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Volume 271, Number 29,
Issue of July 19, 1996
pp. 17512-17518
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Nuclear Localization Signal of the POU
Domain Protein Tst-1/Oct6*
(Received for publication, March 7, 1996)
Elisabeth
Sock
,
Janna
Enderich
,
Michael G.
Rosenfeld
§¶ and
Michael
Wegner

From the Zentrum für Molekulare Neurobiologie,
Universität Hamburg, Martinistrasse 52, D-20246 Hamburg,
Federal Republic of Germany and the § Howard Hughes Medical
Institute, School of Medicine, University of California at San Diego,
La Jolla, California 92093-0648
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
ABSTRACT
POU domain proteins are important regulators of
development and terminal differentiation based upon their
transcriptional activity in the nucleus. Here, we analyzed the
mechanism underlying the nuclear localization of Tst-1/Oct6, a member
of this family that regulates events during neurogenesis and
myelination. Nuclear localization of Tst-1/Oct6 was dependent on the
POU domain, as its deletion prevented access to the nucleus, whereas
its transfer to the amino terminus of -galactosidase was sufficient
to prompt nuclear accumulation of this normally cytosolic protein.
Interestingly, nuclear localization and high affinity DNA binding were
two independent functions of the POU domain and could be separated in
several mutants. While specific high affinity binding to DNA required
the presence of both the POU-specific and the POU homeodomain, the
POU-specific domain was dispensable for nuclear localization of
Tst-1/Oct6. Rather, the nuclear localization function was selectively
contained within the POU homeodomain. Specifically, a basic cluster
(GRKRKKRT) preceding helix 1 of the homeodomain was shown by deletion
mutagenesis to be involved in the nuclear localization of Tst-1/Oct6.
This sequence, which is highly conserved among POU domain proteins, was
by itself capable of translocating -galactosidase to the nucleus
defining it as the bona fide nuclear localization signal of Tst-1/Oct6
and presumably other POU domain factors.
INTRODUCTION
POU domain proteins constitute a class of transcription factors
that all share a highly conserved DNA-binding domain and are usually
expressed during various critical, tightly controlled phases of
embryonic development or cellular differentiation (for review see Refs.
1, 2, 3, 4, 5). A typical expression pattern for POU domain proteins is
displayed by the class III member Tst-1, which is also known as Scip or
Oct6 (6, 7, 8, 9, 10, 11, 12). In the mouse, it is transcribed from an intronless gene
at the distal end of chromosome 4 that exhibits the features of an
expressed retroposon (13, 14). During development, expression of this
protein, which hereafter will be referred to as Tst-1/Oct6, is detected
in embryonic stem cells, skin, neuronal subpopulations, and in
precursors of myelinating glia (6, 11, 15, 16, 17). In the adult,
expression of Tst-1/Oct6 persists in pyramidal neurons of layer 5 in
the cerebral cortex and in skin (1, 6, 18).
Schwann cells which represent the myelinating glia of the peripheral
nervous system have been used extensively to study the function of
Tst-1/Oct6. In these cells, transient expression of Tst-1/Oct6 can be
induced by axons and by agents that raise the intracellular cAMP level
(10, 19). Expression of Tst-1/Oct6 correlates with a period of rapid
cell division which immediately precedes the onset of myelination (17).
In line with its supposed role during differentiation of myelinating
glial cells, targeted expression of a dominant negative form of
Tst-1/Oct6 in Schwann cells led to severe disturbances in the normal
myelination program (20).
Like all other POU domain proteins characterized so far (2, 3, 5),
Tst-1/Oct6 has to exert its function in the nucleus. Nuclear proteins
obligatorily enter this cellular compartment via the nuclear pore
complex after being synthesized in the cytoplasm (21). In general, they
are actively transported through the nuclear pore. This active
transport requires energy (22, 23), soluble factors, such as
importin- , importin- , and Ran/TC4 (24, 25, 26, 27), and a nuclear
localization signal (NLS)1 within the
protein targeted for nuclear import (for review see Ref. 28). NLSs were
first discovered in the yeast Mat 2 protein (29, 30) and in SV40
large T-antigen (31, 32) and are recognized by NLS-binding proteins
such as the importin- ·importin- complex. After the initial NLS
recognition in the cytosol which is mainly mediated by importin- ,
the complex docks to the nuclear pore via importin- , before it gets
translocated through the pore in a Ran/TC4-dependent manner
(25). NLSs have been identified in a variety of nuclear proteins
ranging in size from less than a hundred to more than a thousand amino
acids (33), including polymerases (34), kinases and phosphatases (35,
36), transcription factors (37, 38, 39), histones (40), growth factors (41,
42), tumor suppressors (43, 44), and various viral proteins (31, 32,
45, 46, 47, 48). Here, we have characterized the nuclear localization signal of
Tst-1/Oct6, which is localized in the protein's DNA-binding domain as
a structure highly conserved among POU domain proteins in general. Its
identification allows insights into the evolution of POU domain
proteins and points to ways of regulating the access of POU domain
proteins to the nucleus.
MATERIALS AND METHODS
Plasmid Constructs
Tst-1/Oct6 expression plasmid pCMV/Tst-1
as well as the mutants pCMV/Tst-1 N (missing amino acids 4-240),
pCMV/Tst-1 POU (missing amino acids 241-395), pCMV/Tst-1
POUHD (missing amino acids 336-395), pCMV/Tst-1 C
(missing amino acids 396-448), pCMV/Tst-1 NC (missing amino acids
4-240 and 396-448), and pCMV/Tst-1 mt (carrying a double point
mutation at amino acids 383 and 384 in the recognition helix of the POU
homeodomain) have been described before (49, 50). Additional point
mutants of Tst-1/Oct6 were created as follows. In pCMV/Tst-1 PM1 amino
acids 262 and 263 were changed from two arginines to aspartic acid and
serine; in pCMV/Tst-1 PM2 amino acids 269 and 270 were changed from
threonine and glutamine to methionine and glutamic acid; in pCMV/Tst-1
PM3 amino acids 286 and 287 were changed from serine and glutamine to
valine and aspartic acid; in pCMV/Tst-1 PM4 amino acids 292 and 294 were changed from arginine and glutamic acid to glutamic acid and
glutamine; in pCMV/Tst-1 PM5 amino acids 338-340 were changed from two
lysines and one arginine to three alanines; in pCMV/Tst-1 PM6 amino
acids 387 and 388 were changed from two arginines to valine and
aspartic acid; and in pCMV/Tst-1 dn amino acids 389 and 393 were
changed from a glutamine and an arginine to a glycine and a tryptophan.
The control plasmid pCMV/asTst-1 contained the region coding for
Tst-1/Oct6 inserted in antisense orientation relative to the
cytomegalovirus promoter. pRSVluc and pHSVoct-luc which contain the
firefly luciferase gene under the control of the Rous sarcoma
virus-long terminal repeat and a combination of herpes simplex virus
octamer element (5 -GCATGCTAATGATATTCTTT-3 ) and rat prolactin minimal
promoter, respectively, have been described before (50, 51).
All -galactosidase expression vectors were based on pCMVlacZ (a gift
of Dr. G. E. DiMattia, London Regional Cancer Centre, London, Canada)
which contained the lacZ gene under the control of the CMV
promoter. This lacZ gene lacked the first eight nonessential
amino acids and instead contained a eukaryotic translation consensus
sequence at its 5 end, and the SV40 small t intron A and
polyadenylation signal at its 3 end. A short sequence encompassing the
nuclear localization signal from the T-antigen of SV40 (32) was added
to the 5 end of the lacZ gene, yielding pCMVlacZ(SV40T
NLS). Similarly, fragments from the tst-1/oct6 gene were
added as NcoI fragments to the 5 end of lacZ.
pCMVlacZ(POU) contained sequences coding for amino acids 239-404 of
Tst-1/Oct6; pCMVlacZ(POUS) contained sequences coding for
amino acids 239-324; and pCMVlacZ(POUHD) contained
sequences coding for amino acids 328-404. Plasmid pCMVlacZ(Tst-1 NLS)
was obtained by inserting a short sequence corresponding to amino acids
334-341 of Tst-1/Oct6 (GRKRKKRT) between NcoI and
NruI sites directly behind the start methionine.
Tissue Culture, Transfection, and Preparation of Protein
Extracts
CV1 cells were maintained in Dulbecco's modified
Eagle's medium and U138 glioblastoma cells in RPMI 1640, both
supplemented with 10% fetal calf serum. One day prior to transfection,
CV1 or U138 cells were plated at a density of 2 × 105
per 60-mm plate in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum. Cells were transfected with 2 µg of
Tst-1/Oct6 expression vector and 2 µg of luciferase plasmid using the
calcium-phosphate technique as described (49, 52). Cells were harvested
48 h posttransfection for luciferase assays (49) or for the
preparation of extracts. Extracts were prepared as described (53) with
minor modifications (52). Cells from two 60-mm plates yielded 300 µl
of cytosolic extract and 150 µl of nuclear extract.
Western Blot Analysis
Western blot analysis was performed
as described (52). Detection of luciferase and Tst-1/Oct6 in
approximately 50 µg of cytoplasmic extract and 25 µg of nuclear
extract was achieved using rabbit polyclonal antisera against either
luciferase (gift of Dr. S. Subramani, La Jolla) or against full-length
Tst-1/Oct6 purified from baculovirus-infected Sf9 cells. The amount of
cytoplasmic and nuclear extract corresponded to the equivalent of
1.3 × 105 cells.
Electrophoretic Mobility Shift Assay
Double-stranded
oligonucleotides containing the herpes simplex virus octamer motif
(5 -GCATGCTAATGATATTCTTT) were generated and radiolabeled with Klenow
enzyme and [ -32P]dCTP. For electrophoretic mobility
shift assays, 0.5 ng of labeled probe was incubated with nuclear
extracts of cells transfected with Tst1/Oct6 expression plasmids for 20 min at room temperature in a 20-µl reaction containing 10 mM Hepes, pH 8.0, 5% glycerol, 100 mM NaCl, 5 mM MgCl2, 2 mM dithiothreitol, 0.1 mM EDTA, 2 µg of poly(dI-dC), and 0.5 µg of bovine
serum albumin. One-third of each reaction was loaded onto a 5%
nondenaturing acrylamide gel. Electrophoresis was in 0.5 × TBE at
200 V for 3 h.
Immunocytochemistry and -Galactosidase
Histochemistry
CV1 cells were seeded on chamber slides (Lab-Tec,
Nunc Inc.) and transfected as described above. 48 h
posttransfection, the medium was removed, and the cells were washed
twice with PBS. Cells were then fixed with 3% formaldehyde in PBS for
20 min and treated with 1% Triton X-100 in PBS for 5 min. After
washing the cells twice with PBS, cells were incubated for 20 min with
the polyclonal anti-Tst1 antiserum (diluted 1:1000) in PBS containing
0.1% Tween 20 (PBST). After washing three times with PBST, cells were
incubated 20 min with Cy3-conjugated goat anti-rabbit
antibodies (Dianova) diluted 1:500 in PBST. Cells were washed
extensively with PBST, mounted, and analyzed on an Axiovert microscope
(Zeiss).
-Galactosidase histochemistry was carried out as described (54).
Shortly, cells were fixed for 5 min at 4 °C in PBS containing 2%
formaldehyde and 0.2% glutaraldehyde. After washing, cells were
stained in PBS containing 1 mg/ml X-Gal, 5 mM potassium
ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2.
RESULTS
Tst-1/Oct6 Is a Predominantly Nuclear Protein
Using
electrophoretic mobility shift assays as well as Western blot analysis,
Tst-1/Oct6 has been shown in previous studies to be present in nuclear
extracts from embryonic stem cells, embryonic carcinoma cells,
proliferating Schwann cells, oligodendrocyte precursors, and stably
transformed fibroblasts (9, 11, 13, 15). To be able to study the
cellular distribution of Tst-1/Oct6 in more detail, we used rabbit
polyclonal antibodies raised against full-length Tst-1/Oct6 protein
that contained a 6xHIS-tag fused to its amino terminus and was purified
from baculovirus-infected Sf9 cell extracts. When tested on nuclear and
cytosolic extracts prepared from embryonic stem cells or U138 glial
cells transiently transfected with Tst-1/Oct6, this antibody recognized
specifically and at high dilution a protein of 47,000 that was enriched
in the nucleus (Fig. 1, and data not shown). This
protein was absent from U138 cells not transfected with an expression
vector for Tst-1/Oct6 showing that this protein was indeed Tst-1/Oct6.
A small fraction of Tst-1/Oct6 was also found in the cytosolic
extracts. It is unclear at present whether this is an artifact of the
extract preparation or represents truly cytosolic Tst-1/Oct6.
Fig. 1.
Nuclear localization of Tst-1/Oct6 maps to
the POU domain. A, Western blot analysis was performed on
cytosolic and nuclear extracts prepared from U138 cells transfected
transiently with wild-type (wt) Tst-1/Oct6 or various
mutants. The protein present in each pair of extracts is depicted
above the lanes. For the detection of Tst-1/Oct6
(upper panels), nitrocellulose filters were incubated with
anti-Tst-1 antiserum at a dilution of 1:3000. To evaluate
cross-contamination of extract fractions, the distribution of
luciferase in both cytosolic (Cyt) and nuclear
(NE) fractions was analyzed by reprobing the same
nitrocellulose filters with anti-luciferase antiserum at a dilution of
1:3000 (lower panels). Horseradish peroxidase-coupled
protein A was used in each case to develop the blot. Molecular weight
markers are shown. B, electrophoretic mobility shift
analysis of some of the extracts tested in A. In addition to
the free herpes simplex virus octamer probe on the bottom of the gel,
complexes of different mobility were detected for mutants C and
NC, corresponding to their sizes.
The POU Domain Targets Tst-1/Oct6 to the Nucleus
To be able
to map the region of Tst-1/Oct6 responsible for its predominantly
nuclear localization, we used a series of mutants in which single
domains of the Tst-1/Oct6 protein had been removed (Table
I). Their DNA binding activity and ability for
transcriptional activation were assessed in standard electrophoretic
mobility shift assays and transient transfection assays. As summarized
in Table I, the results correlated well with previously published data
mapping DNA binding to the POU domain and transactivation function to
the amino-terminal part of the protein (50, 55, 56).
To determine the intracellular localization of Tst-1/Oct6 deletion
mutants, we cotransfected Tst-1/Oct6 and the luciferase expression
plasmid pRSVluc into various cell types and performed cell
fractionation studies. The results obtained in U138 glial cells are
shown in Fig. 1, but identical results were also obtained in CV1 and
HeLa cells (data not shown). The quality of cytoplasmic and nuclear
extracts was assessed in each case by Western blot analysis with a
rabbit polyclonal antiserum directed against luciferase. Because
luciferase is known to be a cytoplasmic protein present in the
peroxisomes, it should preferably be detected in the cytoplasmic
fraction, while it should be absent from the nuclear fraction. As shown
in the bottom panel of Fig. 1, most of the luciferase is
indeed localized in the cytoplasm, showing that contamination of the
nuclear fraction with cytosol was minimal. Contamination of cytosol
with nuclear proteins was equally low as evidenced by the distribution
pattern of a high molecular weight band that exhibited weak
cross-reactivity with the anti-Tst-1/Oct6 antiserum (data not shown).
U138 cells transfected with wild-type Tst-1/Oct6 contained a 47,000 protein not present in mock-transfected cells, which was highly
enriched in the nuclear fraction (Fig. 1). A similar pattern of
distribution was also found for mutant Tst-1/Oct6 proteins that either
had all sequences carboxyl-terminal ( C) or amino-terminal ( N) of
the POU domain deleted.
When a mutant was assayed that just contained the POU domain of
Tst-1/Oct6 ( NC), no immunoreactivity could be observed in Western
blot analysis (Fig. 1A). The fact that this mutant was not
detected was explained by the specificity of the antiserum that reacted
with epitopes in the regions both amino- and carboxyl-terminal of the
POU domain, but not with the POU domain itself. A bacterially expressed
POU domain of Tst-1/Oct6, for instance, was not recognized by the
antiserum (data not shown). Electrophoretic mobility shift analysis, on
the other hand, clearly revealed a specific complex of high mobility,
indicative of this mutant, that was predominantly found in the nuclear
extract and comigrated with the complex formed by the bacterially
expressed POU domain of Tst-1/Oct6 (Fig. 1B and data not
shown).
To corroborate the importance of the POU domain for nuclear
localization and address the possibility of additional nuclear
localization signals outside the POU domain, we deleted the POU domain
from the protein and analyzed the cellular distribution of this mutant
( POU; Fig. 1A). Contrary to all other mutants analyzed,
this mutant was predominantly found in the cytosolic fraction. These
results imply that the POU domain is both necessary and sufficient to
target Tst-1/Oct6 to the nucleus.
The POU Homeodomain Is Essential for Nuclear
Localization
Analysis of nuclear localization by cell
fractionation has been shown in the past to be susceptible to artifacts
(57). During extract preparation, nuclear proteins that were not bound
to nuclear constituents leaked from the nucleus and were recovered in
the cytosol despite carrying a nuclear localization signal. Thus it is
conceivable that the POU mutant was retrieved in the cytosolic
fraction merely because of its inability to bind to DNA not, however,
because of the true absence of a nuclear localization signal.
Therefore, we tried to corroborate and extend our cell fractionation
studies by immunocytochemistry on transiently transfected CV1 cells.
The antiserum raised against Tst-1/Oct6 yielded only a weak background
staining when employed to mock-transfected cells (Fig.
2A). Transfection with an expression plasmid
for wild-type Tst-1/Oct6, on the other hand, led to an intense staining
of cell nuclei (Fig. 2B). Comparable nuclear staining was
also detected with mutants N and C, indicating that these regions
of Tst-1/Oct6 are dispensable for nuclear localization (Fig. 2,
C and F). In contrast, the POU mutant was
present predominantly in the cytosol, leading to a uniform, dispersed
labeling of the entire cell (Fig. 2D). This obvious
agreement between cell fractionation studies and immunocytochemistry
strongly confirms the role of the POU domain in targeting Tst-1/Oct6 to
the cell nucleus.
Fig. 2.
Immunocytochemistry studies on various
Tst-1/Oct6 mutants. CV1 cells transfected with expression plasmids
for various Tst-1/Oct6 mutants were analyzed by indirect
immunofluorescence using rabbit anti-Tst1 antiserum (diluted 1:1000) as
primary and Cy3-conjugated goat anti-rabbit antibodies as
secondary antibodies (diluted 1:500). Approximately 1 in 10 cells were
transfected. A, control of mock-transfected CV1 cells;
B, cells transfected with expression plasmids for Tst-1 wild
type; C, Tst-1 N; D, Tst-1 POU;
E, Tst-1 POUHD; and F, Tst-1
C.
Intriguingly, a comparable loss of nuclear localization was observed
with a mutant, which had only the POU homeodomain removed but still
retained the POU-specific domain ( POUHD, Fig.
2E). This clearly points to the importance of the POU
homeodomain for nuclear localization.
The POU Homeodomain Is Sufficient to Direct -Galactosidase to
the Cell Nucleus
For a closer inspection we transferred parts of
Tst-1/Oct6 to a heterologous protein and evaluated the localization of
the corresponding fusions. The fusion partner was cytosolic
-galactosidase (Fig. 3B). By the
amino-terminal addition of a short stretch of amino acids, which
represents the nuclear localization signal (NLS) of SV40 T-antigen,
-galactosidase could, however, be directed to the cell nucleus as
shown before (Fig. 3C) (58). When the POU domain of
Tst-1/Oct6 was placed onto LacZ instead of the NLS of SV40 T-antigen,
it was also able to target the fusion protein to the cell nucleus (Fig.
3D). In contrast, nuclear localization was not achieved by
fusing the POU domain coding sequences in the opposite orientation to
the lacZ gene, although a protein of the correct size was
expressed in transfected cells (data not shown). This transfer
experiment further corroborated the importance of the POU domain in the
nuclear localization of Tst-1/Oct6.
Fig. 3.
Cellular localization of -galactosidase
fusion proteins. CV1 cells were transfected with expression
plasmids for various -galactosidase fusion proteins. Localization of
-galactosidase in transfected cells was determined by histochemical
X-Gal staining as described under ``Materials and Methods.''
A, control of mock-transfected CV1 cells; B,
cells transfected with expression plasmids for -galactosidase;
C, SV40T-NLS -galactosidase; D, Tst-1 POU
-galactosidase; E, Tst-1 POUS
-galactosidase; F, and Tst-1 POUHD
-galactosidase.
In addition to the intact POU domain, we also transferred isolated
regions of it to LacZ. The LacZ(POUHD) fusion contained the
POU homeodomain of Tst-1/Oct6 as well as seven amino acids from the
preceding linker region, whereas the LacZ(POUS) fusion
carried the POU-specific domain of Tst-1/Oct6 and most of the adjacent
linker region which joins it to the POU homeodomain. Nuclear
localization was only observed for the fusion protein between the POU
homeodomain and -galactosidase (Fig. 3F). The
POU-specific domain, on the contrary, proved to be incapable of
directing -galactosidase to the nucleus as the fusion between LacZ
and the combination of POU-specific domain and adjacent linker remained
cytosolic (Fig. 3E).
A Basic Cluster in the POU Homeodomain Is Involved in the Nuclear
Localization of Tst-1/Oct6
To further define the nuclear
localization signal present in the POU domain, we substituted highly
conserved amino acids both in the POU-specific and the POU homeodomain
(Table II). These regions were chosen because of their
proven relevance for the function of the POU domain (59, 60, 61, 62). With the
exception of mutant dn which affected a glutamine and an arginine
residue in helix 3 of the POU homeodomain, all other mutants had lost
their ability to bind to DNA. Concomitant with their loss of DNA
binding these mutants failed to stimulate transcription from an
octamer-containing promoter (Table II).
Table II.
Characteristics of Tst-1/Oct6 point mutants
|
Mutated
amino acids |
Localization of mutation |
Activation of HSV
oct-luca |
DNA bindingb |
Subcellular
localizationc
|
|
| PM1 |
262R-D,
263R-S |
POUS,
helix
1 |
 |
 |
Nuclear
|
| PM2 |
269T-M, 270Q-E |
POUS, helix
2 |
 |
 |
Nuclear |
| PM3 |
286S-V,
287Q-D |
POUS, helix 3 |
 |
 |
Nuclear
|
| PM4 |
292R-E, 294E-Q |
POUS, helix
3 |
 |
 |
Nuclear |
| PM5 |
338K-A, 339K-A,
340R-A |
POUHD, basic
cluster |
 |
 |
Cytoplasmic |
| PM6 |
387R-V,
388R-D |
POUHD, helix 3 |
 |
 |
Nuclear
|
| mt |
383W-C, 384F-S |
POUHD, helix
3 |
 |
 |
Nuclear |
| dn |
389Q-G,
393R-W |
POUHD, helix 3 |
+ |
+ |
Nuclear |
|
|
a
Transcriptional activation of HSV oct luc was
determined by transient transfection in U138 cells as described
previously in at least three independent experiments, each performed in
duplicate.
|
|
b
DNA binding was determined in standard electrophoretic
mobility shift assays with the HSV octamer element as a probe and whole
cell extract from transiently transfected U138 cells as a protein
source.
|
|
c
Cellular localization was determined in immunocytochemistry
studies on CV1 cells transiently transfected with the respective
Tst-1/Oct6 mutants.
|
|
All mutants that affected the POU-specific domain (PM1, PM2, PM3, and
PM4) were still found to be nuclear in immunocytochemistry studies,
although none of them exhibited significant binding to an octamer DNA
element. This finding is particularly noteworthy for the mutation
present in PM1, which led to a disruption of the cluster of basic amino
acids within helix 1 of the POU-specific domain that on the basis of
its positive charge would have been a good candidate for a nuclear
localization signal (28). In agreement with our transfer experiments,
these results argue against a participation of the POU-specific domain
in the nuclear localization of Tst-1/Oct6.
Similar to the mutations in the POU-specific domain, most homeodomain
mutants remained unaffected in their nuclear localization despite being
defective in their DNA binding activity and transcriptional activation
function. None of the proteins with mutations in helix 3 of the POU
homeodomain were impaired in their nuclear localization function,
although at least two of them (PM6 and dn) disrupted the basic cluster
present in this DNA recognition helix. Therefore, it seemed unlikely
that helix 3 of the POU homeodomain was involved in nuclear
localization. This conclusion was also supported by the finding that
helix 3 when transferred to a cytosolic form of luciferase was not
able to target this protein to the nucleus (data not shown).
In marked contrast to all other analyzed mutants, PM5 exhibited a
strong cytoplasmic staining (Fig. 4A). PM5
had three positive charges removed from the basic cluster at the
beginning of the POU homeodomain. Thus, while there are three basic
clusters in the POU domain, only the one preceding helix 1 of the POU
homeodomain was involved in nuclear localization.
Fig. 4.
Functional analysis of the NLS from
Tst-1/Oct6. A, immunocytochemistry on cells transiently
transfected with Tst-1/Oct6 mutant PM5 using rabbit anti-Tst1 antiserum
(diluted 1:1000) as primary and Cy3-conjugated goat
anti-rabbit antibodies as secondary antibodies (diluted 1:500).
B, histochemical X-Gal staining of -galactosidase in
cells transfected with Tst-1 NLS -galactosidase.
A Basic Cluster from the POU Homeodomain of Tst-1/Oct6 Is
Sufficient to Direct -Galactosidase to the Cell Nucleus
To
analyze whether this basic cluster alone represented the nuclear
localization signal of Tst-1/Oct6 or functioned only in the context of
the POU homeodomain as part of a more complex signal, we transferred
the isolated basic cluster onto the amino terminus of LacZ and analyzed
the resulting -galactosidase fusion for its cellular localization.
As shown in Fig. 4B, this cluster was sufficient to direct
-galactosidase to the nucleus of transfected cells. Indeed, this
short stretch of amino acids from Tst-1/Oct6 was as efficient in
targeting a heterologous protein to the cell nucleus as the NLS from
SV40 T-antigen and might be regarded as the NLS of Tst-1/Oct6.
DISCUSSION
Most proteins that enter the nucleus do so by an active transport
through the nuclear pore that requires the presence of an NLS in the
transported protein. Although there is no consensus sequence, NLS are
usually short sequences that contain a high proportion of positively
charged amino acids. They occur at various locations within the protein
in single or multiple, functionally redundant copies. They can be
contained within a contiguous stretch of amino acids or may be bi- or
multipartite instead (28, 33). Furthermore, NLS can be strongly
dependent in their function on the exact flanking sequences (63, 64).
Deletion of an NLS from a nuclear protein leads to its redistribution
to the cytoplasm, whereas its addition to a heterologous cytoplasmic
protein often results in an accumulation of this protein in the
nucleus.
The present study shows that the POU domain of Tst-1/Oct6 harbors a
signal that fulfills the above-mentioned criteria of an NLS.
Consequently, deletion of the POU domain caused the resulting
Tst-1/Oct6 mutant to localize to the cytoplasm, whereas its addition to
the usually cytoplasmic -galactosidase caused it to become nuclear.
Thus, the POU domain is not only involved in DNA binding (62), and
interaction with a whole series of cellular and viral coactivators (49,
65, 66, 67, 68, 69), it also contains the protein's nuclear localization function.
Our study, therefore, lends further proof to the multifunctional
character and the tremendous importance of the POU domain in this class
of transcription factors.
Mapping of the NLS was refined in consecutive studies to the POU
homeodomain. Transfer of the POU-specific domain to -galactosidase,
on the other hand, did not cause the fusion protein to become nuclear.
The absence of a nuclear localization function from the POU-specific
domain was emphasized by the fact that none of the amino acid changes
introduced into the four -helices of the POU-specific domain (59,
60) disrupted nuclear localization, although severely impeding other
functions of Tst-1/Oct6. Not even the removal of two positive charges
from the basic cluster within helix 1 of the POU-specific domain in PM1
prevented Tst-1/Oct6 from becoming nuclear. Our conclusions are also
supported by the fact that a naturally occurring splice variant of the
POU domain protein Pit-1 which did not contain a POU-specific domain
was found to enter the nucleus and serve as a dominant repressor of
Pit-1 function (70, 71).
Taking the accumulation of positively charged amino acids within
nuclear localization signals into account, the POU homeodomain contains
two candidate NLS. One of these clusters of basic residues is located
in front of helix 1 of the homeodomain and the other within helix 3, the DNA recognition helix. When these basic clusters were disrupted,
strongly divergent results were obtained for each cluster. Whereas the
basic cluster within helix 3 of the POU homeodomain was dispensable for
nuclear localization, destruction of the basic cluster that precedes
helix 1 of the POU homeodomain caused an effective redistribution of
Tst-1/Oct6 from the nucleus into the cytosol. A peptide (GRKRKKRT)
corresponding to the same basic cluster (position 334-341) conferred
nuclear localization onto a heterologous cytosolic protein. By
targeting -galactosidase to the nucleus, this region was clearly
defined as the bona fide NLS of Tst-1/Oct6. To our knowledge, this is
the first NLS mapped in any POU domain protein.
A prerequisite for the function of nuclear localization signals is
their proper exposure on the surface of the protein. The basic cluster
preceding helix 1 of the POU homeodomain fulfills this criterion.
Although being involved in DNA binding through a series of contacts
with the phosphate backbone (Lys338, Arg340,
Thr341), and a base in the minor groove of DNA
(Arg340), this region is solvent-exposed and readily
accessible for NLS-binding proteins (62).
As shown in Table III, this basic cluster is highly
conserved among POU domain proteins. In fact, there is only one
exception known so far. In I-Pou, a Drosophila class IV POU
domain protein, the number of basic residues is reduced to only three
(72). That I-Pou still enters the nucleus is probably best explained by
the fact that it is also the only POU domain protein that contains
three consecutive basic residues in the linker region preceding the
residual cluster by 12 amino acids. The resulting sequence
KRRDPDAPSVLPAGEKKRT conforms to the consensus for
bipartite NLS that consists of two clusters of basic residues
separated by 10-12 amino acids (28). This peculiarity of the I-Pou
sequence might very well explain why I-Pou is the only POU domain
protein that could afford a disruption of the basic cluster in front of
helix 1 of its homeodomain.
Table III.
Comparison of the NLS from Tst-1/Oct6 with corresponding regions of
other POU domain proteins
| POU class |
POU protein |
Amino acid sequence
|
|
| I |
Pit-1 |
ERKRKRRTT
|
| II |
Oct-1 |
SRRRKKRTS
|
|
Oct-2 |
GRRRKKRTS
|
|
Skn-1 |
GRKRKKRTS
|
| III |
Brn-1 |
GRKRKKRTS
|
|
Brn-2 |
GRKRKKRTS
|
|
Tst-1/Oct6 |
GRKRKKRTS
|
|
Brn-4 |
GRKRKKRTS
|
| IV |
Brn-3.0 |
GEKKRKRTS
|
|
Brn-3.1 |
SERKRKRTS
|
|
Brn-3.2 |
AEKKRKRTS
|
| V |
Oct-3/4 |
QARKRKRTS
|
|
Sprm-1 |
QARKRRRAS
|
| VI |
Emb/Brn-5 |
SKKRKRRTS
|
|
| Homeo |
En |
NDEKRPRTA |
|
Mat
2 |
STKPYRGHR |
|
The high degree of conservation of the basic cluster is even more
striking in light of the significant differences in the flanking
regions between the various classes of POU domain proteins. It strongly
argues in favor of a model in which this basic cluster functions as a
nuclear localization signal in most POU domain proteins. Therefore, we
have most likely identified a general mechanism of nuclear entry for
POU domain proteins. We expect our results for Tst-1/Oct6 to be
prototypic for other members of the whole family of POU domain
proteins.
Interestingly, the high conservation of this basic region within the
POU domain family contrasts sharply with its apparent absence in
several classic homeodomain proteins. The yeast Mat 2 protein, for
instance, has two NLS sequences that function independently of each
other and are both localized at positions different from the NLS of
Tst-1/Oct6 (29, 30). Thus, the NLS seems to be a recent acquisition of
POU domain proteins during evolution. It deserves to be noticed that
the presence of an NLS in the DNA-binding domain of the protein
is a principle also realized in other classes of transcription
factors, including HMG box, HLH, and bZip proteins (37, 38, 39). Although
DNA binding and nuclear localization are functionally separable,
colocalization of both functions might have evolved as a consequence of
exon shuffling in higher eukaryotes.
The NLS of Tst-1/Oct6 is structurally very similar to the NLS of SV40
T-antigen (PKKKRKV) (31). Unlike the majority of nuclear proteins that
contain bipartite NLS (28), Tst-1/Oct6 and T-antigen have single
contiguous stretches of positively charged residues that serve as their
NLS. The analogy reaches even further. The NLS of SV40 T-antigen is in
close proximity to phosphorylation sites for casein kinase II and
cdk/cdc2. Phosphorylation of these sites has been shown to increase or
reduce the rate of nuclear import, respectively (73, 74).
Interestingly, the NLS of Tst-1/Oct6 overlaps with a potential
phosphorylation site for protein kinase A and an as yet unidentified
M-phase-specific kinase, which similar to the NLS itself is strongly
conserved among POU proteins and has been shown to be the target of
phosphorylation in the related POU domain proteins Pit-1 (75, 76) and
Oct-1 (77). Although not observed so far, it is intriguing to speculate
that phosphorylation at this site could regulate nucleocytoplasmic
transport of POU domain proteins in a manner similar to other proteins
such as T-antigen (for review see Ref. 78). This would provide yet
another means for regulation of POU domain protein activity.
As mentioned above, the POU domain is also the main region for
interactions between members of this class of transcription factors and
other cellular proteins (49, 65, 66, 67, 68, 69, 79). It is easily conceivable that
such interactions also influence the accessibility of the NLS of
Tst-1/Oct6. The POU domain might be an excellent target for
interactions with cytosolic retention factors similar to the ones
identified in other classes of transcription factors (80). Our
characterization of the NLS of Tst-1/Oct6 should therefore open new
directions in the functional analysis of POU domain proteins.
FOOTNOTES
*
This work was supported by Grant We 1326/5-1 from the
Deutsche Forschungsgemeinschaft (to M. W.) and a grant from the
National Institutes of Health (to M. G. R.). The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
``advertisement'' in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Investigator with Howard Hughes Medical Institute.
To whom correspondence should be addressed: ZMNH, Interim I,
Pav. 22, Martinistr. 52, D-20246 Hamburg, Germany. Tel.: 49 40 4717 4708; Fax: 49 40 4717 4774.
1
The abbreviations used are: NLS, nuclear
localization signal; X-Gal, 5-bromo-4-chloro-3-indolyl
-D-galactoside; PBS, phosphate-buffered saline.
Acknowledgments
We thank Dr. G. DiMattia for the gift of
pCMV-lacZ and Dr. S. Subramani for providing pRSVluc and
rabbit polyclonal antiserum directed against luciferase. We are
especially grateful to Dr. S. Rhodes for his help with the production
of the polyclonal antibodies against Tst-1/Oct6.
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Development,
March 15, 2007;
134(6):
1133 - 1140.
[Abstract]
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I. Miyata, S. Vallette-Kasic, A. Saveanu, M. Takeuchi, H. Yoshikawa, A. Tajima, K. Tojo, R. Reynaud, M. Gueydan, A. Enjalbert, et al.
Identification and Functional Analysis of the Novel S179R POU1F1 Mutation Associated with Combined Pituitary Hormone Deficiency
J. Clin. Endocrinol. Metab.,
December 1, 2006;
91(12):
4981 - 4987.
[Abstract]
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S. Kellerer, S. Schreiner, C. C. Stolt, S. Scholz, M. R. Bosl, and M. Wegner
Replacement of the Sox10 transcription factor by Sox8 reveals incomplete functional equivalence
Development,
August 1, 2006;
133(15):
2875 - 2886.
[Abstract]
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S. Wissmuller, T. Kosian, M. Wolf, M. Finzsch, and M. Wegner
The high-mobility-group domain of Sox proteins interacts with DNA-binding domains of many transcription factors.
Nucleic Acids Res.,
January 1, 2006;
34(6):
1735 - 1744.
[Abstract]
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C. Baranek, E. Sock, and M. Wegner
The POU protein Oct-6 is a nucleocytoplasmic shuttling protein
Nucleic Acids Res.,
October 31, 2005;
33(19):
6277 - 6286.
[Abstract]
[Full Text]
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R. P. Friedrich, B. Schlierf, E. R. Tamm, M. R. Bosl, and M. Wegner
The Class III POU Domain Protein Brn-1 Can Fully Replace the Related Oct-6 during Schwann Cell Development and Myelination
Mol. Cell. Biol.,
March 1, 2005;
25(5):
1821 - 1829.
[Abstract]
[Full Text]
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H. Kasahara and D. W. Benson
Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies
Cardiovasc Res,
October 1, 2004;
64(1):
40 - 51.
[Abstract]
[Full Text]
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U. Vijapurkar, N. Fischbach, W. Shen, C. Brandts, D. Stokoe, H. J. Lawrence, and C. Largman
Protein Kinase C-Mediated Phosphorylation of the Leukemia-Associated HOXA9 Protein Impairs Its DNA Binding Ability and Induces Myeloid Differentiation
Mol. Cell. Biol.,
May 1, 2004;
24(9):
3827 - 3837.
[Abstract]
[Full Text]
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S. Weiss, I. Gottfried, I. Mayrose, S. L. Khare, M. Xiang, S. J. Dawson, and K. B. Avraham
The DFNA15 Deafness Mutation Affects POU4F3 Protein Stability, Localization, and Transcriptional Activity
Mol. Cell. Biol.,
November 15, 2003;
23(22):
7957 - 7964.
[Abstract]
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M. Kishimoto, Y. Okimura, K. Yagita, G. Iguchi, M. Fumoto, K. Iida, H. Kaji, H. Okamura, and K. Chihara
Novel Function of the Transactivation Domain of a Pituitary-specific Transcription Factor, Pit-1
J. Biol. Chem.,
November 15, 2002;
277(47):
45141 - 45148.
[Abstract]
[Full Text]
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K. Hosoda, A. Imamura, E. Katoh, T. Hatta, M. Tachiki, H. Yamada, T. Mizuno, and T. Yamazaki
Molecular Structure of the GARP Family of Plant Myb-Related DNA Binding Motifs of the Arabidopsis Response Regulators
PLANT CELL,
September 1, 2002;
14(9):
2015 - 2029.
[Abstract]
[Full Text]
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M. Ilia, C. Beasley, D. Meijer, R. Kerwin, D. Cotter, I. Everall, and J. Price
Expression of Oct-6, a POU III Domain Transcription Factor, in Schizophrenia
Am J Psychiatry,
July 1, 2002;
159(7):
1174 - 1182.
[Abstract]
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H. Niwa, S. Masui, I. Chambers, A. G. Smith, and J.-i. Miyazaki
Phenotypic Complementation Establishes Requirements for Specific POU Domain and Generic Transactivation Function of Oct-3/4 in Embryonic Stem Cells
Mol. Cell. Biol.,
March 1, 2002;
22(5):
1526 - 1536.
[Abstract]
[Full Text]
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N. Bondurand, M. Girard, V. Pingault, N. Lemort, O. Dubourg, and M. Goossens
Human Connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10
Hum. Mol. Genet.,
November 1, 2001;
10(24):
2783 - 2795.
[Abstract]
[Full Text]
[PDF]
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B. Brockmann, M. W. Smith, A. G. Zaraisky, K. Harrison, K. Okada, and Y. Kamiya
Subcellular Localization and Targeting of Glucocorticoid Receptor Protein Fusions Expressed in Transgenic Arabidopsis thaliana
Plant Cell Physiol.,
September 1, 2001;
42(9):
942 - 951.
[Abstract]
[Full Text]
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Y. Fei and T. E. Hughes
Nuclear Trafficking of Photoreceptor Protein Crx: The Targeting Sequence and Pathologic Implications
Invest. Ophthalmol. Vis. Sci.,
September 1, 2000;
41(10):
2849 - 2856.
[Abstract]
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R. I. Peirano and M. Wegner
The glial transcription factor Sox10 binds to DNA both as monomer and dimer with different functional consequences
Nucleic Acids Res.,
August 15, 2000;
28(16):
3047 - 3055.
[Abstract]
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R. I. Peirano, D. E. Goerich, D. Riethmacher, and M. Wegner
Protein Zero Gene Expression Is Regulated by the Glial Transcription Factor Sox10
Mol. Cell. Biol.,
May 1, 2000;
20(9):
3198 - 3209.
[Abstract]
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E. E. Tuerk, J. Schreiber, and M. Wegner
Protein Stability and Domain Topology Determine the Transcriptional Activity of the Mammalian Glial Cells Missing Homolog, GCMb
J. Biol. Chem.,
February 18, 2000;
275(7):
4774 - 4782.
[Abstract]
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J. Bryan and M. Morasso
The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1
J. Cell Sci.,
January 11, 2000;
113(22):
4013 - 4023.
[Abstract]
[PDF]
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H. Kasahara and S. Izumo
Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5
Mol. Cell. Biol.,
January 1, 1999;
19(1):
526 - 536.
[Abstract]
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K. Kuhlbrodt, C. Schmidt, E. Sock, V. Pingault, N. Bondurand, M. Goossens, and M. Wegner
Functional Analysis of Sox10 Mutations Found in Human Waardenburg-Hirschsprung Patients
J. Biol. Chem.,
September 4, 1998;
273(36):
23033 - 23038.
[Abstract]
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A. Spit, R. H. Hyland, E. J. C. Mellor, and L. A. Casselton
A role for heterodimerization in nuclear localization of a homeodomain protein
PNAS,
May 26, 1998;
95(11):
6228 - 6233.
[Abstract]
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K. Kuhlbrodt, B. Herbarth, E. Sock, I. Hermans-Borgmeyer, and M. Wegner
Sox10, a Novel Transcriptional Modulator in Glial Cells
J. Neurosci.,
January 1, 1998;
18(1):
237 - 250.
[Abstract]
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J. Schreiber, E. Sock, and M. Wegner
The regulator of early gliogenesis glial cells missing is a transcription factor with a novel type of DNA-binding domain
PNAS,
April 29, 1997;
94(9):
4739 - 4744.
[Abstract]
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G. E. Parker, R. M. Sandoval, H. A. Feister, J. P. Bidwell, and S. J. Rhodes
The Homeodomain Coordinates Nuclear Entry of the Lhx3 Neuroendocrine Transcription Factor and Association with the Nuclear Matrix
J. Biol. Chem.,
July 28, 2000;
275(31):
23891 - 23898.
[Abstract]
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T. M. Sugihara, E. I. Kudryavtseva, V. Kumar, J. J. Horridge, and B. Andersen
The POU Domain Factor Skin-1a Represses the Keratin 14 Promoter Independent of DNA Binding. A POSSIBLE ROLE FOR INTERACTIONS BETWEEN Skn-1a AND CREB-BINDING PROTEIN/p300
J. Biol. Chem.,
August 24, 2001;
276(35):
33036 - 33044.
[Abstract]
[Full Text]
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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.
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