and activator of transcription
(STAT) pathway is regarded as a paradigmatic model f or
such a direct signal transduction, because it transmits
information received from e xtracellular polypeptide signals
without the interplay of second messengers directly to target
promoters in the nucleus [1].
The STAT proteins comprise a family of evolutionarily
conserved t ranscription factors and in mammalian cells
seven known STAT proteins were identified, den oted
STAT1, STAT2, STAT3, STAT4, STAT5a, S TAT5b, and
STAT6, all of which are activated by a distinct set of
cytokines a nd growth factors [1]. T hese proteins consist o f
several conserved functional domains. The amino terminal
N-domain is responsible for t etramerization of all STATs
(with the probable exception of STAT2), and this domain
also regulates receptor recognition and phosphatase
recruitment for some STATs [2–5]. The N-domain is
followed by a coiled-coil domain implicated in protein–
protein interactions [6], a DNA binding domain [7], a
linker domain that participates in DNA binding [8], an
SRC homology 2 (SH2) domain that mediates dimeriza-
tion and receptor binding [9], and a carboxy-terminal
transactivation domain [10].
Best characterized is the role of STAT proteins in
cytokine signaling. Upon binding of extracellular ligands
such as interferons or interleukines to their cognate

receptors, re ceptor-asso ciated Janus kinases, of w hich four
have been described in mammalian cells (JAK1, JAK2,
JAK3 and T YK2), undergo t yrosine a utophosphorylation
and transphosphorylate tyrosine-containing motifs on the
intracellular receptor chains, thus creating docking sites for
the SH2 domain of STAT molecules [11]. Subsequently,
the JAKs catalyze the phosphorylation o f a single tyrosine
residue in the c arboxy t erminus of STAT proteins [10,12].
The tyrosine-phosphorylated STATs detach from the
intracellular receptor tail and homo- or heterodimerize
due to reciprocal phosphotyrosine-SH2 interaction ([1] and
Fig. 1). Before exposure of cells to cytokines the STAT
molecules are nontyrosine phosphorylated, but may assem-
ble into dimeric and higher order complexes [13,14].
Structurally and functionally these aggregates remain
sparsely characterized. Therefore, throughout this review
we will use the term ÔdimerÕ as shorthand for Ôtyrosine-
phosphorylated dimerÕ.
A characteristic but until recently poorly understood
phenomenon associated with cytokine stimulation o f cells
is the inducible and transient accumulation of STAT
proteins [10]. Once in the nucleus, STAT dimers can
directly bind to nonameric DNA sequences known as
gamma-activated sites (GAS) in the promoter region of
cytokine-responsive genes resulting in gene transcription
[7]. Several years ago, Yoneda and coworkers s howed that
cytokine stimulation with concomitant dimerization of
tyrosine-phosphorylated STATs induces their association
with importin t ransport factors [15]. Next, we will describe
what is presently known about the m olecular basis o f t his

process.
Correspondence to U. Vinkemeier, Abteilung Zellula
¨
re Signalverar-
beitung, Leibniz-Forschungsinstitut f u
¨
r Molekulare Pharmakologie,
Freie Universita
¨
t Berlin, Robert-Ro
¨
ssle-Str. 10, 13125 Berlin,
Germany. Fax: +49 30 94793 179, Tel.: +49 30 94793 171,
E-mail:
Abbreviations: CRM1, chromosomal region maintenance 1; dsNLS,
dimer-specific nuclear localization signal; GAS, gamma-activated
sites; JAK, Janus kinase; NLS, nuclear localizati on signal; NPC,
nuclear pore complex; S H2, SRC homology 2; STAT, signal
transducer and activator of transcription.
(Received 18 August 2004, accepted 7 October 2004)
Eur. J. Biochem. 271, 4606–4612 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04423.x
Requirements for cytokine-induced nuclear
import of STATs
Macromolecules and ions alike have to traverse the nuclear
membrane through specialized structures called nuclear
pore complexes (NPCs) [16]. The NPCs constitute high-
order octagonal c hannels that are an integral part of the
nuclear envelope. They are composed of proteins called
nucleoporins which are present in multiples, and s ome of
them contain hydrophobic phenylalanine/glycine (FG)-rich

repeat motifs [16]. Macromolecules exceeding a molec ular
mass of  40 kDa are generally barred from freely crossing
the nuclear membrane by random d iffusion [17]. Thus,
the NPCs function as selectivity filters by r estricting the
transport of some mac romolecules, while allowing the rapid
translocation of o thers.
Detailed mechanistic insight has been acquired into
translocation mechanisms that rely on tra nsport receptors
of the karyopherin superfamily of proteins [18]. Karyophe-
rins mediate either import into or export from the nucleus
and t hey are therefore also c alled i mportins or exportins,
respectively. They recognize loosely conserved sequence
motifs on the surface of their substrates (also called cargoes).
These signals allow the association with cargo proteins and
the subsequent passage of the complex through the nuclear
pore. Importins and e xportins, although structurally rela-
ted, differ in their sequence r equirements for cargo associ-
ation, as nuclear localization signals (NLS) are usually rich
in basic residues, while nuclear export signals are charac-
terized by the presence of hydrophobic residues, usually
leucines [19]. It is believed that the karyopherins act as
chaperones during nucleocytoplasmic translocation. Pas-
sage through the pore appears to require weak and transient
binding to th e nucleoporin FG repeats, an inter action that
by itself was shown to occur independently o f metabolic
energy [20,21]. Energy consumption, however, confers
directionality to this process, which therefore was also
termed active transport. The driving force behind the active
translocation is created by Ran-GTPase nucleotide
exchange factors, which are distributed asymmetrically

between cytosol and nucleus [22]. Nucleotide hydrolysis
by RanGAP, the cytoplasmically localized RanGTPase-
activating protein, r esults in high levels of RanGDP in the
cytosol. In the presence o f RanGDP, i mportins are loaded
with substrates and may translocate through the NPC into
the nucleus, wh ile the export receptors are liberated from
their cargo molecules in this environment. The reverse
reactions take place in the nucleus. Here, a h igh RanGTP/
RanGDP ratio is maintained by the guanine nucleotide
exchange factor RCC1, which catalyzes t he conversion of
RanGDP to RanGTP. RanGTP was demonstrated to
promote both the disassembly of importin/cargo complexes
and the association of exportins such as chromosomal
region maintenance 1 (CRM1) wit h their cargoes [19].
At present, the overwhelming majority of examples of
protein nucleocytoplasmic shuttling belong to this active
mode of translocation. STAT proteins have also been
demonstrated to utilize components o f this Ran-dependent
nuclear import m achinery [15,23]. The karyopherin i mpor-
tin b (p97) has been identified as the c arrier that transports
importin a complexed with STATs into the nuclear
compartment ([15,23] and Fig. 2A). In interferon-stimula-
ted cells dimerized STAT1 and STAT2 bind directly to
importin a5 (NPI-1/hSrp1), a karyopherin t hat contains
Fig. 2. STATs at the nuclear envelope. (A) Carrier-dependent import.
Phosphorylated S TAT dimers expose a dimer-specific n uclear local-
ization signal and associate w ith importin a. Through importin
b-mediated interactions w ith the interio r of the nuclear po re (NPC)
this complex migrates into the nucleus. The complex disassembles after
the bindung of RanGTP. The exact stoich iometry and order of events

have not b een established. (B) Carrier-dep endent export. Unphos-
phorylated S TATs can bind to the exp ortin C RM1 via leucine-rich
nuclear export signals and traverse the NPC. RanGTP enhances the
interaction of CRM1 with cargo proteins. In the cytoplasm, the nuc-
leotide hydrolysis of RanGTP leads to release of the cargo. (C) Carrier-
independent nucleocytoplasmic translocation. For the STATs, the
majority of translocation events occur via direct interactions with
proteins of the nuclear pore. The resulting nucleocytoplasmic cycling
proceeds independen tly of met abolic energy.
Fig. 1. STATs at the c ell me mbrane . A schematic representation of the
events lead ing to the tyro sine phosphorylation (activation) of S TATs.
The activation of receptor-associat ed JAK kinases after cytokine sti-
mulation results in tyrosine phosphorylation of the receptor. The
STATs dock to these sites via their SH2 domains and become tyrosine
phosphorylated conc omitan tly. The a ctivated STATs de tach and
homo- or heterodim erize.
Ó FEBS 2004 Nucleocytoplasmic shuttling of STAT transcription factors (Eur. J. Biochem. 271) 4607
10 armadillo repeats [15,24,25]. Only the very C-terminal
armadillo repeats 8 and 9 bind to STAT1 homodimers
and STAT1-STAT2 heterodimers, whereas classical NLS
sequences interact wi th repeats 2–4, 7 and 8 [26].
The binding site for importin a5 o n the STAT1 dimer has
been mapped to an unusual dimer-specific nuclear localiza-
tion signal (dsNLS) within the DNA binding domain
[24,25]. The homologous sequence in the DNA binding
region of STAT3 was later r eported to a lso function as an
NLS for the dimer [27]. It is interesting to note that binding
of STATs to importin a5 does not appear to pose an
obstacle to promoter binding and transcription, as STAT-
target DNA can disrupt the i mportin a5 complex with

STAT1 [25]. The dsNLS differs from conventional import
signals in some respects (Fig. 3). First, it does not resemble
the consensus sequence of classical mono- or bip artite
NLSs, which consist of one or two arginine/lysine-rich
clusters of basic a mino acids s eparated by a spacer region
ranging from 10 to up to about 40 residues [28,29]. The
STAT1 dsNLS, in contrast, contains only a few positively
charged residues. Another distinguishing feature of the
STAT dsNLS i s its nontransferability, because i t functions
only i n t he context of t he STAT dimer, but not autonom-
ously as is typical for conventional NLSs [28,29]. In
addition, the STAT a mino termini a lso a ppear to p rovide
signals for the c ytokine-inducible nuclear localization as
judged from the inability of a mino terminal deletion
mutants to a ccumulate in the nucleus [30]; and residues i n
the coiled-coil domain seem to contribute to carrier-
dependent nuclear import of some S TATs [27].
The c anonical m odel of the JAK-STAT pathway stated
that unphosphorylated STATs a re cytoplasmic and do not
participate in nucleocytoplasmic shuttling. However, this
model has been challenged by the observation that some
STAT family members undergo constitutive shuttling
between the nuclear and cytosolic compartments even in
the absence of cytokine stimulation. A growing body of
evidence indicates that the nucleocytoplasmic cycling of
STAT proteins is much more dynamic than initially
thought. In the following we will describe and discuss the
recent a dvances, which make necessary a fresh look at the
principles of cytokine signaling.
Continuous nucleocytoplasmic cycling

of STATs
Loss-of-function mutations of the STAT1 dsNLS block
nuclear entry of tyrosine-phosphorylated STAT1 [29]. As
anticipated, the dsNLS mutants failed t o activate interferon-
inducible STAT target genes despite their unperturbed
dimerization and DNA binding abilities. Moreover, the
import defect was associated also with the loss of cytokine-
induced nuclear accumulation. Despite that, ample amounts
of unphosphorylated dsNLS m utants of STAT1 were found
in the nucleus of unstimulated cells [29]. This w as taken a s
the first indication that unphosphorylated STATs used
nuclear import mechanism(s) that deviated from the
importin-dependent translocation described for the phos-
phorylated dimer. Further hints came from the observation
of nuclear pools o f monomeric STAT1 and STAT3 in a
variety o f unstimulated primary cells or established cell lines
[31,32]. P oint mutations in either the SH2 domain or the
tyrosine residue in position 701 that completely prevented
the signal-dep endent dimerization had no effect on the
intracellular STAT1 localization in resting cells [31,32]. The
direct visualization of STAT1 nucleocytoplasmic shuttling
in re sting cells was made possible by the intracellular
microinjection of precipitating anti-STAT1 IgG [29]. Strik-
ingly, upon the microinjection of a specific antibody, but not
of an unspecific immunoglobulin, STAT1 was depleted
from the noninjected compartment [29]. This assay was used
to perform time-course experiments to assess the nucleo-
cytoplasmic flux rates of e ndogenous STAT1 i n unstimu-
lated cells [33]. It was found that the antibody-induced
STAT1 clearance was rapid and complete in about 30 min,

irrespective of w hether the a ntibody was i njected into the
cytoplasm or the nucleus (Fig. 4A–C). Moreover, while
energy-depletion of cells precluded nucleocytoplasmic trans-
port of karyopherin-dependent cargo proteins, the unphos-
phorylated STAT1 continued to exchange between nucleus
and cytosol under this condition [33]. Thus, constitutive
nucleocytoplasmic shuttling continued in the absence of
metabolic energy and an intact RanGTP gradient. High
exchange rates between the nuclear and cytoplasmic STAT
pools were r eported also for STAT3 a nd STAT5 [ 34,35].
These findings were complemented by import assays with
digitonin-permeabilized cells that retain an intact nuclear
envelope, but which are devoid of cytoplasmic proteins such
as importins [36]. These experiments revealed that exclu-
sively unphosphorylated STAT1 c ould enter the nucleus in
the absence of cytosolic proteins, whereas tyrosine-phos-
phorylated STAT1 dimers required both metabolic energy
and added cytosol for nuclear import. Identical observa-
tions were also made for unphosphorylated STAT3 and
STAT5 [ 33]. Moreover, it was found that the carrier-free
transport i s s aturable and appears to o ccur t hrough d irect
contacts between STAT proteins and FG repeat-containing
nucleoporins [33]. Interestingly, in vitro alkylation with
N-ethyl-maleimide of a single cysteine residue in the STAT1
linker domain precluded the translocation across the
nuclear membr ane, s uggesting that the functionally poorly
characterized linker domain plays a fundamental role in
carrier-independent nucleocytoplasmic shuttling [33].
Although the structural details that determine the carrier-
free passage of STATs through t he nuclear pore r emain to

Fig. 3. The dimer-specific nuclear import signal (dsNLS) of STAT1. A
short stretch from the DNA binding domain of STAT1 harbors
overlapping e xport and import activities. Notably, the import activity
is observed only in the native STAT dimer, whereas the exp ort activity
is readily observable in the isolated peptide. Residues that were dem-
onstrated to b e imp ortant for e xport (of isolated peptides) are depicted
in a white box, residues that are required on ly for import (of th e dimer)
are boxed in dark grey. Residues, mutation of which affected both
import and export, are shown in a ligh t grey box. For comparison, the
homologous sequence s of other STATs are listed: D, Drosophila;
h, human.
4608 T. Meyer and U. Vinkemeier (Eur. J. Biochem. 271) Ó FEBS 2004
be established, it was s hown that truncated STAT mutants
that lack the amino- and carboxy-termini entered the
nucleus with identical kinetics as the full-length molecule.
The nuclear export rate of these truncation mutants, on the
other hand, was reduced [33], which indicated that the
structural requirements are complex and possibly affect
transport in a direction-specific manner. Taken together,
STATs use two different import pathways: before cytokine
stimulation, unphosphorylated STATs migrate via a car-
rier-free mechanism that involves direct interactions with
nucleoporins. Nuclear import of tyrosine-phosphorylated
STAT dimers, on the other hand, is dependent on impor-
tins, Ran, and metabolic energy. Both pathways operate
simultaneously in cytokine-stimulated cells and it a ppears
that phospho rylation-induced dimerization is the s witch
from fac ilitated diffusion to carrier-mediated t ranslocation
(Fig. 2). Notably, only one third of the STAT1 molecules
are tyrosine phosphorylated at any moment during cytokine

stimulation [37].
Work in our l aboratory identified a functional leucine-
rich nuclear e xport signal i n S TAT1 and d emonstrated its
role in vivo, t hus showing that nuclear export of STAT1 was
occurring [38]. In the me antime, further putative leucine-
rich nuclear export signals have been identified in varying
locations in STAT1 [39], STAT3 [ 40], and STAT5 [35], a s
well as in Dictyostelium STATa [41], and STATc [ 42]. Of
note is the fact that characterization of the STAT export
signals remains inc omplete, as export a ctivity in the full
length molecule has not been demonstrated yet for some of
them. Interestingly, a biphasic regulation was described for
STATa in which extracellular cAMP initially directs nuclear
import of tyrosine-phosphorylated STATa and phosphory-
lation of amino terminal serine residues catalyzed by
glycogen synthase kinase-3 promotes its subsequent export
[41]. This raises the intriguing possibility of flux modulations
via post-translational modifications also for mammalian
STATs. However, the respective phosphorylation sites are
not conserved.
While the CRM1-mediated nuclear export was initially
implicated only in the termination o f cytokine-induced
nuclear accumulation of STATs, it is now clear that t his
export pathway operates constitutively [33]. P reincubation
of resting cells with the CRM1 inh ibitor leptomycin B
did not cause the nuclear accumulation of STAT1, which
by some was taken as an indication that STATs do not
shuttle i n r esting cells [39]. I n addition, it was noted that
leptomycin merely attenuated the cytoplasmic r elocation
after cytokine-induced nuclear accumulation, but did not

cause a complete block [38]. As described above, th is
phenotype is explained by the existence of a carrier-
independent an d h ence leptomycin-insensitive nuclear
export mechanism [33]. STATs are predominantly cyto-
plasmic in resting cells, although STAT- and cell type-
specific differenc es were reported [32]. For STAT1, the
underlying molecular mechanism was d etermined t o e ntail
the cooperative action of both the carrier-free and the
CRM1-dependent translocation mechanism (Fig. 2B,C).
It was found that inactivation specifically of CRM1 or
STAT1
Hoechst
FITC-BSA
A
B
C
D
Fig. 4. Nucleocytoplasmic s huttling o f STAT1
in resting and cytokine-stimulated cells. Anti-
body microinjection assays with an un specific
STAT3 antibody (A) or a specific STAT1
antibody (B–D). After antibo dy injection the
cells were incubated for 30 min at 37 °C,
before fixation and immunocytochemical
detection of endogenou s STAT1. The site of
injection was marked by the coinjection of
fluorescine-conjugated bovine serum albumin.
Arrows point at the injected cells. The control
in (A) demonstrated that the ST AT1 distri-
bution is not affected by microinjection of an

unspecific ant ibod y. Th e injectio n of a
STAT1-specific a ntibody reveale d the consti-
tutive cycling of STAT1 in resting cells (B,C).
Cytoplasmic injection of anti-STAT1 depleted
endogenous STAT1 from the nucleus (B),
whereas nuclear delivery of anti-STAT1
caused STAT1 accum ulation in the n ucleus
(C). In (D) the cells were treated w ith inter-
feron-c for 60 min to induce the nuclear
accumulation of STAT1, before anti-S TAT1
was injected into the cytosol of th e indicated
cell. After another 30 min, nuclear STAT1
was substantially diminished in the injected
cell. Note the continued nuclear accumulation
in the neighboring cells.
Ó FEBS 2004 Nucleocytoplasmic shuttling of STAT transcription factors (Eur. J. Biochem. 271) 4609
generally of energy-consuming transport p athways caused
a nuclear relocation, resulting in a pancellular STAT1
distribution [33]. Whether retention mechanisms such as
the complexation w ith cytoplasmic anchoring f actors also
contribute to the cytoplasmic a ccumulation in resting cells
is currently unclear.
As was mentioned already, c ytokine s timulation of cells
triggers a dramatic translocation of STATs into the nucleus.
This phenomenon, which depending on the stimulus and its
intensity can last for several hours, was initially believed t o
reflect an exclusively nuclear residence of STATs. However,
nuclear accumulation was recognized to be a highly
dynamic process, as the rapid nucleocytoplasmic cycling
of STATs continues even during the accumulation phase. In

the following we will outline how dimerization, the STAT/
DNA dissociation rate, and the activity of a nuclear
phosphatase were identified as the crucial p layers that
control retention and accumulation of STATs in the
nucleus.
The STAT/DNA dissociation rate is a central
integrator of cytokine signaling
Novel i nsight into the readily observable cytokine-stimula-
ted nuclear accumulation of STATs has been gained in the
recent past. It was long known t hat dimerization of
phosphorylated STATs is an absolute r equirement for an
observable accumulation in the nucleus [10]. However, it has
become clear that t he concurrent switch to carrier-dep end-
ent transport is not the cause of nuclear accumulation, as
mutants were generated that were imported normally in
response to cytokine stimulation, but that nevertheless were
not capable of nuclear retention [ 43]. Based o n in vivo
labeling experiments and subcellular fractionations, it was
previously proposed that the duration of STAT nuclear
accumulation was influenced by the activity of tyrosine
phosphatases [37]. Several phosphatases, some of them
nuclear, have been demonstrated to affect the rate o f STAT
dephosphorylation in vivo [44]. Alternatively, ubiquitination
followed by d egradation was p roposed to terminate S TAT
signaling in the nucleus [45].
Recent work unambiguously demonstrated that tyro-
sine-phosphorylated STAT1 is incapable of nuclear exit
and has to be depho sphorylated in order to leave the
nuclear compartment [4,43]. T his f act c onstitutes t he basis
of the cytokine-induced nuclear accumulation of STATs.

The i mportance o f r educed export for the induced nuclear
accumulation was also shown for a STAT protein from
Dictyostelium [42]. While the nuclear accumulation can last
for s everal hours, the nuclear phosphatase activity results
in almost instantaneous dephosphorylation. Therefore t he
question arises as to the mechanisms that defer tyrosine
dephosphorylation. Surprisingly, this mechanism was
determined to be DNA binding. It was found that the
sequence-specific off-rate from DNA was correlated with
the half-life of the phosphorylated protein [43]. STAT
dimers that were bound to high-affinity GAS sites resisted
dephosphorylation better, as compared to STAT molecules
bound to non-GAS sites (Fig. 5 ). Thus, contrary to the
previous assum ption that dephosphorylation releases
STATs from DNA, it was the other way around, and
DNA binding protected STATs from the enzyme activity.
This conclusion was supported by measurements of the
intranuclear mobility of STAT1 in the presence and
absence of phosphatase activity [4,43,46]. Even if the
phosphatase activity was blocked, the mobility of S TAT1
remained close to the diffu sion limit. Normally, however,
owing to t heir high D NA o ff-rate [2], t he protection from
dephosphorylation conferred by DNA binding does not
last for the entire time of nuclear accumulation. In vivo,the
half-life of phosphorylated STAT1 and STAT3 was shown
to not exceed 15–30 min even on a target promoter [37,47].
Thus, the apparently constant level of nuclear accumulated
STAT molecules is maintained b y constant nuclear export
and successive re-import [48,49]. The r esulting nucleocyto-
plasmic cycling during nuclear accumulation was clearly

demonstrated by cytoplasmic trapping of STAT1 after
antibody microinjection ([43] and Fig. 4D). The central
role of dimerization for nuclear retention of STATs was
confirmed by a STAT1 mutant that had l ost its ability to
recruit the inactivating phosphatase TC45 [43,50]. Ex-
change of a single amino acid residue in the amino terminal
domain could reverse the defective nuclear accumulation of
a DNA binding mutant without rescuing the DNA binding
phenotype [4]. These observations also contradicted a
competing model for nuclear accumulation, which stated
that DNA binding was a necessary prerequisite for nuclear
accumulation [39].
Thus, the coupling of dephosphorylation and nuclear
retention to t he sequence-specific DNA of f-rate constitutes
a regulatory mechanism that integrates at least three
important determinants of cytokine signaling. These are
the half-life of the transcriptionally active STAT dimer, the
duration o f p romoter o ccupancy, and finally the ability to
link nuclear activity to the a ctivity of c ytok ine receptors i n
the cell membrane.
Fig. 5. STATs in the nucleus. STAT binding
sites on DNA differ strongly in terms of their
DNA off-rate, which is lowest at optimal tar-
get sites (GAS). Enzymatic dephosphorylation
of STATs is po ssible only when the molecule is
off DNA. Thus, the activity of the STAT
dimer is extended at promoters with op timal
STAT binding site(s).
4610 T. Meyer and U. Vinkemeier (Eur. J. Biochem. 271) Ó FEBS 2004
STAT nucleocytoplasmic transport in disease

It is increasingly becoming clear that nucleocytoplasmic
cycling of signal transducers is an intricate process that
affects signaling in many ways. It is therefore not surprising
that several viral proteins, such as the V proteins from
Nipah and Hendra viruses, both of which cause zoonotic
diseases in animals and humans, have been shown to
interfere with the nucleocytoplasmic translocation o f STAT
proteins ([51–53]; reviewed in [54]). The interferon antag-
onistic activity of these paramyxovirus V proteins included
the cytoplasmic sequestration of STAT1 and STAT2 in high
molecular mass c omplexes. It was shown that Nipah and
Hendra V proteins alter the subcellular d istribution of
STAT1 in resting cells and prevent nuclear import of both
STAT1 and STAT2 in interferon-stimulated cells. Thus,
inhibition of nucleocytoplasmic shuttling constitutes a viral
strategy to evade the antiviral effects of interferons. In
addition, impaired interleukine-12-dependent nuclear trans-
location of STAT4 was reported in a patient with recurrent
mycobacterial infection [55]. These first examples demon-
strate already that nucleocytoplasmic transportation of
STATs can offer novel possibilities also for medical
intervention.
Acknowledgements
The authors’ research on t his subject is funded by grants from the
Deutsche Forschungsgemeinschaft, the EMBO-Young-Investigator-
Program and the Bundesministerium fu
¨
r Bildung und Forschung
(BioFuture).
References

1. Levy, D.E. & Darnell, J.E. Jr (2002) Stats: transcriptional c ontrol
and biological impact. Nat. Rev. Mol. Cell Biol. 3, 651–662.
2. Vinkemeier, U., Cohen, S.L., Moarefi, I., Chait, B.T., Kuriyan, J.
& Darnell, J.E. Jr (1996) DNA binding of in vitro activated Stat1
alpha, Stat1 beta and truncated Stat1: interaction between NH
2
-
terminal domains stabilizes binding of two dimers to tandem
DNA sites. EMBO J. 15, 5616–5626.
3. Shuai, K., Liao, J . & S ong, M.M. (1996 ) Enhancement of anti-
proliferative activity of gamma interferon by the specific inhibition
of tyro sine dephosphorylation of Stat1. Mol. Cell. Biol. 16, 4932–
4941.
4. Meyer, T., Hendry, L., Begitt, A., John, S. & Vink emeier, U.
(2004) A s ingle residue modulates t yrosine dephosphorylation,
oligomerization, and nuclear accumulation of St at t ranscription
factors. J. Biol. C hem . 279, 18998–19007.
5. Ota,N.,Brett,T.J.,Murphy,T.L.,Fremont,D.H.&Murphy,
K.M. (2004) N-domain-dependent nonphosphorylated STAT4
dimers required for cytokine-driven activation. Nat. Immu nol. 5,
208–215.
6. Horvath, C.M., Stark, G.R., Kerr, I.M. & Darnell, J.E. Jr (1996)
Interactions between STAT and non- STAT proteins in the inter-
feron-stimulated gene factor 3 transcription complex. Mol. Cell.
Biol. 16, 6957–6964.
7. Horvath, C.M., Wen, Z. & Darnell, J.E. Jr (1995) A STAT p rotein
domain that determines DNA sequence r ecognition suggests a
novel DNA-bindin g domain. Genes Dev. 15, 984–994.
8. Yang, E., Henriksen, M.A., Schaefer, O., Zakharova, N. & Dar-
nell, J.E. Jr (2002) Dissociation time from DNA determines

transcriptional function in a STAT1 linker mutant. J. Biol. Chem.
277, 13455–13462.
9. Sh uai, K., Horvath , C.M., H uang, L.H., Q ureshi, S.A., Cowbu rn,
D. & D arnell, J.E. Jr (1994) Interferon activation of t he tran-
scription factor Stat91 involves dimerization through SH2–phos-
photyrosyl peptide interaction s. Cell 76, 821–828.
10. Shuai, K., Stark, G.R., Kerr , I.M. & Darnell, J.E. Jr (1993) A
single phosphotyrosine r esidu e of Stat91 required fo r gene a c ti-
vation by interferon-gamma. Science 261, 1744–1746.
11. Stark, G.R., K err, I.M., Williams, B.R., Silverman, R.H. &
Schreiber, R.D. (1 998) H ow cells respond to interferons. Annu.
Rev. Biochem. 67, 227–264.
12. Sc hindler, C., Sh uai, K., Prezio so, V.R. & Darnell, J.E. Jr (1992)
Interferon-depende nt tyrosine phosph orylation of a latent cyto-
plasmic transcription factor. Science 257, 809–813.
13. Ndubuisi, M.I., Guo, G.G., Fried, V.A., Etlinger, J.D. & Sehgal,
P.B. (1999) Cellular physiology of STAT3: Where’s the cytoplas-
mic monomer? J. Biol. Chem. 274, 25499–25509.
14. Haan, S., Kortylewski, M., Behrmann, I., Mu
¨
ller-Esterl, W.,
Heinrich, P.C. & Schaper, F. (2000) Cytoplasmic STAT proteins
associate prior to activation. Biochem. J. 34 5, 417–421.
15. Sekimoto, T., Imamoto, N., Nakajima, K., Hirano, T. & Yoneda,
Y. (1997) E xtracellular signal-d ependent nuclear import of Stat1 is
mediated by nuclear pore-targeting complex formation with NPI-
1, but not Rch1. EMBO J. 16, 7067–7077.
16. Fahre nkrog, B., K o
¨
ser, J. & Aebi, U. (2004) The nu clea r p ore

complex: a jack of all trades? Trends Biochem. Sci. 29, 175–182.
17. Paine, P.L. & Feldherr, C.M. (1972) Nucleocytoplasmic exchange
of macromolec ules. Exp. Cell Res. 74, 81–98.
18. Rout, M.P., Aitchison, J.D., Magnasco, M.O. & Chait, B.T.
(2003) Virtual gating and nuclear transport: the hole picture.
Trends Cell Biol. 13, 622–628.
19. Mattaj, I.W. & Englmeier, L. (1998) Nucleocytoplasmic transport:
the soluble phase. Annu.Rev.Biochem.67, 265–306.
20. Ko se, S., Imamoto , N., Tachibana, T., Shimamoto , T. & Yoneda,
Y. (1997 ) Ran-unassisted n uclear m igration of a 97-kD co mpo-
nent of nuclear pore-targeting complex. J. Cell Biol. 139, 841–849.
21. Sc hwoebel, E.D., Talcott, B., Cushman, I. & Moore, M.S. (1998)
Ran-dependent signal-mediated n uclear import do es not r equire
GTP hydrolysis by Ran. J. Biol. Che m. 273, 35170–35175.
22. Bischoff, F.R., Scheffzek, K. & Ponstingl, H. (2002) How Ran is
regulated. ResultsProbl.CellDiffer.35, 49–66.
23. Se kimoto, T., Nakajima, K., T achibana, T., Hirano , T. & Yone-
da, Y. (1996) I nterferon-gam ma-dependen t nuclear import of
Stat1 is med iated b y t he G TPase activity of Ran/TC4. J. Biol.
Chem. 271, 31017–31020.
24. Fagerlund, R., M ele
´
n, K., K innunen, L. & Julkunen, I. (2002)
Arginine/lysine-rich nuclear l ocalization signals mediate interac-
tions between dimeric STATs and importin alpha 5. J. Biol. Chem.
277, 30072–30078.
25. McBride, K.M., Banninger, G ., McDonald, C. & Reich, N.C.
(2002) Regulated nuclear import of the STAT1 transcription fac-
tor by direct binding of importin-alpha. EMBO J. 21, 1754–1763.
26. Mele

´
n, K., Fagerlund, R., Franke, J., Ko
¨
hler,M.,Kinnunen,L.&
Julkunen, I. (2003) Importin a nuclear localization signal binding
sites for STAT1, STAT2, and i nfluenza A viru s nuc leoprotein.
J. Biol. Chem. 278, 28193–28200.
27. Ma, J., Zhang, T., Novotny-Diermayr, V., Tan, A.L. & Cao, X.
(2003) A novel se quence in the coiled-coil domain of Stat3
essential for its nuclear translocation. J. Biol. Chem. 278, 29252–
29260.
28. Mele
´
n, K ., Kinnunen, L. & J ulkunen, I. (2001) Arginine/lysine-
rich structural element i s involved in interferon-induced n uclear
import of STATs. J. Biol. Chem. 276, 16447–16455.
29. Meyer, T., Begitt, A., Lo
¨
dige, I., van Rossum, M. & Vinkemeier,
U. (2002) Constitutive and I FN-gamma-induc ed nuclear import
of STAT1 proceed through independe nt pathways. EMBO J. 21 ,
344–354.
Ó FEBS 2004 Nucleocytoplasmic shuttling of STAT transcription factors (Eur. J. Biochem. 271) 4611
30. Stre hlow, I. & Schindler, C. (1998) Amino-terminal signal trans-
ducer a nd activator of transcription (STAT) domains regulat e
nuclear translocation and STAT d eactiv ation. J. Biol. Chem. 273,
28049–28056.
31. Chatterjee-Kishore, M., Wright, K.L., T ing, J.P. & Stark, G.R.
(2000) How S tat1 mediates constitutive gene expression: a com-
plex of unphosphorylated Stat1 and IRF 1 supports transcriptio n

of the LMP2 gene. EMBO J. 19, 4111–4122.
32. Meyer, T., Gavenis, K. & Vinkemeier, U. (2002) Cell type-specific
and tyrosine phosphorylation-independent nuclear presence of
STAT1 and STAT 3. Exp. Cell Res. 272, 45–55.
33. Marg, A., Shan, Y., Meyer, T., Meissner, T., Brandenburg, M. &
Vinkemeier, U. (2004) N ucleocytoplasmic shuttling by nucleo-
porins Nup153 and Nu p214 and CRM1-dependent nuclear exp ort
control t he sub cellu lar d istributio n o f l atent Stat1. J. Cell Biol.
165, 823–833.
34. Pranada, A.L., M etz, S., Herrmann, A., Heinrich, P.C. & Mu
¨
ller-
Newen, G. (2004) Real time analysis of S TAT3 nucleocytoplasmic
shuttling. J. Biol. Chem. 279, 15114–15123.
35. Zeng, R., Aoki, Y., Yoshida, M., Arai, K. & Watanabe, S. (2002)
Stat5B shuttles between cytoplasm and nucleus in a cytokine-
dependent and -independent m anner. J. Immunol. 168 , 4567–4575.
36. Adam, S.A., Marr, R.S. & Gerace, L. (1990) N uclear protein
import in permeabi lized mammalian cells re quires soluble cyto-
plasmic factors. J. Cell Biol. 111, 807–816.
37. Haspel, R .L., Salditt-Georgie ff, M. & Darnell, J.E. Jr (1996) The
rapid inactivation of nuclear tyrosine phosphorylated Stat1
depends u pon a protein tyrosine phosphatase. EMBO J. 15, 6262–
6268.
38. Begitt, A., Meyer, T., van Rossum, M. & Vinkemeier, U. (2000)
Nucleocytoplasm ic translocation of Stat1 is regulated b y a leucine-
rich export signal in the coiled-coil domain. Proc. Natl Acad. Sci.
USA 97, 10418–10423.
39. McBride, K.M., McDonald, C. & R eich, N.C. (2000) Nuclear
export signal located within the DNA-binding domain of the

STAT1 transcription fact or. EMBO J. 19, 6196–6206.
40. Bhattacharya, S. & Schindler, C. (2003) Regulation of Stat3
nuclear export. J. Clin. Invest. 111, 553–559.
41. Ginger, R.S., Dalton, E.C., Ryves, W.J., Fukuzawa, M., Williams,
J.G. & Harwood, A.J. ( 2000) Glycogen synthase kinase-3 e n-
hances nuclear expo rt of a Dictyostelium STAT protein. EMBO J.
19, 5483–5491.
42. Fukuzawa, M., Abe, T. & Williams, J.G. (2003) The Dictyostelium
prestalk cell inducer DIF regulates n uclear accumulation of a
STAT protein by controlling its rate of export from the nucleus.
Development 130, 797–804.
43. Meyer, T., Marg, A., L emke, P., Wies ner, B. & Vinkemeier, U .
(2003) DN A binding co ntrols inactivation and nuclear ac cumu-
lation of the transcription factor Stat1. Genes Dev. 17, 1992–2005.
44. Shuai, K. & Liu, B. (2003) Regulation of J AK-STAT signalling in
the immune sy stem. Nat. Rev. Immunol. 3, 900–911.
45. Kim, T.K. & M aniatis, T. (1996) Re gulation of inte rferon-gam-
ma-activated ST AT1 by the ubiquitin-proteasome p athway. Sci-
ence 273, 1717–1719.
46. Lillemeier, B.F., Ko
¨
ster, M. & Kerr, I.M. ( 2001) STAT1 from the
cell membrane to the DNA. EMBO J. 20, 2508–2517.
47. Lerner,L.,Henriksen,M.A.,Zhang,X.&Darnell,J.E.Jr(2003)
STAT3-depen dent e nhan ceosome assembly and disassemb ly:
synergy w ith GR for full transcript ional increase o f the alp ha
2-macroglobulin gene. Genes Dev. 17, 2564–2577.
48. Andre ws, R.P., Ericksen, M.B., Cunningham, C.M., Daines,
M.O. & Hershey, G.K. (2002) Analysis of the life cycle of STAT6.
Continuous cycling of STAT6 is required for IL-4 signaling.

J. Biol. Chem. 277, 36563–36569.
49. Swameye, I., Mu
¨
ller, T.G., Timmer, J., Sandra, O. & Klingmu
¨
ller,
U. (2003) Identification of nucleocytoplasmic cycling as a remote
sensor in cellular signaling by databased modeling. Proc . Natl
Acad. Sci. USA 100, 1028–1033.
50. ten Hoeve, J., de Jesus Ibarra-Sanchez, M., Fu, Y., Zhu, W.,
Tremblay, M., David, M. & Shuai, K. (2002) Identification of a
nuclear Stat1 protein tyrosine pho sphatase. Mol. Cell. Biol. 22,
5662–5668.
51. Rodriguez, J.J., Parisien, J.P. & Horvath, C.M. (2002) Nipah virus
V protein evades alpha and gamma interferons by preventing
STAT1 and STAT2 activation and nuclear accumulation. J. Virol.
76, 11476–11483.
52. Rodriguez, J.J., Wang, L.F. & Horvath, C.M. (2003) He ndra virus
V protein inhibits interferon signalingbypreventingSTAT1and
STAT2 nuclear acc umulation. J. Virol. 77 , 11842–11845.
53. Shaw, M.L., Garcia-Sastre, A., Palese, P. & B as ler, C.F. (2004)
Nipah virus V a nd W p rote ins have a c o mmon STAT1-binding
domain yet inhibit S TAT1 activation from the cytoplasmic an d
nuclear compartments, respectively. J. Virol. 78, 5633–5641.
54. Horvath, C.M. (2004) Weapons of STAT destruction. Interferon
evasion by Paramyxovirus V proteins. Eur. J. Bi och em. 271, 4621–
4628.
55. To yoda, H., Id o, M., H ayashi, T ., Gabazza, E.C., S uzuki, K.,
Bu, J., Tanaka, S., Nakano, T., Kamiya, H., Chipeta, J., Kisenge,
R.R., Kang, J., Hori, H. & Komada, Y. (20 04) I mpairment of

IL-12-dependent STAT4 nuclear translocation i n a p atient with
recurrent Myco bacterium avium infec ti on . J. Immunol. 172,
3905–3912.
4612 T. Meyer and U. Vinkemeier (Eur. J. Biochem. 271) Ó FEBS 2004