From the Department of Chemistry and Biochemistry and
§ Howard Hughes Medical Institute, University of Colorado,
Boulder, Colorado 80309 and ¶ Promega Corporation,
Madison, Wisconsin 53711
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ABSTRACT |
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Stimulation of mammalian cells results in
subcellular relocalization of Ras pathway enzymes, in which
extracellular signal-regulated protein kinases rapidly translocate to
nuclei. In this study, we define conditions for nuclear localization of
mitogen-activated protein kinase kinase 1 (MKK1) by examining effects
of perturbing the nuclear export signal (NES), the regulatory
phosphorylation sites Ser218 and Ser222,
and a regulatory domain at the N terminus. After disrupting the NES
( Among the key signaling pathways regulating mammalian cell growth
and differentiation is the
MAP1 kinase cascade,
comprising MAP kinases, ERKs 1 and 2, and MAP kinase kinases MKK1 and 2 (for review, see Refs. 1 and 2). This pathway is activated by many
different extracellular stimuli through p21 Ras-coupled mechanisms.
Enhancement of MKK or ERK activity in response to cell stimulation
involves phosphorylation at residues located within the activation lip
of each kinase. In the case of human MKK, phosphorylation at two serine
residues (Ser218 and Ser222 in MKK1;
Ser222 and Ser226 in MKK2) by upstream protein
kinases, Raf-1, c-Mos, or MAP/ERK kinase kinase (MEKK) leads to
maximal enzyme activation.
In addition to kinase activation, several studies have demonstrated
that the components in this pathway undergo regulated subcellular
relocalization. After cell stimulation, ERKs are taken up into nuclei
within 5-30 min and are retained for several hours (3-5). This
enables transmission of signaling to the nucleus, where an important
end result is transcriptional control. The involvement of ERK in
phosphorylation and regulation of a number of nuclear factors suggests
that redistribution of ERK from cytosolic to nuclear compartments is
necessary for signaling (for review, see Ref. 6). In addition, stable
nuclear localization of ERK has been correlated with differentiation of
PC12 cells, a process involving phosphorylation of nuclear
transcription factors involved in neuronal gene expression
(7-9).
Initial studies failed to show a similar translocation of MKK in
response to signaling (5, 10). This was later modified by the discovery
of a leucine-rich sequence located between residues 32 and 42 of MKK
(ALQKKLEELEL), which
behaves as a functional nuclear export signal (NES) (11). Removal or
mutation of the NES leads to constitutive nuclear localization of MKK,
and cross-linking of peptides containing this sequence to heterologous
proteins facilitates their nuclear export. Sequences of this type have been shown to be recognition sites for binding to Crm1/exportin 1, a
nuclear protein that facilitates export of cellular components including PKI Interestingly, nuclear import of MKK mutants lacking the NES appears to
be promoted in response to serum treatment. In serum-starved COS or
HEK293 cells, an MKK mutant with deletion of residues 32-51, which
disrupts the NES, a K97A mutation rendering the enzyme inactive, and
S218E/S222E mutations at the activation lip, was predominantly cytosolic but distributed into nuclei after 10 min of serum treatment (16). This suggests that mechanisms for nuclear import of MKK are
regulatable, either through enhanced import or inhibition of export.
Presumably, such mechanisms could require activation of signaling
pathways, such that phosphorylation and/or activation of MKK might be
important conditions for stabilizing its nuclear distribution.
In this study, we examined the requirements for nuclear localization of
MKK1 by first comparing the behavior of MKK mutants with respect to
serum-stimulated nuclear import. Our results show that enhanced
negative charge at the activation lip promotes import, although
elevated kinase activity is not required, indicating that
phosphorylation, not MKK activation, is a key event necessary for
nuclear import. However, translocation is sensitive to a cell-permeable MKK inhibitor, indicating that signaling events downstream of MKK are
also important for stimulation of import. We also show that wild type
MKK redistributes to nuclei early in mitosis, before nuclear envelope
breakdown, demonstrating that nuclear uptake of MKK containing a
functional NES can be regulated normally during the cell cycle.
Comparison of the mitotic nuclear localization of various MKK mutants
corroborates the behavior seen on serum stimulation. We conclude that
phosphorylation of MKK at the activation lip and activation of
downstream targets are necessary events regulating nuclear
translocation of wild type MKK.
Cell Culture and Transfection--
Mouse NIH 3T3 cells were
grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with
penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal
bovine serum (Gemini). Cells were grown to 50% confluence and then
transfected using LipofectAMINE (Life Technologies, Inc.) mixed with 1 µg of cDNA constructs driving expression of various human MKK1
mutants under the cytomegalovirus promoter (plasmid pMCL; Ref. 17), or
constitutively active Raf-1 under the Rous sarcoma virus long terminal
repeat promoter (BXB-Raf-1, a gift of Reinhold Krug and Ulf Rapp). The
transfection efficiency was ~10%, scored by anti-HA staining of the
tagged MKK. Different forms of MKK1 used in these studies include: wild
type (WT), KM (mutation of Lys97 to Met), AA (mutation of
Ser218 and Ser222 to Ala),
Twenty-four hours after transfection, some cells were serum starved by
exchanging the media with DMEM and 0% serum and further incubation for
20 h. Unless otherwise noted, cells were stimulated by addition of
fetal bovine serum and phorbol 12-myristate 13-acetate (PMA, Sigma) to
final concentrations of 10% and 100 nM, respectively, treated for 2 h, and harvested for immunoblotting or
immunofluorescence experiments. In some experiments, MKK and ERK
signaling was inhibited by treating cells with 20 µM
1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126,
Promega) for 30 min at 37 °C before stimulation with 5% serum and
50 nM PMA. U0126 was dissolved in dimethyl sulfoxide to a
stock concentration of 10 mM and then diluted directly into culture medium (final dimethyl sulfoxide, Immunofluorescence, Immunoblotting, and Protein Kinase
Assays--
Affinity-purified primary antibodies used in
immunochemical studies included anti-phospho-MEK1/2, a rabbit
polyclonal antibody recognizing phosphorylated MKK1 and MKK2 (New
England Biolabs); C-18, a rabbit polyclonal antibody recognizing the C
terminus of MKK1 (Santa Cruz); anti-ACTIVE MAP kinase, a rabbit
polyclonal antibody recognizing diphosphorylated ERK1 and ERK2
(Promega); C-14, a rabbit polyclonal antibody recognizing the C
terminus of ERK2 (Santa Cruz); 12CA5, a mouse monoclonal antibody to
hemagglutinin tag sequence (BabCo); and M-20, a goat polyclonal
antibody to lamin B (Santa Cruz).
For immunofluorescence, cells were grown on glass coverslips and
transfected as above. After transfection and treatment, coverslips were
rapidly rinsed in cold (4 °C) phosphate-buffered saline, immediately
fixed by addition of 0.1% glutaraldehyde and 2% formaldehyde in
phosphate-buffered saline for 5 min, and then permeabilized with cold
(
Primary antibodies were used at dilutions of 1:100, with the
exception of 12CA5, which was used at 1:500. Controls for nonspecific staining of phosphorylated MKK were performed by preincubating anti-phospho-MEK1/2 with 0.1 mg/ml diphosphorylated
CVSGQLIDS(P)MANS(P)FVGTRSY, synthesized by Macromolecular Resources
(Ft. Collins, CO). Controls for nonspecific staining of HA-MKK1 were
performed by preincubating 12CA5 antibody with 0.1 mg/ml YPYDVPDYA (a
gift of James Goodrich). Cells were viewed and photographed using a
Zeiss Axioplan fluorescence microscope with a Photometrics Sensys
digital charge-coupled device camera system, and images were
manipulated using IP-LAB Spectrum software.
For immunoblots, cells were grown in 60-mm dishes and treated as above.
Cells were harvested by scraping into lysis buffer containing 50 mM 2-glycerol phosphate, 1 µM microcystin,
0.2 mM sodium orthovanadate, 1.5 mM EGTA, 1 mM benzamidine, 2 µg/ml pepstatin A, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, and 1 mM dithiothreitol, followed by centrifugation (17,000 × g, 10 min).
Proteins in extract supernatants (20 µg/lane) were separated by
SDS-polyacrylamide gel electrophoresis (15% acrylamide) and
transferred to Immobilon P (Millipore). Blots were reacted for 1 h
with primary antibody (1:1000 dilution for each antibody) followed by
0.8 µg/ml donkey anti-rabbit or anti-mouse IgG coupled to horseradish
peroxidase (Jackson Laboratories) and visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Kinase assays were performed as described previously (19). HA-tagged
MKK1 was immunoprecipitated from extract supernatants with 12CA5
antibody, followed by phosphorylation of bacterially expressed
recombinant ERK2-K52R (1 µM) (a gift of Melanie Cobb; Ref. 20) using 10 mM MgCl2 and 0.1 mM ATP.
HA-WT-MKK1 was overexpressed in NIH 3T3 cells and probed by
indirect immunofluorescence using antibody 12CA5 to the HA tag. The
localization of WT-MKK was cytosolic in starved cells and unaffected by
stimulation with serum-PMA or coexpression with constitutively active
BXB-Raf-1 (Fig. 1, A-C). In
contrast, disruption of the NES by deletion of residues 32-37 resulted
in a mutant (32-37), nuclear uptake of MKK was enhanced when quiescent cells
were activated with serum-phorbol 12-myristate 13-acetate or BXB-Raf-1
cotransfection. Uptake was enhanced by mutation of Ser218
and Ser222 to Glu and Asp, respectively, and blocked by
mutation of these residues to Ala, although mutation of
Lys97 to Met, which renders MKK catalytically inactive, did
not interfere with uptake. Therefore, nuclear uptake of MKK requires
incorporation of phosphate or negatively charged residues at the
activation lip but not enzyme activity. On the other hand, uptake of an
active MKK mutant with disrupted NES (
32-51) was elevated in
quiescent as well as stimulated cells, and pretreatment of cells with
the MKK inhibitor
1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene blocked
nuclear uptake. Thus, signaling downstream of MKK is also necessary for
translocation. Finally, wild type MKK containing an intact NES
translocates to nuclei during mitosis before envelope breakdown.
Comparison of mutants with Ser to Glu and Asp or Ala substitutions
indicates that Ser phosphorylation is also required for mitotic nuclear
uptake of MKK.
INTRODUCTION
Top
Abstract
Introduction
References
, I
B, Rev, and Dsk1, in addition to MKK (12, 13).
The fungal antibiotic leptomycin B binds to Crm1 and interferes with
its association with NES-containing proteins, thus blocking their
export (13-15). Accordingly, the distribution of MKK can be shifted
from cytosolic to nuclear within 1 h of leptomycin B treatment in
COS cells (14), suggesting that although nuclear localization of MKK is
not stable, import is dynamic with rapid rates of influx and efflux.
EXPERIMENTAL PROCEDURES
N6 (deletion of
residues 32-37),
N6/KM (
N6 with mutation of Lys97 to
Met),
N6/AA (
N6 with mutation of Ser218 and
Ser222 to Ala),
N6/ED (
N6 with mutation of
Ser218 and Ser222 to Glu and Asp,
respectively),
N3 (deletion of residues 32-51),
N3/AA (
N3
with mutation of Ser218 and Ser222 to Ala),
N3/ED (
N3 with mutation of Ser218 and
Ser222 to Glu and Asp), and
N4/ED (deletion of residues
44-51, with mutation of Ser218 and Ser222 to
Glu and Asp, also named G1C). Most of these constructs were described
previously (17-19).
N6/KM and
N3/KM/ED were respectively constructed from
N6 and
N3/ED, introducing a K97M mutation by site-directed mutagenesis using the Chameleon double-stranded mutagenesis kit (Strategene).
N3/AA and
N6/AA were constructed by
EcoRI digestion of MKK1-AA and
N3 or
N6, respectively,
and purification of the 944-bp fragment from the AA digest and the vector fragments from the
N3 or
N6 digests, followed by ligation. Mutants were constructed in pRSET, and mutations were confirmed by
dideoxy chain termination DNA sequencing. The MKK coding sequence was
then excised from pRSET with BamHI and HindIII
and ligated between the corresponding sites in pMCL (17).
0.2%, v/v). In experiments examining MKK nuclear accumulation during mitosis, NIH 3T3 cells were
grown in DMEM and 10% serum without further treatment, and mitotic
cells were identified by the appearance of condensed chromatin in
4',6-diaminino-2-phenylindole (DAPI) staining.
20 °C) 100% methanol for 5 min. Similar results were obtained
when samples were fixed with 10% neutral buffered formalin for 5 min
and then permeabilized with cold (
20 °C) 100% methanol for 5 min.
After permeabilization, cells were incubated in Tris-buffered saline
(50 mM Tris, pH 7.6, 0.15 M NaCl), 0.1% Tween
20, and 3% bovine serum albumin for 1 h. Coverslips were
incubated with primary antibody for 1 h, washed four times with
Tris-buffered saline and 0.1% Tween 20, incubated for 1 h with
fluorescein isothiocyanate- or Texas Red-conjugated donkey anti-rabbit,
anti-mouse, or anti-goat secondary antibody (0.8 µg/ml, Jackson
Laboratories), washed four times with Tris-buffered saline and 0.1%
Tween 20, and finally counterstained with DAPI (0.4 µg/ml in
phosphate-buffered saline).
RESULTS
N6-MKK) that localized to the cytosol in starved cells
but redistributed to nuclei in response to serum-PMA or coexpression
with BXB-Raf (Fig. 1, D-F). Thus, regulated nuclear uptake
of MKK can be observed with a mutant lacking the NES. This supports and
extends previous reports using a
N3/K97M/S218E/S222D-MKK1 mutant
(16) and establishes that deletion of the NES is sufficient to reveal
serum-stimulated nuclear uptake of MKK. The fact that redistribution
can be observed by coexpression with BXB-Raf in the absence of
serum-PMA further indicates that the activation of the Raf-MKK-ERK
pathway is a key part of the stimulatory signal for nuclear uptake.
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Fig. 1.
Nuclear translocation of N6-MKK is
stimulated in response to serum-PMA. NIH 3T3 cells were
transfected with plasmids driving expression of HA-WT-MKK1
(A-C) or HA-
N6-MKK1 (D-F) as described under
"Experimental Procedures." After transfection, cells were serum
starved for 22 h (A and D) or 20 h
(B and E) and then treated with 10% serum and
100 nM PMA for 2 h. C and F,
alternatively, cells were cotransfected with MKK and a plasmid driving
expression of constitutively active BXB-Raf-1. Cells were fixed with
0.1% glutaraldehyde and 2% formaldehyde, permeabilized with methanol,
stained with 12CA5 primary antibody to the HA tag followed by goat
anti-mouse secondary antibody coupled to Texas Red, and visualized by
fluorescence microscopy. Weakly staining cells apparent in E
and F are attributable to background staining with the 12CA5
antibody, as determined in peptide competition controls (data not
shown). Experiments in A, B, D, and E were
repeated six times, and experiments in C and F
were repeated three times, with similar results.
Examination of the phosphorylation state of the N6-MKK mutant by
immunoblotting with anti-phospho-MEK1/2 antibodies showed that this
mutant is phosphorylated in response to serum-PMA stimulation (Fig.
2A, lanes 1 and 2),
conditions under which its specific activity is enhanced (Fig.
2F, lanes 1 and 2). We therefore asked whether
the regulated nuclear import of this mutant is sensitive to the
increased phosphorylation state at the activation lip versus the activation state of MKK. Two variants of
N6-MKK were designed to
distinguish these possibilities.
N6/KM contains a mutation of
Lys97 to Met and is inactive but phosphorylatable at the
activation lip.
N6/AA contains mutations in Ser218 and
Ser222 to Ala and is inactive and not phosphorylatable at
the activation lip. A third variant,
N6/ED, contains S218E and S222D
mutations and is not phosphorylatable but is constitutively activated
due to the introduction of negative charge at the activation lip, with
55-fold greater activity than WT-MKK in vitro (18). In response to transfection with
N6/ED, MKK and ERK activity were elevated in serum-starved cells (Fig. 2, C and F,
lanes 7 and 8).
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Each mutant was expressed and examined under serum-starved or
serum-PMA-treated conditions. Like WT and N6, both
N6/KM and
N6/AA were cytosolic in serum-starved cells (Fig.
3, A, C, E, and G).
Like
N6, the
N6/KM mutant redistributed to nuclei in response to
serum (Fig. 3, D and F), although under this
condition
N6/KM was phosphorylated, but not activated (Fig. 2,
A and F, lanes 3 and 4). In contrast,
N6/AA showed little nuclear uptake in response to serum-PMA (Fig.
3H), a condition under which it was unphosphorylated and
inactive (Fig. 2, A and F, lanes 5 and 6). This suggests that the presence of phosphorylatable
residues is needed for nuclear accumulation, although the activation of MKK by mutation is not sufficient for this process. Consistent with
this hypothesis, the
N6/ED mutant was constitutively nuclear even in
starved cells (Fig. 3, I and J). Thus, negatively
charged residues at the activation lip appear to stabilize the nuclear localization of MKK.
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The results with N6-MKK were tested further by examining the nuclear
uptake of MKK with residues 32-51 deleted (
N3-MKK), which both
enhances basal activity as well as removes the NES (11, 17). Variants
of
N3 were constructed, containing mutations of S218A/S222A
(
N3/AA) or S218E/S222D (
N3/ED). As expected by removal of the
NES, nuclear uptake of
N3 was significant in quiescent cells,
although uptake was enhanced in the presence of serum-PMA (Fig. 3,
K and L). Likewise, whereas
N3/ED was
constitutively nuclear,
N3/AA was excluded in the presence or
absence of stimulation (Fig. 3, O-R). This corroborates
results above with the
N6 mutants, suggesting that phosphorylation
or incorporation of negative charge at the activation lip is critical
for uptake. A final MKK mutant was tested, combining deletion of
residues 44-51 (
N4) with S218E/S222D, which has 630-fold greater
activity than WT-MKK (17) but retains the NES. This mutant is
constitutively cytoplasmic even under stimulated conditions (Fig. 3,
S and T), indicating that activation of MKK is
not sufficient for nuclear import.
A possibility not excluded by these experiments is that facilitated
nuclear uptake of MKK involves regulation of import or export factors
targeted by signaling pathways. This is supported by evidence showing
that the combinatorial mutant, N3/KM/ED, is excluded in quiescent
cells but nuclear in response to serum-PMA (16). We performed the same
experiment and obtained similar results (Fig. 3, M and
N). Although this mutant is negatively charged at the
activation lip, it is inactive as a consequence of the K97M mutation.
Thus, although phosphorylation at the activation lip may be a necessary
condition for nuclear uptake, uptake may also require activation of
downstream signaling targets. One possibility, consistent with the
nuclear redistribution of MKK in response to BXB-Raf coexpression, is
that such factors may be targeted by components downstream in the
MKK-ERK pathway. Therefore, we tested the effect of blocking signaling
downstream of MKK on its nuclear uptake.
U0126 is a phenylthiobutadiene compound recently described as a
selective inhibitor of MKK1 and MKK2 (21). Pretreatment of NIH 3T3
cells with 20 µM U0126 blocked ERK activation 90% in
response to serum-PMA (Fig.
4A). Similar results were
observed in cells transfected with
N6-MKK or
N3-MKK before
inhibitor treatment (Fig. 4, B and C). Under
these conditions, nuclear uptake of
N6-MKK in response to serum-PMA
was inhibited (Fig. 5, A-D). Interestingly, nuclear uptake of
N3-MKK was also suppressed under conditions of serum starvation as well as serum-PMA treatment (Fig. 5,
E-H). U0126 has previously been shown to inhibit the constitutively active mutant
N3/ED-MKK (21); therefore, it should
also inhibit
N3-MKK. The resulting behavior indicates that
suppressing the activity of the
N3 mutant interferes with its
nuclear accumulation. We conclude that ERK signaling downstream of MKK
is needed to promote nuclear uptake of MKK mutants in response to cell
stimulation.
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Although different forms of MKK show clear differences in acute nuclear uptake, all experiments were performed using mutant enzymes in which the NES is deleted. However, in cycling NIH 3T3 cells growing in 10% fetal bovine serum (as well as all other mammalian cell lines we have so far examined), we have observed that endogeneous MKK becomes activated and localized to nuclei during mitosis, revealed by immunocytochemical staining with an antibody that recognizes phosphorylated MKK (Fig. 6, A-C; 34). We therefore examined the distribution of HA-tagged WT-MKK1 after expression in cycling NIH 3T3 cells and observed nuclear localization in mitotic cells, despite the fact that the NES was retained (Fig. 6, D-F). This relocalization occurs early in prophase, before nuclear envelope breakdown, as demonstrated by staining of the nuclear matrix using an antibody probe to lamin B (Fig. 6E). A similar distribution of endogeneous WT-MKK was observed when probing cells with antibody recognizing the C terminus of MKK1 (data not shown).
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The importance of phosphorylation on the nuclear uptake was then tested by examining mitotic cells for distribution of expressed HA-tagged MKK mutants, all of which contained an intact NES sequence. As observed with WT-MKK, S218E/S222D and K97M mutants showed nuclear staining in prophase cells identified by DAPI staining of partially condensed chromatin (Fig. 7, D, F, J, and M). Under these conditions, significant phosphorylation of endogeneous and expressed MKK was observed in nuclei, revealed using the anti-phospho-MEK1/2 antibody (Fig. 7, E and K). In contrast, nuclear localization of the S218A/S222A mutant was reduced substantially compared with the other mutants, although nuclear staining of endogeneous phosphorylated MKK could still be observed (Fig. 7, G and H). This indicates that removal of phosphorylatable residues at the activation lip suppresses MKK uptake during mitosis in a manner similar to the behavior in response to acute stimulation by serum-PMA.
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DISCUSSION |
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In this study, we present evidence demonstrating that, like ERK, nuclear uptake of MKK in mammalian cells occurs in a regulatable manner. Our findings demonstrate that the dependence of uptake on acute cell stimulation reflects a requirement for at least two events involved in the Raf-MKK-ERK pathway. Incorporation of negative charge at the activation lip of MKK is a necessary condition for nuclear uptake, demonstrated by the correlation of translocation with phosphorylation, the ability to mimic this effect by substituting the phosphorylatable serine residues with acidic amino acids, and the interference with uptake by disrupting these phosphorylatable residues with alanine.
Importantly, nuclear uptake of MKK is also observed with catalytically
inactive mutants, indicating that the enzymatic activity of MKK is not
important for signal-induced import. Nevertheless, the contrast in
behavior of N3/ED and
N3/KM/ED mutants, in which the inactive
N3/KM/ED mutant is cytosolic but imported in response to
stimulation, whereas constitutively active
N3/ED is nuclear even
under unstimulated conditions, suggests at least some dependence on MKK
activity, perhaps through the activation of downstream signaling
events. Consistent with this hypothesis, serum-PMA-stimulated nuclear
import of MKK was blocked by the MKK inhibitor U0126 under conditions
in which ERK activation was strongly inhibited. This block in uptake
was observed with
N3-MKK as well as
N6-MKK, indicating that
nuclear localization of the constitutively active
N3 and
N3/ED
under serum-starved conditions is most likely attributable to elevated
ERK activity in cells expressing these mutants.
Mechanisms involved in ERK translocation are instructive toward understanding events regulating MKK translocation. A nuclear localization signal is not apparent in the primary sequence of ERK, and the available evidence indicates that elevated nuclear localization of ERK occurs after its overexpression, suggesting that ERK import may occur through passive diffusion (5). In addition, ERK import is blocked by overexpression of MKK, whereas MKK mutants that are unable to form stable MKK-ERK interactions have no effect, suggesting that ERK translocation occurs after dissociation of an MKK·ERK complex (23). On the other hand, recent studies demonstrate that ERK import requires phosphorylation at the activation lip, an event that promotes homodimerization, and ERK mutants deficient in dimerization are not retained efficiently in nuclei (24). Nuclear localization of ERK may be stabilized through dimerization, which occludes a potential NES located at the dimerization interface, or through new interactions with nuclear targets.
Taken together, a working hypothesis for translocation events after cell stimulation can be developed, based on previous work and the findings of our study. In unstimulated cells, MKK and ERK exist in the cytosolic compartment bound within low affinity complexes. Cell stimulation leads to phosphorylation and activation of MKK and ERK, concomitant with disruption of MKK-ERK interactions. ERK is then imported by a mechanism that is still unknown, possibly involving passive diffusion or facilitated uptake. Dimerization of ERK or complex formation with nuclear targets may then suppress the rate of export, enabling stable nuclear localization for several hours. Phosphorylation of MKK enhances its uptake through mechanisms that depend on its phosphorylation as well as activation of signaling components downstream, including ERK. Because acute uptake is observed only with MKK mutants disrupted in the export recognition sequence, the regulation by MKK phosphorylation and ERK activation most likely occurs at the level of import. Furthermore, the import rate after stimulation must still be slow relative to the rate of export, which presumably involves interaction of MKK with export factors, including Crm1 and Ran-GTP. Thus under normal conditions, the rate of export precludes substantial accumulation of MKK in nucleus.
The importance of nuclear uptake of MKK for cell regulation has been substantiated by studies showing that constitutively active mutants of MKK are most potent as inducers of NIH 3T3 cell transformation when they lack the NES (25). This suggests that transformation is enhanced by stable nuclear localization of MKK. This effect was correlated with increased levels of activated ERK in the nucleus and was blocked by MAP kinase phosphatase 1 (25). Thus, the role of nuclear MKK in promoting cell growth requires activation of its downstream target, implying that a primary function of nuclear MKK is to maintain nuclear ERK in an active form.
An alternative function for nuclear import or export of MKK is to
facilitate inward or outward translocation of ERK, perhaps through
MKK-ERK interactions. A similar mechanism has been reported for PKI,
in which NES-mediated nuclear export of PKI functions to remove the
free C subunit of cAMP-dependent protein kinase from nuclei
and to enable reformation of the inactive R2C2
holoenzyme (26). Although this seems less likely in the case of MKK
regulation, given the evidence for dissociation of MKK and ERK after
cell stimulation (23), the mechanism has not been tested directly and
remains a viable possibility.
One of the ambiguities confounding the issue of MKK translocation is that nuclear localization of wild type MKK is never observed in response to acute stimulation under normal cellular conditions (5, 10, 11). Thus, the striking nuclear redistribution we observe during prophase suggests a mitotic role for MKK in the regulation of normal growing cells. Our results indicate that mitotic nuclear localization of NES-intact MKK also requires phosphorylation at the activation lip, similar to the characteristics for serum-dependent uptake of the NES deletion mutants. This most likely involves activation of known upstream components Src and Raf-1, which have been shown to also be active early in mitosis (27-29). We hypothesize that these events also regulate translocation at the level of import, in analogy to serum-PMA stimulation. However, the fact that nuclear localization is observed with MKK containing an intact NES indicates that a mechanism for retarding export of MKK must occur during mitosis, thus stabilizing nuclear pools of active MKK and ERK.
An important function of MKK activation during mitosis may be to
promote M phase entry in somatic cells, similar to its role during M
phase progression in meiosis. This is suggested by recent experiments
showing that the MKK inhibitor PD98059 or dominant negative mutants of
MKK both delay M phase entry in synchronized NIH 3T3
cells.2 Later in mitosis, ERK
activation appears to inhibit anaphase entry. This is suggested by
studies in cell free extracts of Xenopus embryos
demonstrating that active ERK promotes metaphase arrest and blocks
cdc2-cyclin B inactivation (30-33). We have also found that active ERK
appears to regulate a phosphoepitope recognized by the 3F3/2 antibody
(34), which is believed to regulate kinetochore attachment in metaphase
cells (22). Thus, available evidence supports both positive and
negative roles for the MKK-ERK pathway during mitotic progression.
Presumably, nuclear uptake of MKK during mitosis is involved in
controlling the activity state of nuclear pools of ERK to regulate the
timing of events in mitosis.
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ACKNOWLEDGEMENTS |
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We are grateful to Loree Kim and James Goodrich for providing HA synthetic peptide, Reinhold Krug and Ulf Rapp for plasmids for mammalian expression of BXB-Raf-1, and Jocelyn Wright and Edwin Krebs for sharing results before publication. Special thanks to Timothy Lewis and Anne Whalen for help and advice with the construction of MKK mutants and to Katheryn Resing for many helpful discussions.
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FOOTNOTES |
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* This work was supported by Grants R01-GM48521 (to N. G. A.) and F32 GM18151 (to P. S. S.) from the National Institutes of Health.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.
To whom correspondence should be addressed: Dept. of Chemistry
and Biochemistry, Campus Box 215, University of Colorado, Boulder, CO
80309. Tel.: 303-492-4799; Fax: 303-492-2439; E-mail:
ahnn{at}spot.colorado.edu.
2 J. H. Wright, E. Munar, P. Andreassen, R. Margolis, R. Seger, and E. G. Krebs, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated protein kinase; MKK, MAP kinase kinase; NES, nuclear export signal; HA, hemagglutinin; DAPI, 4',6-diaminino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; WT, wild type; PMA, phorbol 12-myristate 13-acetate; U0126, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene.
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REFERENCES |
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