School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK
Author for correspondence (e-mail:
j.g.williams{at}dundee.ac.uk)
Accepted 25 November 2002
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SUMMARY |
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Key words: STAT proteins, DIF, Dictyostelium discoideum, prestalk differentiation
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INTRODUCTION |
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STAT proteins were discovered as the transcriptional regulators that
mediate interferon action but are now known to have many other cellular and
developmental functions (Bromberg,
2000; Bromberg and Darnell,
2000
; Horvath,
2000
; Levy, 1999
;
Luo and Dearolf, 2001
;
Watson, 2001
;
Zeidler et al., 2000
). Despite
their importance the mechanisms that regulate STAT nuclear accumulation are
relatively poorly understood. STATs are activated by tyrosine phosphorylation
and dimerise via reciprocal SH2 domain:phosphotyrosine interactions
(Shuai et al., 1993
).
Generally, dimerisation seems a necessary and sufficient trigger to induce
nuclear accumulation and or biological activity
(Bromberg et al., 1999
;
Milocco et al., 1999
).
However, these sometimes seem to occur without an apparent need for
dimerisation (Johnson et al.,
1999
; Kumar et al.,
1997
). Initially, we reported that mutant forms of Dd-STATc,
wherein the site of tyrosine phosphorylation and/or the SH2 domain were
destroyed, remain DIF inducible (Fukuzawa
et al., 2001
). However, we subsequently discovered that, when gene
repair by homologous recombination is prevented, a mutation in the tyrosine
phosphorylation site is non-inducible (M. F. and J. G. W., unpublished data)
(Fukuzawa et al., 2001
). Hence
Dd-STATc conforms to the general pattern of metazoan STAT behaviour,
dimerisation triggers nuclear accumulation.
Here we analyse the Dd-STATc protein to delineate the mechanisms that direct its nuclear accumulation. We show that DIF regulates the nuclear accumulation of Dd-STATc by controlling its rate of export from the nucleus and we map nuclear import and export signals within the protein.
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MATERIALS AND METHODS |
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Photobleaching asssay of nuclear export
Dictyostelium cells transformed with GFP:STATc were rendered
competent to respond to DIF as described above and then allowed to spread on a
Petri dish (Petriperm hydrophilic, In Vitro Systems and Services, GmbH). Even
in an uninduced cell there is always a level of nuclear fluorescence that is
slightly higher than in the cytoplasm and this fluorescence difference was
used to locate the position of the nucleus using a confocal microscope (Leica
DMRBE model TCS-SP2). The nucleus was then masked to protect it from
irradiation. The cytoplasm was irradiated for 5 seconds at a wavelength of 478
nm and this usually proved to be a level of photobleaching sufficient to
reduce the GFP fluorescence by at least 90%. Cells with 10% or less residual
cytoplasmic GFP fluorescence were then incubated further and the fluorescence
signal within the nuclei was determined at different time points.
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RESULTS |
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When the majority of isolated cells within a population (the behaviour is
different for cells in clumps and for a minority of the single cells, see
legend to Fig. 1A) are
photobleached and incubated in the absence of added DIF, 50% of the GFP:STATc
protein exits from the nucleus with a t1/2 of about 1.5 minutes
(Fig. 1B). In contrast, in the
presence of DIF, the majority (approx. 65%) of the GFP:STATc protein remains
in the nucleus over the entire 5 minute incubation period. The inhibitory
effect of DIF on Dd-STATc nuclear efflux is not due to non-specific inhibition
of all nuclear export, because the nuclear efflux of Dd-STATa, a
Dictyostelium STAT that is regulated by cAMP signalling
(Araki et al., 1998;
Kawata et al., 1997
), is not
inhibited by DIF (Fig. 1C).
The above results show that DIF inhibits the nuclear export of Dd-STATc but DIF could act to control both the export rate and the import rate of Dd-STATc. In principle, photobleaching could be used to distinguish these possibilities. However, technical limitations, imposed by the very small size of the nucleus, the rapid movement of the cells and the toxic effect of the photobleaching radiation on the nucleus made it impossible to perform the reverse experiment: i.e. to photobleach the nucleus and then determine the rate at which GFP:STATc moves into the nucleus in the absence or presence of DIF. Hence we used a molecular genetic approach to further analyse the process.
Dd-STATc contains a sub-region that directs nuclear export
Because the photobleaching experiments suggested that DIF induces Dd-STATc
nuclear accumulation by inhibiting its nuclear export, we searched for nuclear
export signals (NESs) within Dd-STATc. The region of Dd-STATc between residues
505 and 554 is partially homologous in sequence to a region of Dd-STATa that
directs nuclear export when fused to GFP
(Fig. 2A)
(Ginger et al., 2000). This
entire region of Dd-STATc, which we term the EXP region, is very Leu/Ile-rich
and this is the characteristic feature of NESs. There is a region (B) near the
C terminus of EXP that fits well to the HTV1-rex/rad24 type (LXXXLXL) of NES
and another (A), nearer the N terminus, which is also a reasonable match
(Fig. 2A). We showed that the
EXP region functions as a nuclear export mediator both by deleting it from the
intact protein and by fusing it to GFP.
|
GFP:STATcEXP, a GFP fusion protein containing an internal deletion
that removes only the EXP region, is constitutively localised within the
nucleus (Fig. 2B). We employed
photobleaching to determine whether GFP:STATc
EXP is constitutively
localised within the nucleus because it is defective in export from the
nucleus (Fig. 3). Comparison of
Fig. 3 with
Fig. 2A shows that
GFP:STATc
EXP-expressing cells incubated in the absence of
DIF behave very much like GFP:STATc-expressing cells incubated in the
presence of DIF, i.e. the majority of the GFP fusion protein
remains in the nucleus over the entire 5-minute incubation period. This
observation readily explains the constitutive nuclear accumulation of
GFP:STATc
EXP. The fact that the GFP:STATc
EXP fusion protein is
itself unaffected by the addition of DIF
(Fig. 3) also shows that the
EXP region is essential for DIF-induced inhibition of nuclear export.
|
The EXP region can also act in isolation as an export mediator. This was
shown by fusing EXP to GFP, to generate EXP:GFP. GFP is small enough to
diffuse into the nucleus freely but the EXP:GFP fusion protein is enriched in
the cytoplasm over the nucleus (Fig.
4), indicating that the EXP region contains one or more functional
NESs. The best-characterised nuclear export process is that mediated by CRM1
(exportin-1) and the binding sites for CRM1 are, as stated, loosely conserved,
leucine-rich sequences (Fig.
2A). In the case of Dd-STATa
(Ginger et al., 2000) nuclear
export directed by the NES-containing region is sensitive to leptomycin B
(LMB), a drug that binds to and inhibits CRM1. When cells expressing the
EXP:GFP fusion protein are treated with LMB, nuclear exclusion is lost and the
protein accumulates in both the nucleus and the cytoplasm
(Fig. 4). Thus the EXP region
contains at least one functional, CRM1-dependent NES.
|
In the absence of the EXP region the N-terminal-proximal half of
Dd-STATc directs constitutive nuclear accumulation
The constitutive accumulation of GFP:STATcEXP, the GFP fusion
protein containing an internal deletion that removes just the EXP region
(Fig. 2B), suggests that there
are cryptic nuclear import signals within Dd-STATc. Dd-STATa and Dd-STATc are
very differently regulated but they are highly conserved in the proximal
regions of their C termini. Hence we first searched for import activity in the
N-terminal-proximal half of Dd-STATc. GFP:STATc1-504 encodes a GFP fusion
protein containing the region of Dd-STATc extending from the N terminus to a
point just upstream of the EXP region. The GFP:STATc 1-504 fusion protein is
constitutively enriched in the nucleus
(Fig. 5A), showing that there
are one or more nuclear import signals in the N-terminal half of Dd-STATc.
|
In construct GFP:STATc1-554, a 50 residue longer N-terminal fragment fusion wherein the EXP region is retained, the GFP fusion protein is excluded from the nucleus (Fig. 5B). Thus the NESs contained within the EXP region are dominant over the import signals within the N-terminal region. Moreover, the fact that LMB treatment of cells transformed with GFP:STATc1-554 causes nuclear accumulation (Fig. 5B) shows that exclusion from the nucleus depends upon the activity of CRM1.
Essential nuclear import signals are located within the N-terminal 46
amino acids
We next mapped the presumptive NLSs contained within the N-terminal region
by performing N to C deletion analysis of the Dd-STATc protein. A construct
with five N-terminal amino acids deleted retains DIF-inducible nuclear
accumulation but all smaller constructs, including a construct
(GFP:STATc47-929) with only 46 amino acids deleted, yield GFP fusion proteins
that are equally distributed between the cytoplasm and the nucleus and non-DIF
inducible (Fig. 6A). Thus the
N-terminal 46 amino acids (we will term this the `IMP region') are necessary
for DIF-inducible nuclear accumulation. [NB We believe that there is at least
one other weakly active nuclear import region, located just downstream of EXP,
between residues 555 and 607 (M. F. and J. G. W., unpublished data). The
presence of this second import domain probably explains why the N-terminally
deleted proteins, where IMP is absent, are not excluded from the nucleus,
despite the presence of the EXP region.]
|
When fused to GFP, in IMP:GFP, the IMP region directs constitutive nuclear
accumulation (Fig. 6B). Thus,
in addition to being necessary for DIF-inducible nuclear accumulation of the
Dd-STATc protein, the IMP region can function as an autonomous NLS. Within the
IMP region there is a stretch of basic amino acids
(1MSNNNPKKRPLD12), that could conceivably
form part of a `classical', importin- binding NLS. However, when the
three basic residues are mutated to alanine, within the context of the whole
protein, nuclear accumulation in response to DIF is unaffected (data not
shown). This is in accord with previous work on NLSs within STAT1, where
importin-
5 is believed to mediate nuclear import but via a totally
distinct mechanism from that utilised for classical NLSs
(Sekimoto et al., 1997
;
McBride et al., 2002
).
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DISCUSSION |
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The notion of Dd-STATc nuclear accumulation as a balance between competing
import and export processes accords with the now generally held view of
regulated nuclear translocation. One reagent that has been valuable in
arriving at this perspective in other systems is LMB, because it can be used
to selectively inhibit nuclear export and thus reveal any constitutive import.
However, we find that LMB treatment does not induce nuclear accumulation of
the intact Dd-STATc protein (M. F. and J. G. W., unpublished data). A similar
observation has been made for STAT1 (Begitt
et al., 2000). Again, the intact STAT1 protein failed to
accumulate in the nucleus of cells not treated with interferon, in the
presence of LMB, although molecular dissection showed that it contains an
LMB-sensitive NES. In this case, however, LMB did slow the efflux of STAT1
after adaptation to the interferon stimulus. We find no such effect in the
case of DIF-treated cells (M. F. and J. G. W., unpublished data).
LMB resistance of the intact Dd-STATc protein may be the result of
co-operative interaction between EXP and sequences in the C-terminal half of
the protein. Multiple nuclear export signals within a protein can act
co-operatively to direct nuclear export by CRM1 (Gaubatz, 2001) and there are
additional, weak nuclear export signals in the region of Dd-STATc between
residue 716 and the C terminus at residue 929 (M. F. and J. G. W., unpublished
data). Perhaps, therefore, co-operating NESs in the intact Dd-STATc protein
produce a substrate with a high relative affinity for CRM1, that can function
at sub-saturating doses of LMB. This effect would be compounded by the
relative non-susceptibility of Dictyostelium cells to LMB; at 20 nM
the concentration used here for Dd-STATc only 30% of cells transformed with a
nuclear excluded mutant of Dd-STATa (Dd-STATaAcidpep) showed nuclear
accumulation and higher concentrations of LMB blocked both export and import
(Ginger et al., 2000). A
similar mechanism has been proposed to explain the LMB insensitivity of a
mutant form of the ICP27 protein of Herpes Simplex Virus
(Murata et al., 2001
).
Deletion of a region containing LMB-sensitive NESs leads to
constitutive nuclear accumulation
Although LMB does not affect the intact Dd-STATc protein, we were able to
verify the photobleaching result by mutating the Dd-STATc protein. The fact
that deletion from Dd-STATc of the LMB-sensitive nuclear export mediators
located in the EXP region leads to constitutive nuclear accumulation
complements the conclusion from the photobleaching results very well; in cells
transformed with this deletion construct the fusion protein accumulates in the
nucleus without the need for DIF treatment. This again suggests that the
cellular processes directing nuclear import of the intact protein are
constitutively active and that the rate of export from the nucleus, partly or
wholly mediated by the EXP region, acts to determine the [cytoplasm]/[nucleus]
ratio for Dd-STATc. In support of this suggestion we showed, using
photobleaching, that deletion of the EXP region drastically reduced its rate
of efflux from the nucleus and rendered it DIF insensitive.
There is a nuclear import signal very near the N terminus that falls
under the control of the EXP region
We localised nuclear import signals by showing that the 46 amino acid
residue region at the N terminus, the IMP region, is essential for the nuclear
accumulation of Dd-STATc. We also showed that the IMP region itself will
direct nuclear accumulation when fused to GFP. The nuclear export signals
contained within the EXP region are, however, dominant to the IMP region; so
that a fragment containing the entire N-terminal half of the protein,
including EXP and IMP, is excluded from the nucleus. This result again
indicates the existence of an equilibrium between the nuclear import and
export of Dd-STATc. Analysis of the LMB sensitivity of this construct also
supports the notion discussed above, that co-operative interaction between EXP
and C-terminal-proximal export signals leads to the LMB
insensitivity of the whole Dd-STATc protein; because this fragment,
which contains only the approximate N-terminal half of the Dd-STATc protein,
accumulates in the nucleus in the presence of LMB.
A model for the DIF-induced nuclear accumulation of Dd-STATc
The C-terminal-proximal half of Dd-STATc is essential for the regulation of
nuclear accumulation by DIF. This region contains the DNA binding site, the
SH2 domain and the site of tyrosine phosphorylation and we now know that
tyrosine phosphorylation is essential for DIF-induced nuclear accumulation of
Dd-STATc. We therefore suggest that the DIF-induced dimerisation of Dd-STATc
in some way masks EXP from CRM1 (Fig.
7). This allows the nuclear import signals in the IMP region to
become dominant over the NESs in the EXP region, and as a result Dd-STATc
accumulates in the nucleus.
|
Regulated export controls the nuclear accumulation of other transcription
factors, e.g. p53, YAP1p, NF-AT (reviewed by
Fonseca, 2002) but has not,
thus far been reported for STAT proteins. While the efflux of STAT1 from the
nucleus after cessation of interferon signalling
(McBride et al., 2000
), and of
Dd-STATa upon adaptation to cAMP signalling
(Ginger et al., 2000
), are
both mediated by CRM1 this is, to our knowledge, the first case in which the
initial nuclear accumulation of a STAT protein has been shown to be
regulated at the level of nuclear export. Indeed, in the best characterised
STAT induction system, the activation of STAT1 by interferon
,
regulation appears to be at the level of nuclear import
(McBride et al., 2002
).
Although STAT1 does not seem to contain a classical nuclear localisation
signal (NLS), importin
5 binds to STAT1 via sequences near its C
terminus (Sekimoto, 1997). Activation, by intereferon treatment, increases the
degree of importin
5 binding to STAT1 and this increase correlates
quantitatively with the formation of STAT1 dimers
(McBride et al., 2002
). The
mechanism suggested by the STAT1 study is in one respect similar to that
suggested here; dimerisation state governs the balance between import and
export signals. The significant difference is that STAT1 dimerisation seems to
unmask a latent NLS while we suggest that dimerisation of Dd-STATc masks an
active NES.
Several different nuclear export signals have been mapped within STAT1
(Begitt et al., 2000;
Ginger et al., 2000
;
McBride et al., 2002
;
McBride et al., 2000
;
Melen et al., 2001
) but the
EXP region of Dd-STATc is not conserved in position with respect to any of
these. Also, as stated, regulated nuclear translocation of STAT1 appears to
occur by a quite different mechanism than that of Dd-STATc. However, the
mammalian STAT protein family has 6 other members that could, in principle, be
regulated differently from STAT1. It remains possible therefore that nuclear
export-based regulation features in mammalian STAT signalling.
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ACKNOWLEDGMENTS |
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Footnotes |
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