From the Institut für Biochemie und Molekulare Zellbiologie der Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany
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ABSTRACT |
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DNA binding activity and nuclear transport of
B-Myb in Xenopus oocytes are negatively regulated. Two
distinct sequence elements in the C-terminal portion of the protein are
responsible for these different inhibitory activities. A C-terminal
Xenopus B-Myb protein fragment inhibits the DNA binding
activity of the N-terminal repeats in trans, indicating
that intramolecular folding may result in masking of the DNA binding
function. Xenopus B-Myb contains two separate nuclear
localization signals (NLSs), which, in Xenopus oocytes,
function only outside the context of the full-length protein. Fusion of
an additional NLS to the full-length protein overcomes the inhibition
of nuclear import, suggesting that masking of the NLS function rather
than cytoplasmic anchoring is responsible for the negative regulation
of Xenopus B-Myb nuclear transfer. During
Xenopus embryogenesis, when inhibition of nuclear import is
relieved, Xenopus B-myb is preferentially
expressed in the developing nervous system and neural crest cells.
Within the developing neural tube, Xenopus B-myb
gene transcription occurs preferentially in proliferating,
non-differentiated cells.
The Myb-type DNA binding repeat motif defines a growing family of
proteins in animals, plants, and lower eukaryotes. Three members of
this family, termed c-Myb, A-Myb, and B-Myb, have been described in
vertebrates (reviewed in Ref. 1). Though structurally related, these
three vertebrate Myb variants appear to fulfill very different
biological functions. On the basis of their expression characteristics,
it has been proposed that c-Myb and A-Myb exert cell type-specific
activities, whereas B-Myb seems to serve a more general function.
B-myb expression in Xenopus is maternal, and
transcripts can be detected throughout embryogenesis as well as in
several adult tissues (2). The human and murine B-myb
encoding genes are also widely expressed in different tissues during
embryogenesis, with differential expression during testis development
(3-5). Interestingly, B-myb expression in mammals has been
linked to proliferative activity (5). Earlier B-Myb promoter studies have already identified a negative regulatory element that is responsible for repression of B-myb transcription in
G0 via the activity of the E2F DNA-binding protein (6).
B-myb expression is induced at the G1/S
transition of the cell cycle (7-10). Furthermore, ectopic expression
of B-myb can induce DNA synthesis in certain cell types
(11). Taken together, these observations strongly suggest that B-Myb
has a general function in cell division.
Mapping of functional domains in human B-Myb identified a DNA-binding
domain located at the N terminus, followed by a transcriptional activation domain; it has been reported that the carboxyl portion of
the human protein contains two separate nuclear localization signals
(12, 13). B-Myb can activate transcription through two very different
mechanisms. The first is independent from its DNA-binding domain and
operates, probably indirectly, via the heat shock element (14), whereas
the second depends on the B-Myb DNA-binding domain and works on
promoters that contain B-Myb DNA-binding sites (15). The latter
mechanism has been found to depend on the cell-type utilized in
transient transfection studies, and this has been suggested to reflect
the requirement for a specific cofactor in transactivation (3). The
direct physical interaction of B-Myb with another protein may also
negatively regulate its activity; inhibition of
B-Myb-dependent transactivation correlates with specific
binding of p107, an Rb-related protein (11).
More recently, it has been demonstrated that the transactivation
function of B-Myb is subject to post-translational regulation in a cell
cycle-dependent manner. Phosphorylation by cdk2 in the C-terminal portion of the protein is required to activate the transactivation function of full-length B-Myb (16-18). A similar effect in transient transfection can be reproduced by C-terminal truncations (17, 18). It therefore appears that the B-Myb C terminus
negatively regulates transactivation and that this repression is
relieved by cell cycle-dependent phosphorylation. However,
the exact molecular events that are initiated by this modification
remain to be solved.
We have previously described that both DNA binding activity and nuclear
transfer of the full-length B-Myb protein are repressed in stage V/VI
Xenopus oocytes and that DNA binding activity is relieved
upon C-terminal truncation of the protein. Furthermore, we were able to
demonstrate that maternal B-Myb becomes phosphorylated upon meiotic
maturation, but that these phosphorylation events were not sufficient
to activate DNA binding of the full-length Xenopus B-Myb
protein (2, 19).
This study elucidates the molecular mechanisms that are responsible for
the negative regulation of DNA binding activity and nuclear transport
of Xenopus B-Myb. Deletion mutagenesis identifies two
distinct elements within the C terminus of Xenopus B-Myb
that are responsible for repression of DNA binding and nuclear
transport, respectively. Two independent, functional nuclear
localization signals were mapped in the C-terminal portion of B-Myb. We
also demonstrate that the C terminus of the Xenopus B-Myb
protein can inhibit the DNA binding activity of the N-terminal portion
in trans. These and other observations suggest that
intramolecular folding may be involved in masking DNA binding and
nuclear transport activities. Analysis of the spatial expression
characteristics of B-myb during Xenopus
embryogenesis reveals neural specific expression and links
B-myb gene transcription to proliferative activity within
the developing neural tube.
Plasmids and Cloning Procedures--
The plasmid pSP64T XB-Myb
(2) was used to generate XB-Myb C-terminal mutants with the
ExoIII/mung bean nuclease deletion system (Stratagene, La
Jolla, CA). The corresponding mutant proteins were generated by
in vitro transcription/translation for use in the
electrophoretic mobility shift assays.
A BamHI site upstream of the XB-myb translation
start ATG was generated by
PCR1 using sense (5' GTC
CGG ATC CGC AGA ATG TCC CGG CGG 3') and
antisense (5' TAT TTC AGC CCA TCG ATT TCC CAA TAC 3')
primers. The resulting PCR fragment spanning nucleotides 46-540 of
XB-myb cDNA was cloned into the pGEM-TTM vector
(Promega, Madison, WI) and sequenced. From this plasmid a DNA fragment
derived by cutting with BamHI and ClaI was
generated. In parallel a DNA fragment was generated from full-length
and truncated pSP64T XB-Myb by restriction digestion using
ClaI and SalI. Both fragments
(BamHI-ClaI and ClaI-SalI)
were used for simultaneous ligation into pGEX-5X-1 vector (Pharmacia,
Uppsala, Sweden). The resulting chimera of GST and full-length or
truncated XB-Myb were sequenced to confirm the in-frame fusion. These
constructs were expressed in XL-1 blue Escherichia coli,
followed by purification of the fusion proteins according to the
procedure of Kirov (20).
In order to prepare proteins for oocyte microinjection, the full-length
and truncated DNAs were transferred into the Myc-tag vector pCS2MT (21)
by cutting the GST clones with BamHI/SalI, performing a fill-in reaction, and cloning into
XhoI/filled-in pCS2MT vector. Site-directed mutation of the
NLS1 was generated by PCR with the sense primer (5' TCT GTG CTG AAA CAA
CAC AAC AAC AGA AAC ATT ACC CTG TCA
CCT GTT ACA G 3') and the antisense primer (5' CTG TAA CAG GTG ACA GGG
TAA TGT TTC TGT TGT TGT GTT GTT TCA GCA CAG A 3') using the Quick Change system (Stratagene, La Jolla, CA).
All the constructs were manually sequenced using either Sequenase
(U. S. Biochemicals) or an Applied Biosystems sequencing system, using
Taq dye terminator cycle
sequencing.2
Electrophoretic Mobility Shift Assays--
A double-stranded DNA
fragment containing the Myb-specific DNA-binding motif (2) was labeled
by fill-in reaction using the Klenow fragment of DNA polymerase and
[ In Vitro Protein Expression--
The combined in
vitro transcription/translation (TNT) system (Promega) was used to
generate the 35S-labeled in vitro translated
proteins. Reactions were performed according to the Promega TNT
protocol, and [35S]methionine (Amersham, Buckinghamshire,
UK) was used for radiolabeling. In vitro translation
products were analyzed by SDS-PAGE and phosphorimaging (Molecular
Dynamics, Krefeld, Germany).
Microinjection of Xenopus Oocytes and Analysis of Nuclear
Transport--
Oocytes were removed from adult Xenopus
laevis. Stage V/VI oocytes (23) were isolated manually or by
collagenase treatment (Worthington, Freeholds, NJ) and kept in normal
strength modified Barth saline, 1 × MBSH (24). Approximately 50 nl of
each of the different 35S-labeled fusion protein solutions
(in the rabbit reticulocyte lysate) were injected into the cytoplasm of
oocytes. After injection, the oocytes were incubated for between 2 and
20 h in 1× MBSH at 18 °C. Nuclei and cytoplasmic fractions
were manually dissected in ice-cold NET-2 buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Nonidet P-40
supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin), collected
on ice (20 cytoplasmic and nuclear fractions per sample), and
homogenized. The homogenates were centrifuged 3-4 times in a
microcentrifuge (14,000 × g, 3 min) and the
supernatant used for immunoprecipitation analysis.
Immunoprecipitation--
Nuclear and cytoplasmic fractions were
subjected to immunoprecipitation using monoclonal antibody against the
9E10 epitope of human c-Myc protein (Santa Cruz Biotech, Santa Cruz,
CA). The myc antibody was bound to Protein G-GammaBind Plus Sepharose
(Pharmacia), for 2 h at room temperature in NET-2 buffer. The PGS
antibody pellets were washed three times with NET-2 buffer.
Subsequently, supernatants of homogenized cytoplasmic or nuclear
fractions of injected oocytes (see above) were added and incubated for
90 min at 4 °C. The immunoprecipitate was washed four times with
NET-2 buffer, dried, and dissolved in 40 µl of SDS sample buffer. The samples were heated to 100 °C, loaded on a 10% SDS-polyacrylamide gel, and analyzed using a PhosphorImager.
Whole-mount in Situ Hybridization and Histology--
Whole-mount
in situ hybridization was carried out according to the
procedure of Harland (25), using a digoxigenin-labeled antisense
XB-myb RNA probe. RNA was generated using
Bsp106I-linearized pT7T3 XB-Myb and T7 RNA polymerase.
For sectioning, stained and postfixed embryos were gelatin-embedded and
Vibratome-sectioned at 30 µm thickness and photographed under a
phase-contrast microscope.
Western Blot Analysis--
The myc-tagged in vitro
translated proteins were resolved on a 10% SDS-polyacrylamide gel,
electroblotted to nitrocellulose membrane, probed with the human c-myc
monoclonal antibody (Santa Cruz Biotech) and detected using the ECL
chemiluminiscent detection system (Amersham). The x-ray films were
quantified using a Bio-Rad densitometer (Bio-Rad Laboratories, Munich, Germany).
Spatial Expression Characteristics of B-myb during Xenopus
Embryogenesis--
B-myb is expressed during
Xenopus embryogenesis, as revealed by Northern and Western
blot analysis (2, 19). In order to characterize the spatial
distribution of B-myb encoding transcripts, staged
Xenopus embryos were subjected to whole-mount in
situ hybridization with a B-myb-specific antisense RNA
probe (Fig. 1). During neurula stages,
B-myb is found to be exclusively expressed within the developing central nervous system. At the open neural plate stage, B-myb-specific signals are most prominent in the eye
anlagen, as well as in the anterior portion of the neural plate (Fig.
1A). Upon closure of the neural tube, B-myb
continues to be expressed in fore-, mid-, and hindbrain, as well as in
the entire optic vesicle and in neural crest cells (Fig. 1,
C and E). During further development up to the
tadpole stage, this pattern is generally maintained (Fig. 1,
B and D). In the embryonic eye, B-myb
expression becomes restricted to the ciliary margin (Fig.
1F), a group of undifferentiated, proliferative cells that
give rise to all major cell types of the retina (Fig. 1F)
(26). A transverse section at the level of the hindbrain reveals that,
within the neural tube, B-myb is preferentially expressed in
the ventricular zone, which also contains proliferating,
non-differentiated cells (Fig. 1G).
In summary, B-myb expression in the developing
Xenopus embryo is specific to the developing central nervous
system and to structures derived therefrom, such as the eye and neural
crest/branchial arches. As development proceeds, B-myb in
both the tadpole eye and the tadpole hindbrain is found to be
preferentially expressed in proliferating, non-differentiated cells.
C-terminal Elements of B-Myb Inhibit DNA Binding in Cis and in
Trans--
We have previously reported that full-length
Xenopus B-Myb, either as a recombinant protein isolated from
bacteria, or in its native form from Xenopus oocytes and
embryos, is inhibited in its DNA binding capacity. Removal of the
C-terminal portion of the protein relieves this inhibition (2, 19).
Extending these studies, we have now generated a more systematic set of C-terminal deletion mutants of B-Myb and have assayed for their DNA
binding activity in order to map the inhibitory domain more precisely.
Truncated versions of XB-Myb were produced by in vitro translation and analyzed in electrophoretic mobility shift experiments with a radiolabeled oligonucleotide containing the Myb DNA-binding site
(Fig. 2). Assays were performed both in
the presence and in the absence of a competitor oligonucleotide
encompassing the B-Myb recognition site, in order to test for
specificity of complex formation. C-terminal truncations of up to 275 amino acids do not relieve inhibition of DNA binding, but deletion of
308 amino acids recovers maximal DNA binding activity. Thus, a protein
element located between the amino acids in position 425 and 458 is
sufficient to fully inhibit the specific DNA binding capacity of the
N-terminal XB-Myb repeats; we refer to this region as the DNA-binding
regulatory domain.
The fact that both full-length XB-Myb from Xenopus
oocytes/embryos and the recombinant, bacterially expressed protein are inactive in DNA binding had already suggested that the inhibitory mechanism does not rely on a specific corepressor protein.
Alternatively, inhibition of DNA binding could be a consequence of
intramolecular folding and interaction between C- and N-terminal
domains of XB-Myb. We therefore tested the possibility that a
C-terminally derived fragment containing the entire DNA-binding
regulatory domain inhibits the DNA binding activity of the N-terminal
fragment in trans. Electrophoretic mobility shift assays
with a constant amount of the N-terminal DNA-binding domain in the
presence of increasing amounts of the C-terminal fragment containing
the DNA-binding regulatory domain result in gradually reduction of
DNA-complex formation (Fig. 3). Taken
together, these results indicate that a sequence element in XB-Myb
located between residues 425 and 458 negatively regulates the DNA
binding activity of XB-Myb by intramolecular folding and mediates
direct interaction between N- and C-terminal domains of XB-Myb.
Positive and Negative Regulatory Elements for the Nuclear Transport
of B-Myb in Xenopus Oocytes--
We previously reported that neither
full-length XB-Myb nor the N-terminal half of the protein are
translocated to the nucleus of Xenopus oocytes (19). For a
more detailed analysis, a systematic series of progressive C-terminal
deletion mutants of XB-Myb was produced by in vitro
translation and injected into the cytoplasm of stage V/VI
Xenopus oocytes. After 18 h of incubation, nuclear and
cytoplasmic fractions were manually separated and analyzed by
SDS-polyacrylamide gel electrophoresis (Fig.
4).
As reported earlier, full-length XB-Myb was not found to be imported
into the nucleus, and only background levels of the protein could be
recovered from the nuclear fraction (19). Our work shows that a
C-terminal deletion, spanning amino acids 655-733, partially activates
nuclear transport. A bigger deletion of the C terminus, encompassing
amino acids 543-733, enhances nuclear transfer activity. Further
removal of sequence elements containing one of two putative basic NLSs
strongly reduces nuclear transport, while additional deletion of the
second putative NLS fully inhibits the same process. A protein fragment
containing both of the putative NLSs but lacking the C-terminal
negative regulatory domain, as well as the N-terminal half of XB-Myb,
carries full nuclear import activity.
The activity of the two putative NLSs was therefore analyzed in more
detail. Kinetic analysis of the nuclear import with XB-Myb lacking the
C-terminal inhibitory domain reveals a significant level of nuclear
transfer even after 2 h (Fig. 5).
Conversely, mutation of NLS1 or deletion of NLS2 results in
significantly reduced import kinetics. Site-directed mutagenesis of
NLS1 in a deletion mutant that already lacks NLS2 leads to a further
reduction of import to background levels. Taken together, these
observations define two physically separate, but functionally
cooperative, nuclear localization signals in XB-Myb, which are
negatively regulated by the C-terminal portion of the protein.
Analysis of the Molecular Mechanism for B-Myb Cytoplasmic Retention
in Xenopus Oocytes--
The experiments on the regulation of B-Myb DNA
binding activity described above argued for an intramolecular
interaction of N- and C-terminal portions of the protein that might
directly involve the DNA-binding N-terminal repeats. If such an
interaction were to occur, it could also be responsible for the
inhibition of nuclear transfer, for example by masking the NLS
function. To test this possibility, the XB-Myb internal fragment
sufficient for efficient nuclear transfer (residues 349-545, Fig. 4)
was characterized in further detail. The molecular mass of the
corresponding protein (roughly 46 kDa) is within the protein size range
that is believed to allow passive nuclear import by diffusion. However, its import is strongly temperature-dependent (Fig.
6A), arguing for the existence
of an active import mechanism. Fusion of the C-terminal transport
regulatory domain completely ablates import in a manner similar to
TFIIIA, which is used as a cytoplasmic retention control (Fig.
6B). This finding demonstrates that the N-terminal half of
XB-Myb bearing the DNA-binding domains is not required for the
inhibition of nuclear import and is therefore not likely to be involved
in masking of the XB-Myb NLS function.
We further investigated the molecular mechanism responsible for the
negative regulation of Xenopus B-Myb nuclear transport. Two
principal modes of inhibition were considered, one involving interaction of the nuclear transport regulatory domain with a cytoplasmic anchor, and a second one relying on either masking of the
NLS function via interaction with a molecule that is physically distinct from B-Myb, or via intramolecular folding.
If an anchoring mechanism is responsible for cytoplasmic retention of
B-Myb, transfer of the nuclear transport regulatory domain to a
different nuclear protein constitutively imported in Xenopus
oocytes should result in its cytoplasmic retention. In order to test
this possibility, ribosomal protein L5, which is constitutively
transported to the nucleus in Xenopus oocytes (27), was
fused to either full-length or truncated versions of Xenopus
B-Myb. All of these constructs are imported into the nucleus after
injection into the cytoplasm of Xenopus oocytes (Fig.
7). These findings demonstrate the
dominance of a functional NLS over the cytoplasmic retention domain. It
is therefore unlikely that interaction of the nuclear transport
regulatory domain with a cytoplasmic anchor is responsible for the
cytoplasmic sequestration of full-length Xenopus B-Myb as
observed in Xenopus oocytes.
We report herein on the expression of B-myb during
Xenopus embryogenesis and on the protein domains involved in
DNA binding and nuclear transport, two aspects related to the
regulation of XB-Myb function in transcription. The C-terminal portion
of Xenopus B-Myb contains two distinct sequence
elements responsible for the negative regulation of DNA
binding and nuclear transport activities, respectively. The region
between amino acid residues 425 and 458 is involved in the
inhibition of DNA binding, presumably through intramolecular
interaction between the N and C termini. The last 88 amino acid
residues of the C terminus (TRD) negatively regulate nuclear transfer;
this inhibition is likely to involve masking of the two separate NLSs,
either by intra- or by intermolecular interactions.
Expression of XB-myb is preferentially detected in
proliferating, non-differentiated neural cells in the developing
Xenopus embryo. This correlates with the bona fide function
of XB-Myb in cell division, as has been proposed for mammalian B-Myb
(28). In mouse, B-myb is also mainly expressed in the
developing central nervous system, especially in highly proliferating
cells (5). XB-myb is expressed initially as a continuous
unit in the anterior neural plate, later becoming more strongly
expressed in the lateral regions of the anterior neural plate and
fading from the median region. These two domains of expression will
give raise to the eye primordia (29). The expression of
XB-myb in the developing eye anlage correlates with those of
Pax 6 (30) and ET (29). Studies involving these genes have shown the
existence of a single retina field, which splits into two distinct
primordia (29). Sections of neurula-stage embryos show that
XB-myb is initially detected in the whole retinal
neuroepithelium. At later stages it becomes restricted to the cells of
the retinal ciliary margin, the multipotent retinal progenitor cells
(26). It remains to be demonstrated whether XB-Myb also regulates
retinal proliferation.
We show that a serine/threonine-rich region (amino acids 369-545) that
includes two NLSs is required for efficient nuclear import of
XB-Myb and the negative regulation of DNA binding. This region is
highly conserved among Myb family members, including human, mouse,
Xenopus, and chicken B-Myb, implying that it has a conserved
function. Such a domain is a potential target of phosphorylation, and
it displays two ankyrin-like repeats (31). The ankyrin motif has been
related to both cell cycle control and differentiation (31), and these
repeats have also been implicated in protein-protein interactions (32).
Such interactions have indeed been shown in mammalian cells in culture
for c-Myb (33), and in Xenopus oocytes for
B-Myb.3
The region responsible for DNA binding inhibition lies at the C
terminus of XB-Myb (amino acids 425-458). Several observations on the
activation of B-Myb correlate with results obtained for the regulation
of c-Myb activities. Deletion of the C terminus of c-Myb increases its
transcriptional activation capacity (34-36). In addition, the
suppressor activity of the negative regulatory domain in c-Myb
functions in cis and in trans (36). The negative regulatory function of the C terminus in B-Myb has been detected at
different levels of B-Myb activity. The transactivation potential of
B-Myb in transient transfection assays has been found to be inhibited
in several cell lines. The inhibition can be relieved either by
introducing C-terminal truncations or by coactivation of the cell
cycle-regulated protein kinase cdk2, which appears to use the C
terminus of B-Myb as a direct substrate (16-18). We previously
reported that maternal Xenopus B-Myb became
hyperphosphorylated upon meiotic maturation of oocytes, but that this
did not affect binding to DNA (19). Our finding that the C-terminal
portion of Xenopus B-Myb inhibits the DNA binding activity
of the N-terminal portion in trans supports a model whereby
a direct intramolecular interaction is responsible for the negative
regulation of DNA-complex formation observed. This mechanism would not
require additional factors interacting with the DNA-binding regulatory
domain, although some of the interacting factors observed for XB-Myb in
oocytes3 might function in the relief of inhibition and
promote transcriptional activation later on in embryogenesis.
Intramolecular inhibition of DNA binding has been described for other
proteins. An excellent example is the study of the Ets family of
transcription factors. DNA binding by Ets-1 is prevented by
intramolecular interactions involving the N terminus, the ETS domain,
and nearby sequences. DNA-binding inhibition is relieved by
conformational changes that occur in the presence of DNA. These changes
are thought to promote cooperative binding of a stabilizing protein
partner (37, 38). Although intramolecular interactions participate in
DNA-binding inhibition of XB-Myb, in contrast to Ets-1, the inhibition
takes place even in presence of the DNA target. Our results open the
way for further investigations about the nature of positive regulation
of B-Myb mediated by a protein partner or by posttranslational modifications.
In full-grown oocytes XB-Myb is cytoplasmic, and during embryogenesis
XB-Myb localizes in the nucleus. We analyzed the mechanism of negative
regulation of XB-Myb in oocytes that would prevent XB-Myb functioning
as a transcription factor. We have shown that the nuclear-cytoplasmic
distribution of XB-Myb involves at least two different protein
elements: the TRD, responsible for the cytoplasmic retention of the
protein, and two NLSs, which are necessary for efficient nuclear import
of XB-Myb. Our data suggest that both NLSs are functionally cooperative
and would be the bona fide domains involved in nuclear import in the
embryo. According to our data, the molecular mechanism responsible for
the negative regulation of XB-Myb nuclear targeting in oocytes is
likely to involve masking of the NLS. We showed the dominance of an
additional NLS over the TRD in the full-length protein, indicating that
the extra signal promotes XB-Myb nuclear entry. This finding excludes
the possibility of a cytoplasmic anchoring mechanism. Thus, the
retention of XB-Myb in the oocyte cytoplasm could be mediated either by intra- or intermolecular interactions that mask the NLS.
If the NLS function in Xenopus B-Myb is inhibited by
interaction with other proteins that mask the B-Myb NLSs, such
interacting proteins should be present in Xenopus oocyte
extracts. As an initial step toward the identification of such
interacting proteins, radiolabeled oocyte proteins were incubated with
full-length and truncated versions of immobilized Xenopus
B-Myb. Putative inhibitors should bind to full-length or shortened
versions of Xenopus B-Myb that were not able to travel to
the nuclear compartment, as analyzed in the oocyte injection assays
(see above). Such experiments do indeed detect proteins that interact
with a recombinant Xenopus B-Myb/GST fusion, but not with
the GST extension alone. However, there was no clear correlation
between inhibition of nuclear transfer and binding of any of these
proteins (data not shown).
B-Myb phosphorylation could be part of the mechanism that regulates DNA
binding and/or nuclear transfer, two activities that define
prerequisites for transcription activation by B-Myb. However, phosphorylation of B-Myb during oocyte maturation was not found to be
sufficient to activate the DNA binding activity of B-Myb (19).
Furthermore, the distribution of B-Myb between nucleus and cytoplasm
does not correlate with cell cycle activities (39). Thus, the
regulation of B-Myb as a transcriptional activator is not likely to be
due to a simple, phosphorylation-mediated switch between binding and
not binding to B-Myb-dependent promoters, or between
cytoplasmic and nuclear localization, but seems to rely on a more
complex mechanism. This mechanism might involve all three processes.
Further investigation will elucidate the role of hyperphosphorylation
in the regulation of XB-Myb activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (22). 6.25 fmol of labeled DNA were
incubated with in vitro translated proteins in binding
buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10 mM dithiothreitol) in a 20-µl final volume for 20 min at
25 °C. Nonspecific DNA binding was diminished by competition with
both 50 ng/ml poly(dI·dC) (Boehringer Mannheim, Germany) and 50 ng/ml
M13 single-stranded DNA. DNA binding activity was competed specifically
with an 800-fold molar excess of non-labeled, double-stranded DNA
fragment containing the DNA-binding motif of Myb. The complex was
resolved in 8% non-denaturing polyacrylamide:bisacrylamide (29:1)
gels, 0.25[time] TBE buffer (44.5 mM Tris borate, pH 8.4, 1 mM EDTA) and analyzed by use of a PhosphorImager
(Molecular Dynamics, Krefeld, Germany).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
XB-myb is expressed in the
developing nervous system during Xenopus
embryogenesis. Whole-mount in situ hybridization
experiments were carried out with XB-myb antisense RNA as a
probe and staged Xenopus embryos. A, neural fold
(stage 17) Xenopus embryo; arrowheads indicate
eye anlagen. B and C, lateral view (B)
and anterior view (C) of a tailbud (stage 24)
Xenopus embryo. o.v., optic vesicle;
n.c., neural crest; mes., mesencephalon;
pro., prosencephalon. D, somite
Xenopus embryo (stage 28); e, eye;
b.a., branchial arches; ot.v., otic vesicle.
E, transverse section of a stage 22 Xenopus
embryo; mes., mesencephalon; op.ves.,
optic vesicle. F and G, transverse sections of a
stage 29/30 Xenopus embryo; mes., mesencephalon;
c.m., ciliary margin; ot.v., otic vesicle;
rom., rhombencephalon. Note preferential staining within the
ventricular zone of the rhombencephalon.
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Fig. 2.
Mapping of the DNA-binding regulatory domain
in Xenopus B-Myb. The DNA binding activity of a
progressive series of C-terminal XB-Myb deletion mutants analyzed by
electrophoretic mobility shift assays with the Myb-specific consensus
DNA recognition site. A, schematic representation of
C-terminal deletion mutants utilized; deletion end points as well as
DNA binding activities are indicated. The positions of the DNA-binding
repeats (DNA-BD) (40, 41) and of the DNA-binding regulatory
domain (DBRD) as determined in this series of experiments
are indicated. B, SDS-polyacrylamide gel electrophoresis of
the in vitro transcription/translation products; numbering
according to the schematic representation as shown in panel
A. Proteins were radiolabeled by incorporation
[35S]methionine. C, electrophoretic mobility
shift assay with the 32P-radiolabeled Myb consensus DNA
recognition site. Proteins were as shown in panels
A and B. Cx denotes the position of
the specific complexes, F the position of the free probe.
Assays were performed either in the presence (+) or absence ( ) of
specific competitor DNA.
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Fig. 3.
The C-terminal XB-Myb fragment inhibits DNA
binding activity in trans. A,
electrophoretic mobility shift assay with full-length (FL)
XB-Myb, an N-terminal fragment (N) that contains the
DNA-binding repeats (DNA-BD), and an internal fragment
(C) that contains the DNA-binding regulatory domain
(DBRD). N + C is a mixture of the two latter
proteins. Cx denotes the position of the specific complex,
F the position of the free probe. B, titration of
the in trans inhibitory effect of the internal fragment
(C) on the DNA binding activity of the N-terminal fragment
(N). C, Western blot of the proteins utilized in
the experiment shown in panel B.
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Fig. 4.
Mapping of the nuclear transport regulatory
domain in Xenopus B-Myb. 35S-Labeled
XB-Myb variants produced in vitro were microinjected into
the cytoplasm of Xenopus oocytes. After 18 h of
incubation, nuclear (N) and cytoplasmic (C)
fractions were separated manually and analyzed for XB-Myb protein
content by immunoprecipitation and SDS-gel electrophoresis. The nuclear
accumulation is indicated as a percentage of total protein recovery in
the nuclear fraction. The schematic representation shows the different
deletion mutants utilized. TRD, transport regulatory domain;
NLS1&2, nuclear localization signals 1 and 2.
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Fig. 5.
XB-Myb carries two physically separate and
functionally cooperative nuclear localization signals.
A, kinetics of the nuclear transfer of N-terminal XB-Myb
fragments containing mutations in either one of the two NLSs, or in
both. Oocyte microinjections and protein recovery were performed as
described in the legend to Fig. 4. The duration of oocyte incubation
after protein microinjection is indicated above the assays.
B, site-directed mutagenesis was performed in the NLS1.
Three lysine residues were replaced by asparagines. C,
quantification of the nuclear transfer as a percentage of total protein
recovery in the nuclear fraction given in the form of a bar diagram for
the four different protein variants as indicated.
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Fig. 6.
The C terminus but not the DNA-binding domain
is involved in the inhibition of XB-Myb nuclear transfer.
A, temperature dependence of the nuclear transport of an
internal XB-Myb protein fragment that contains NLS1 and NLS2.
Microinjected oocytes were incubated for 5 h at either 4 °C or
18 °C prior to separation of nuclear and cytoplasmic fractions.
TFIIIA, which is retained in the cytoplasm, was coinjected with the
XB-Myb variants as an internal control for cytoplasm contamination in
nuclear fractions (A and B). B, the
transport regulatory domain inhibits active transport of the internal
XB-Myb fragment in Xenopus oocytes.
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Fig. 7.
Fusion of an additional NLS function relieves
cytoplasmic retention of XB-Myb in Xenopus
oocytes. Different portions of XB-Myb were fused to
ribosomal protein L5, which is constitutively transported to the
nucleus of Xenopus oocytes; the structure of the different
fusion constructs is indicated. Oocyte microinjections and protein
processing were performed as described in Fig. 4. A and
B, fusion of a partial or of the entire TRD from XB-Myb does
not interfere with nuclear transport of ribosomal protein L5 in
Xenopus oocytes. C, fusion of full-length XB-Myb
to ribosomal protein L5 does not interfere with nuclear transport in
Xenopus oocytes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Andrés Carrasco for continuous interest in and support of the project and Dr. Rolando Rivera-Pomar and Tony Streeter for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Volkswagenstiftung and Grant SFB 523 from the Deutsche Forschungsgemeinschaft.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.
Present address: Max Planck Institut für biophysikalische
Chemie, Am Fassberg 11, 37077 Göttingen, Germany.
§ To whom correspondence should be addressed. Tel.: 49-551-395683; Fax: 49-551-395960; E-mail: tpieler{at}gwdg.de.
2 During the cloning procedure, we noted an error in the published sequence. This change implies an addition of 26 amino acid residues at the C terminus. The corrected sequence has been submitted to GenBank (accession number M75870).
3 G. Humbert-Lan and T. Pieler, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; GST, glutathione S-transferase; NLS, nuclear localization signal; PAGE, polyacrylamide gel electrophoresis; TRD, transport regulatory domain; XB-Myb, Xenopus B-Myb.
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