M I N I R E V I E WNucleocytoplasmic shuttling of STAT transcription factors Thomas Meyer and Uwe Vinkemeier Abteilung Zellula¨re Signalverarbeitung, Leibniz-Forschungsinstitut fu¨r Mole
Trang 1M I N I R E V I E W
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 signal transducer and 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 are constantly shuttling between nucleus and
cyto-plasm irrespective of cytokine 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 an integrated network of
cell–cell communications and humeral interactions to
coordinate complex cellular processes such as proliferation,
differentiation, and homeostasis Cells recognize external
stimuli and transform the signals into a cellular response,
which most often result in an alteration in the pattern of
expressed genes Many signal transducers that function as
transcription factors have to traverse the barrier of the
nuclear envelope in order to gain access to specific target
genes within the nuclear compartment The Janus kinase
(JAK)-signal transducer and activator of transcription
(STAT) pathway is regarded as a paradigmatic model for
such a direct signal transduction, because it transmits
information received from extracellular 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 transcription factors and in mammalian cells
seven known STAT proteins were identified, denoted
STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and
STAT6, all of which are activated by a distinct set of
cytokines and growth factors [1] These proteins consist of
several conserved functional domains The amino terminal
N-domain is responsible for tetramerization 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, receptor-associated Janus kinases, of which four have been described in mammalian cells (JAK1, JAK2, JAK3 and TYK2), undergo tyrosine autophosphorylation 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 of a single tyrosine residue in the carboxy terminus 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 of 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 showed that cytokine stimulation with concomitant dimerization of tyrosine-phosphorylated STATs induces their association with importin transport factors [15] Next, we will describe what is presently known about the molecular basis of this process
Correspondence to U Vinkemeier, Abteilung Zellula¨re
Signalverar-beitung, Leibniz-Forschungsinstitut fu¨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: vinkemeier@fmp-berlin.de
Abbreviations: CRM1, chromosomal region maintenance 1; dsNLS,
dimer-specific nuclear localization signal; GAS, gamma-activated
sites; JAK, Janus kinase; NLS, nuclear localization signal; NPC,
nuclear pore complex; SH2, SRC homology 2; STAT, signal
transducer and activator of transcription.
(Received 18 August 2004, accepted 7 October 2004)
Trang 2Requirements 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 channels that are an integral part of the
nuclear envelope They are composed of proteins called
nucleoporins which are present in multiples, and some of
them contain hydrophobic phenylalanine/glycine (FG)-rich
repeat motifs [16] Macromolecules exceeding a molecular
mass of 40 kDa are generally barred from freely crossing
the nuclear membrane by random diffusion [17] Thus,
the NPCs function as selectivity filters by restricting the
transport of some macromolecules, while allowing the rapid
translocation of others
Detailed mechanistic insight has been acquired into
translocation mechanisms that rely on transport receptors
of the karyopherin superfamily of proteins [18]
Karyophe-rins mediate either import into or export from the nucleus
and they are therefore also called importins 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 exportins, although structurally
rela-ted, differ in their sequence requirements 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 the nucleoporin FG repeats, an interaction that
by itself was shown to occur independently of 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, results in high levels of RanGDP in the cytosol In the presence of RanGDP, importins are loaded with substrates and may translocate through the NPC into the nucleus, while the export receptors are liberated from their cargo molecules in this environment The reverse reactions take place in the nucleus Here, a high RanGTP/ RanGDP ratio is maintained by the guanine nucleotide exchange factor RCC1, which catalyzes the 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) with 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 of this Ran-dependent nuclear import machinery [15,23] The karyopherin impor-tin b (p97) has been identified as the carrier 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 that contains
Fig 2 STATs at the nuclear envelope (A) Carrier-dependent import Phosphorylated STAT dimers expose a dimer-specific nuclear local-ization signal and associate with importin a Through importin b-mediated interactions with the interior of the nuclear pore (NPC) this complex migrates into the nucleus The complex disassembles after the bindung of RanGTP The exact stoichiometry and order of events have not been established (B) Carrier-dependent export Unphos-phorylated STATs can bind to the exportin CRM1 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 independently of metabolic energy.
Fig 1 STATs at the cell membrane A schematic representation of the
events leading to the tyrosine phosphorylation (activation) of STATs.
The activation of receptor-associated 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 concomitantly The activated STATs detach and
homo- or heterodimerize.
Trang 310 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 with repeats 2–4, 7 and 8 [26]
The binding site for importin a5 on 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 reported to also 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 importin 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 bipartite
NLSs, which consist of one or two arginine/lysine-rich
clusters of basic amino acids separated 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 is its nontransferability, because it functions
only in the context of the STAT dimer, but not
autonom-ously as is typical for conventional NLSs [28,29] In
addition, the STAT amino termini also appear to provide
signals for the cytokine-inducible nuclear localization as
judged from the inability of amino terminal deletion
mutants to accumulate in the nucleus [30]; and residues in
the coiled-coil domain seem to contribute to
carrier-dependent nuclear import of some STATs [27]
The canonical model of the JAK-STAT pathway stated
that unphosphorylated STATs are 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 advances, 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 to 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 mutants of STAT1 were found
in the nucleus of unstimulated cells [29] This was taken as 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 of monomeric STAT1 and STAT3 in a variety of unstimulated primary cells or established cell lines [31,32] Point mutations in either the SH2 domain or the tyrosine residue in position 701 that completely prevented the signal-dependent dimerization had no effect on the intracellular STAT1 localization in resting cells [31,32] The direct visualization of STAT1 nucleocytoplasmic shuttling
in resting 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 endogenous STAT1 in unstimu-lated cells [33] It was found that the antibody-induced STAT1 clearance was rapid and complete in about 30 min, irrespective of whether the antibody was injected 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 reported also for STAT3 and 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 could 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 is saturable and appears to occur through direct 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 membrane, suggesting 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 the nuclear pore remain to
Fig 3 The dimer-specific nuclear import signal (dsNLS) of STAT1 A
short stretch from the DNA binding domain of STAT1 harbors
overlapping export and import activities Notably, the import activity
is observed only in the native STAT dimer, whereas the export activity
is readily observable in the isolated peptide Residues that were
dem-onstrated to be important for export (of isolated peptides) are depicted
in a white box, residues that are required only for import (of the dimer)
are boxed in dark grey Residues, mutation of which affected both
import and export, are shown in a light grey box For comparison, the
homologous sequences of other STATs are listed: D, Drosophila;
h, human.
Trang 4be established, it was shown 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 appears
that phosphorylation-induced dimerization is the switch
from facilitated diffusion to carrier-mediated translocation
(Fig 2) Notably, only one third of the STAT1 molecules
are tyrosine phosphorylated at any moment during cytokine
stimulation [37]
Work in our laboratory identified a functional
leucine-rich nuclear export signal in STAT1 and demonstrated its
role in vivo, thus showing that nuclear export of STAT1 was
occurring [38] In the meantime, further putative
leucine-rich nuclear export signals have been identified in varying
locations in STAT1 [39], STAT3 [40], and STAT5 [35], as
well as in Dictyostelium STATa [41], and STATc [42] Of
note is the fact that characterization of the STAT export
signals remains incomplete, as export activity 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 of cytokine-induced nuclear accumulation of STATs, it is now clear that this export pathway operates constitutively [33] Preincubation
of resting cells with the CRM1 inhibitor leptomycin B did not cause the nuclear accumulation of STAT1, which
by some was taken as an indication that STATs do not shuttle in resting cells [39] In addition, it was noted that leptomycin merely attenuated the cytoplasmic relocation after cytokine-induced nuclear accumulation, but did not cause a complete block [38] As described above, this phenotype is explained by the existence of a carrier-independent and hence leptomycin-insensitive nuclear export mechanism [33] STATs are predominantly cyto-plasmic in resting cells, although STAT- and cell type-specific differences were reported [32] For STAT1, the underlying molecular mechanism was determined to entail 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
FITC-BSA
A
B
C
D
Fig 4 Nucleocytoplasmic shuttling of STAT1
in resting and cytokine-stimulated cells
Anti-body microinjection assays with an unspecific
STAT3 antibody (A) or a specific STAT1
antibody (B–D) After antibody injection the
cells were incubated for 30 min at 37 °C,
before fixation and immunocytochemical
detection of endogenous 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 STAT1
distri-bution is not affected by microinjection of an
unspecific antibody The injection of a
STAT1-specific antibody revealed 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 accumulation in the nucleus
(C) In (D) the cells were treated with
inter-feron-c for 60 min to induce the nuclear
accumulation of STAT1, before anti-STAT1
was injected into the cytosol of the indicated
cell After another 30 min, nuclear STAT1
was substantially diminished in the injected
cell Note the continued nuclear accumulation
in the neighboring cells.
Trang 5generally of energy-consuming transport pathways caused
a nuclear relocation, resulting in a pancellular STAT1
distribution [33] Whether retention mechanisms such as
the complexation with cytoplasmic anchoring factors also
contribute to the cytoplasmic accumulation in resting cells
is currently unclear
As was mentioned already, cytokine stimulation 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 to
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 players that
control retention and accumulation of STATs in the
nucleus
The STAT/DNA dissociation rate is a central
integrator of cytokine signaling
Novel insight into the readily observable
cytokine-stimula-ted nuclear accumulation of STATs has been gained in the
recent past It was long known that dimerization of
phosphorylated STATs is an absolute requirement for an
observable accumulation in the nucleus [10] However, it has
become clear that the concurrent switch to
carrier-depend-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 on 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 of STAT
dephosphorylation in vivo [44] Alternatively, ubiquitination
followed by degradation was proposed to terminate STAT
signaling in the nucleus [45]
Recent work unambiguously demonstrated that
tyro-sine-phosphorylated STAT1 is incapable of nuclear exit
and has to be dephosphorylated in order to leave the
nuclear compartment [4,43] This fact constitutes the basis
of the cytokine-induced nuclear accumulation of STATs
The importance of reduced export for the induced nuclear
accumulation was also shown for a STAT protein from
Dictyostelium[42] While the nuclear accumulation can last for several hours, the nuclear phosphatase activity results
in almost instantaneous dephosphorylation Therefore the 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 assumption 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 STAT1 remained close to the diffusion limit Normally, however, owing to their high DNA off-rate [2], the 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 by constant nuclear export and successive re-import [48,49] The resulting 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 lost 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 the sequence-specific DNA off-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 of promoter occupancy, and finally the ability to link nuclear activity to the activity of cytokine receptors in 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 possible only when the molecule is
off DNA Thus, the activity of the STAT dimer is extended at promoters with optimal STAT binding site(s).
Trang 6STAT 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 of 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 complexes It was shown that Nipah and
Hendra V proteins alter the subcellular distribution 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 this 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 control
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 & Song, M.M (1996) Enhancement of
anti-proliferative activity of gamma interferon by the specific inhibition
of tyrosine dephosphorylation of Stat1 Mol Cell Biol 16, 4932–
4941.
4 Meyer, T., Hendry, L., Begitt, A., John, S & Vinkemeier, U.
(2004) A single residue modulates tyrosine dephosphorylation,
oligomerization, and nuclear accumulation of Stat transcription
factors J Biol Chem 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 Immunol 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 protein
domain that determines DNA sequence recognition suggests a
novel DNA-binding 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 Shuai, K., Horvath, C.M., Huang, L.H., Qureshi, S.A., Cowburn,
D & Darnell, J.E Jr (1994) Interferon activation of the tran-scription factor Stat91 involves dimerization through SH2–phos-photyrosyl peptide interactions Cell 76, 821–828.
10 Shuai, K., Stark, G.R., Kerr, I.M & Darnell, J.E Jr (1993) A single phosphotyrosine residue of Stat91 required for gene acti-vation by interferon-gamma Science 261, 1744–1746.
11 Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H & Schreiber, R.D (1998) How cells respond to interferons Annu Rev Biochem 67, 227–264.
12 Schindler, C., Shuai, K., Prezioso, V.R & Darnell, J.E Jr (1992) Interferon-dependent tyrosine phosphorylation 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 345, 417–421.
15 Sekimoto, T., Imamoto, N., Nakajima, K., Hirano, T & Yoneda,
Y (1997) Extracellular signal-dependent nuclear import of Stat1 is mediated by nuclear pore-targeting complex formation with
NPI-1, but not Rch1 EMBO J 16, 7067–7077.
16 Fahrenkrog, B., Ko¨ser, J & Aebi, U (2004) The nuclear pore complex: a jack of all trades? Trends Biochem Sci 29, 175–182.
17 Paine, P.L & Feldherr, C.M (1972) Nucleocytoplasmic exchange
of macromolecules 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 Kose, S., Imamoto, N., Tachibana, T., Shimamoto, T & Yoneda,
Y (1997) Ran-unassisted nuclear migration of a 97-kD compo-nent of nuclear pore-targeting complex J Cell Biol 139, 841–849.
21 Schwoebel, E.D., Talcott, B., Cushman, I & Moore, M.S (1998) Ran-dependent signal-mediated nuclear import does not require GTP hydrolysis by Ran J Biol Chem 273, 35170–35175.
22 Bischoff, F.R., Scheffzek, K & Ponstingl, H (2002) How Ran is regulated Results Probl Cell Differ 35, 49–66.
23 Sekimoto, T., Nakajima, K., Tachibana, T., Hirano, T &
Yone-da, Y (1996) Interferon-gamma-dependent nuclear import of Stat1 is mediated by the GTPase activity of Ran/TC4 J Biol Chem 271, 31017–31020.
24 Fagerlund, R., Mele´n, K., Kinnunen, L & Julkunen, I (2002) Arginine/lysine-rich nuclear localization 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 influenza A virus nucleoprotein.
J Biol Chem 278, 28193–28200.
27 Ma, J., Zhang, T., Novotny-Diermayr, V., Tan, A.L & Cao, X (2003) A novel sequence in the coiled-coil domain of Stat3 essential for its nuclear translocation J Biol Chem 278, 29252– 29260.
28 Mele´n, K., Kinnunen, L & Julkunen, I (2001) Arginine/lysine-rich structural element is involved in interferon-induced nuclear 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 IFN-gamma-induced nuclear import
of STAT1 proceed through independent pathways EMBO J 21, 344–354.
Trang 730 Strehlow, I & Schindler, C (1998) Amino-terminal signal
trans-ducer and activator of transcription (STAT) domains regulate
nuclear translocation and STAT deactivation J Biol Chem 273,
28049–28056.
31 Chatterjee-Kishore, M., Wright, K.L., Ting, J.P & Stark, G.R.
(2000) How Stat1 mediates constitutive gene expression: a
com-plex of unphosphorylated Stat1 and IRF1 supports transcription
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 STAT3 Exp Cell Res 272, 45–55.
33 Marg, A., Shan, Y., Meyer, T., Meissner, T., Brandenburg, M &
Vinkemeier, U (2004) Nucleocytoplasmic shuttling by
nucleo-porins Nup153 and Nup214 and CRM1-dependent nuclear export
control the subcellular distribution of latent Stat1 J Cell Biol.
165, 823–833.
34 Pranada, A.L., Metz, S., Herrmann, A., Heinrich, P.C &
Mu¨ller-Newen, G (2004) Real time analysis of STAT3 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 -incytokine-dependent manner J Immunol 168, 4567–4575.
36 Adam, S.A., Marr, R.S & Gerace, L (1990) Nuclear protein
import in permeabilized mammalian cells requires soluble
cyto-plasmic factors J Cell Biol 111, 807–816.
37 Haspel, R.L., Salditt-Georgieff, M & Darnell, J.E Jr (1996) The
rapid inactivation of nuclear tyrosine phosphorylated Stat1
depends upon a protein tyrosine phosphatase EMBO J 15, 6262–
6268.
38 Begitt, A., Meyer, T., van Rossum, M & Vinkemeier, U (2000)
Nucleocytoplasmic translocation of Stat1 is regulated by a
leucine-rich export signal in the coiled-coil domain Proc Natl Acad Sci.
USA 97, 10418–10423.
39 McBride, K.M., McDonald, C & Reich, N.C (2000) Nuclear
export signal located within the DNA-binding domain of the
STAT1 transcription factor 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
en-hances nuclear export 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 nuclear accumulation of a
STAT protein by controlling its rate of export from the nucleus.
Development 130, 797–804.
43 Meyer, T., Marg, A., Lemke, P., Wiesner, B & Vinkemeier, U (2003) DNA binding controls inactivation and nuclear accumu-lation of the transcription factor Stat1 Genes Dev 17, 1992–2005.
44 Shuai, K & Liu, B (2003) Regulation of JAK-STAT signalling in the immune system Nat Rev Immunol 3, 900–911.
45 Kim, T.K & Maniatis, T (1996) Regulation of interferon-gam-ma-activated STAT1 by the ubiquitin-proteasome pathway 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-dependent enhanceosome assembly and disassembly: synergy with GR for full transcriptional increase of the alpha 2-macroglobulin gene Genes Dev 17, 2564–2577.
48 Andrews, 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 phosphatase 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) Hendra virus
V protein inhibits interferon signaling by preventing STAT1 and STAT2 nuclear accumulation J Virol 77, 11842–11845.
53 Shaw, M.L., Garcia-Sastre, A., Palese, P & Basler, C.F (2004) Nipah virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and 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 Biochem 271, 4621– 4628.
55 Toyoda, H., Ido, M., Hayashi, T., Gabazza, E.C., Suzuki, K.,
Bu, J., Tanaka, S., Nakano, T., Kamiya, H., Chipeta, J., Kisenge, R.R., Kang, J., Hori, H & Komada, Y (2004) Impairment of IL-12-dependent STAT4 nuclear translocation in a patient with recurrent Mycobacterium avium infection J Immunol 172, 3905–3912.