1 Department of Pharmacology, School of Medicine, University of North Carolina
at Chapel Hill, Chapel Hill, NC 27599, USA
2 Center for Cell Biology and Cancer Research, Albany Medical College, 47 New
Scotland Avenue, Albany, NY 12208, USA
* Author for correspondence (e-mail: aplina{at}mail.amc.edu )
Accepted 1 April 2002
![]() |
Summary |
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Key words: Integrins, Nucleus, ERK, JNK, p38, Elk-1
![]() |
Introduction |
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The growth factor-mediated activation of ERKs has been shown by several
groups to be adhesion-dependent (reviewed by
Howe et al., 2002;
Schwartz and Baron, 1999
).
Importantly, additional levels of regulation exist. Upon activation, ERK
translocates to the nucleus, where it phosphorylates several transcription
factors, such as Elk-1 (Chen et al.,
1992
; Lenormand et al.,
1993
; Whitmarsh et al.,
1995
). We and others have recently shown that the translocation of
ERKs from the cytoplasm to the nucleus is also regulated by adhesion
(Danilkovitch et al., 2000
;
Aplin et al., 2001
).
Furthermore, adhesion enhances ERK-mediated Elk-1 phosphorylation and
transactivation potential (Aplin et al.,
2001
). ERK import into the nucleus is dependent upon both its
phosphorylation and homodimerization
(Khokhlatchev et al., 1998
).
The GTPase Ran is important for the transport of a wide variety of molecules
in and out of the nucleus (Gorlich and
Kutay, 1999
). Indeed, Ran is required for import of ERK, although
the import factors for ERK that mediate docking to the nuclear pore complex
have not been identified (Adachi et al.,
1999
). Nuclear export of dephosphorylated ERK is mediated by MEK,
which has entered the nucleus independently from ERK (reviewed by
Cyert, 2001
).
Nuclear localization of MAP kinases is vital for fulfillment of many of
their activities. Translocation of ERK to the nucleus is required for cell
cycle progression and neuronal differentiation
(Brunet et al., 1999;
Kim et al., 2000
;
Robinson et al., 1998
). JNK
and p38 forms of MAP kinases differ from ERK since they are activated more
efficiently in response to stress stimuli, such as UV irradiation and
inflammatory cytokines, rather than by growth factors
(Brunet and Pouyssegur, 1996
;
Ip and Davis, 1998
;
Ono and Han, 2000
).
Subsequently they elicit diverse responses to ERK. Similar to ERK, JNK and
p38-mediated phosphorylation of nuclear targets is essential for the
initiation of responses downstream of these MAP kinases. Fibroblasts, in which
the transcription factor c-Jun has been replaced by a mutant c-Jun deficient
in JNK phosphorylation sites, have defects in proliferation and response to
stress stimuli (Behrens et al.,
1999
). Additionally, expression of the JNK scaffolding molecule,
JNK-interacting protein 1 prevents nuclear translocation of JNK and inhibits
growth factor-induced endothelial cell proliferation
(Dickens et al., 1997
;
Pedram et al., 1998
).
MAP kinase signaling modules consist of three kinase components that act in
sequence. In the classical ERK pathway, Raf phosphorylates MAP/ERK kinases
(MEKs) 1 and 2, which in turn phosphorylate ERKs 1 and 2. JNK activation is
mediated by the MAP kinase kinases (MKKs) 4 and 7
(Derijard et al., 1995;
Lin et al., 1995
;
Sanchez et al., 1994
;
Tournier et al., 1997
). A
variety of activators of MKK4 and MKK7 have been described, including MAP/ERK
kinase kinases (MEKKs) 1-4, members of the mixed-lineage kinase family and the
apoptosis-stimulated kinases (reviewed by
Davis, 2000
;
Garrington and Johnson, 1999
).
Activated JNK translocates to the nucleus
(Cavigelli et al., 1995
;
Kawasaki et al., 1996
;
Mizukami et al., 1997
) and is
able to phosphorylate a variety of transcription factors, including c-Jun,
activating transcription factor 2 (ATF-2), Elk-1 and nuclear factor of
activated T cells (NFAT) (Chow et al.,
1997
; Gille et al.,
1995
; Price et al.,
1996
; Whitmarsh et al.,
1995
). Regulation of p38 isoforms occurs to differing extents
through phosphorylation by MKK3, -4 and -6
(Han et al., 1996
;
Han et al., 1997
;
Moriguchi et al., 1996
;
Ono and Han, 2000
;
Raingeaud et al., 1996
;
Stein et al., 1996
).
Activators of MKKs involved in the p38 pathway include MEKK4,
apoptosis-stimulated kinases (Ichijo et
al., 1997
), and transforming growth factor ß-activating
kinase (Moriguchi et al.,
1996
). In the nucleus, p38 phosphorylates Elk-1 and ATF-2, as well
as substrates that are not targets for other MAP kinases, such as CHOP/GADD153
(growth arrest and DNA damage inducible gene 153)
(Price et al., 1996
;
Wang and Ron, 1996
).
Far less is known about the activation-dependent alterations in the
localization of JNK and p38, and the control of their nucleocytoplasmic
trafficking, than is known about ERK. JNK localizes to the cytoplasm, the
nucleus and, interestingly, focal contacts
(Almeida et al., 2000;
Dickens et al., 1997
;
Read et al., 1997
). Endogenous
p38 in mammalian cells is distributed both in the cytosol and in the nucleus
(Raingeaud et al., 1995
;
Read et al., 1997
) and nuclear
p38 may be exported upon activation
(Ben-Levy et al., 1998
).
Similarly, in budding, and fission yeast the p38 homologs, Hog1 (high
osmolarity glycerol response) and Spc1, respectively, are detected both in the
cytoplasm and nucleus of unstimulated cells
(Ferrigno et al., 1998
;
Gaits and Russell, 1999
). In
budding yeast, nuclear accumulation of Hog 1 is impaired in cells with a
temperature-sensitive allele of the Ran homolog, Gsp1, when cells were
maintained at the restrictive temperature
(Ferrigno et al., 1998
).
Additionally, mutant yeast carrying a deletion of the importin-ß homlog,
NMD5, display impaired nuclear accumulation of Hog1
(Ferrigno et al., 1998
). In
Schizosaccharomyces pombe, nuclear accumulation of Spc1, in response
to osmotic stimuli, requires a functional Pim1, a homologue for the Ran
guanine nucleotide exchange factor, RCC1
(Gaits and Russell, 1999
).
Thus, despite the lack of a consensus nuclear localization sequence in p38,
uptake of MAP kinases in yeast appears to be an active process. Nuclear export
is mediated by exportins, the best characterized of which is CRM1, which
recognizes leucine-rich sequences within the cargo
(Gorlich and Kutay, 1999
). In
fission yeast, the nuclear export of Spc1 is regulated by CRM1
(Gaits and Russell, 1999
).
Since regulation of MAP kinase accumulation in the nucleus has important
biological ramifications, we analyzed whether the adhesion regulation of
nucleocytoplasmic trafficking observed for ERK extended to other MAP kinases,
namely JNK and p38. We show that activation of both JNK and p38 can be
rendered anchorage-independent either by treatment with anisomycin or, in the
case of JNK, by expression of active MEKK1. We find that active JNK and active
p38 accumulate in the nucleus both in suspended and adherent cells.
Furthermore, activated JNK efficiently phosphorylates the transcription
factors, c-Jun and Elk-1, regardless of anchorage. In contrast, ERK-mediated
phosphorylation is anchorage-dependent
(Aplin et al., 2001), a level
of control that is by-passed by expression of an active form of ERK that
accumulates in the nucleus.
![]() |
Materials and Methods |
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Antibodies
Antibodies to phospho(Ser63) c-Jun, phospho(Ser73) c-Jun,
phospho(Thr183/Tyr185) JNK, phospho(Thr180/Tyr182) p38, phospho(Thr202/Tyr204)
ERK and phospho(Ser383) Elk-1 were purchased from Cell Signaling Technology
(Beverly, MA). Other antibodies used in this study were anti-c-Jun
(Transduction labs., Lexington KY); anti-Elk-1 clone I-20, anti-JNK clone
C-17, anti-Raf clone C-12, anti-p38 clone N-20, and anti-MEK1 clone C-22 (from
Santa Cruz Biotechnology, CA); anti-FLAG M2 (from Sigma); and anti-HA clone,
12CA5 and anti-Myc clone 9E10 (from BabCo, Richmond, CA).
Cell culture and transfection
NIH3T3 cells were maintained in Dulbecco's minimal essential medium (DMEM)
containing 10% bovine calf serum and transfected with SuperFect (Qiagen,
Valencia, CA) according to the manufacturer's instructions.
Cell adhesion and preparation of cell lysate
Serum-starved cells were detached with trypsin/EDTA. Trypsin activity was
quenched with DMEM/BSA containing 1 mg/ml soybean trypsin inhibitor. Cells
were washed and rolled for 45 minutes on a rotating platform in DMEM/BSA. At
this time, cells were either maintained in suspension or replated onto dishes
coated with 10 µg/ml fibronectin (BD Biosciences, Bedford, MA) for a
further 2 hours. Cells were stimulated appropriately with anisomycin
(Calbiochem) before lysis in modified RIPA buffer containing 50 mM Hepes, pH
7.5, 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mM sodium
vanadate, 1 mM nitrophenylphosphate, 0.2 µM calyculin A, 1 mM AEBSF and 10
µg/ml aprotinin (Aplin and Juliano,
1999).
Immunoprecipitation and western blotting
For immunoprecipitations, cell lysates were first precleared by incubating
for 30 minutes at 4°C with protein G-sepharose. FLAG-Elk-1 was
immunopreciptiated from precleared lysates with anti-FLAG antibody overnight
at 4°C, followed by the addition of protein G-Sepharose and then further
incubated for 2 hours at 4°C. Precipitates were washed three times with
cold RIPA buffer and boiled with SDS-PAGE sample buffer to dissociate the
proteins.
For analysis by western blotting, samples were separated by SDS-PAGE under
reducing conditions. Immunoreactivity was detected using horse-radish
peroxidase-conjugated secondary antibodies and enhanced chemiluminescence
(Amersham, Arlington Heights, IL), as previously described
(Lin et al., 1997).
In vitro kinase assays
Protein G pellets from immunoprecipitations were washed once in modified
RIPA buffer and twice in high salt buffer
(Lin et al., 1997). Pellets
were then re-suspended in 40 µl of kinase assay buffer containing 10 mM
Tris, pH 7.5, 10 mM MgCl2, 1 mM DTT, 10 µM ATP, 5 µCi
[
-32P]-ATP (370 MBq/ml; Du Pont, Boston, MA) and 2 µg of
recombinant GST-cJun (1-133), and incubated for 30 minutes at room
temperature. Reactions were terminated by the addition of 13 µl of 4x
sample buffer and boiling for 5 minutes. The samples were subjected to
SDS-PAGE, and the gels were dried. The dried gels were exposed to X-ray films,
and the [32P]-labeled substrate bands were quantified using a Storm
840 PhosphorImager with Image-QuaNT software (Molecular Dynamics, Sunnyvale,
CA).
Immunofluorescence microscopy
Cells replated on glass coverslips, pre-coated with fibronectin or
polylysine (Sigma), were fixed in 3.7% formaldehyde in Dulbecco's phosphate
buffered saline (PBS) for 10 minutes, rinsed in PBS, and permeabilized for 5
minutes in PBS containing 0.5% Triton X-100 prior to staining. Nonspecific
staining was blocked with 2% BSA/PBS and all subsequent antibody reactions
were performed in 2% BSA/PBS. Slides were incubated with the indicated primary
antibody overnight at 4°C. Coverslips were rinsed extensively in PBS and
then stained with either FITC-conjugated goat anti-mouse, FITC-conjugated
swine anti-rabbit or TRITC-conjugated goat anti-rabbit IgG for 60 minutes at
ambient temperature, as appropriate. Phospho JNK and phospho p38 were detected
with AlexaFluor 488 anti-rabbit IgG (Molecular Probes, Eugene, OR). Following
the antibody incubations, the coverslips were washed in PBS, rinsed in
deionized water, and mounted in Permafluor (Thomas, Swedesboro, NJ). Nuclei
were stained with Hoechst 33342 reagent (Molecular Probes). Slides were viewed
either on a Zeiss Axioskop or an Olympus BX60 microscope equipped for
epifluorescence and with image capturing software.
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Results |
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|
Active JNK accumulates in the nucleus and efficiently phosphorylates
the nuclear transcription factor c-Jun in suspended cells
Once activated, JNK accumulates in the nucleus and phosphorylates several
transcription factors. c-Jun is selectively phosphorylated at serines 63 and
73 and transcriptionally activated by JNK, but not ERK
(Minden et al., 1994). We
tested whether or not adhesion affected the ability of anisomycin-activated
JNK to accumulate in the nucleus and phosphorylate c-Jun. Treatment with 50
ng/ml anisomycin enhanced anti-phospho JNK staining by immunofluorescence in
both adherent and suspended cells compared with non-treated controls
(Fig. 2A). In both adherent and
suspended cells, the majority of phospho-JNK localized to the nucleus.
Furthermore, we visualized the phosphorylation of c-Jun using
phosphorylation-state-specific antibodies. c-Jun was poorly phosphorylated at
Ser63, when cells were either adherent to fibronectin or maintained in
suspension for 3 hours in the absence of an added stimulus
(Fig. 2B). Under both adherent
and suspended conditions, robust phosphorylation was observed in response to
anisomycin-mediated activation of the JNK pathway
(Fig. 2B). Similarly,
phosphorylation of c-Jun at Ser73, in response to anisomycin treatment, was
detectable under both suspension and adherent conditions (data not shown).
Endogenous c-Jun in NIH 3T3 cells was exclusively localized to the nucleus
(Fig. 2C).
|
We next examined the extent of c-Jun phosphorylation over the range of doses that anisomycin-induced activation of JNK. Cells were either maintained in suspension or allowed to adhere to fibronectin, before stimulation with increasing doses of anisomycin. Phosphorylation of c-Jun at Ser63 and Ser73 was determined by western blotting. Consistent with observations in the immunofluorescence experiments, c-Jun phosphorylation was low in unstimulated cells (Fig. 3A). Robust phosphorylation of c-Jun at both Ser63 and Ser73 was induced in response to treatment with a 20 ng/ml or higher dose of anisomycin. The level of c-Jun phosphorylation was comparable at equivalent anisomycin concentrations both in suspended and adherent cells (Fig. 3A). The level of total c-Jun was unaltered by the state of adhesion and a mobility shift, characteristic of enhanced phosphorylation, was observed at high anisomycin doses (Fig. 3A, lower panel). Additionally, the time course of c-Jun phosphorylation, at both Ser63 and Ser73, was equivalent regardless of the state of adhesion (Fig. 3B). The combination of immunofluorescence and biochemical approaches demonstrates that activated JNK is able to localize efficiently to the nucleus and phosphorylate a nuclear target in an anchorage-independent manner.
|
Adhesion to fibronectin is required for active ERK, but not active
JNK-mediated phosphorylation of Elk-1
The lack of anchorage control of JNK-mediated c-Jun phosphorylation
contrasted with our previous findings in the ERK pathway that demonstrated
that even when ERK is activated, adhesion signals are required for ERK to
accumulate in the nucleus and phosphorylate Elk-1
(Aplin et al., 2001). Thus, we
employed the expression constructs, 22W Raf and
MEKK1, which are active
versions of components of the ERK and JNK pathway, respectively. Overexpressed
active MEKK1 was predominately localized in the cytoplasm and was excluded
from the nucleus with the exception of staining in the nucleolus
(Fig. 4A). 22W Raf also showed
predominantly a cytoplasmic localization
(Fig. 4A). We used a
FLAG-tagged version of the transcription factor, Elk-1, as a common substrate
of ERK and JNK (Gille et al.,
1995
; Janknecht et al.,
1993
; Price et al.,
1996
; Whitmarsh et al.,
1995
; Yang et al.,
1998
). The overexpressed FLAG-Elk-1 localized to the nucleus in
both adherent and suspended cells (Fig.
4B). We have previously shown 22W Raf-mediates equivalent
activation of ERK in suspended and adherent cells
(Aplin et al., 2001
). Despite
anchorage-independent ERK activation, Elk-1 phosphorylation is
anchorage-dependent in cells transfected with 22W Raf
(Fig. 4C, lanes 3,4). However,
when an activated form of MEKK1 was expressed, Elk-1 phosphorylation at Ser383
was anchorage-independent (Fig.
4C, lanes 5,6). Thus, the ability of activated JNK to
phosphorylate nuclear transcription factors is not regulated by adhesion to
the ECM, in contrast to activated ERK, which requires adhesion to efficiently
phosphorylate Elk-1.
|
Anisomycin activates the p38 pathway in an anchorage-independent
manner
We also examined activation of the p38 pathway under adhesion and
suspension conditions. Anisomycin potently activates the p38 MAP kinase
cascade at, or upstream of, MKK6 (Hazzalin
et al., 1996). We tested whether anisomycin activated p38 in an
anchorage-independent manner. Initially, we stimulated cells that were either
in suspension or re-adhered to fibronectin with different doses of anisomycin
for 30 minutes. We probed for p38 activation using a phosphospecific antibody
that detects dually phosphorylated (threonine 180 and tyrosine 182) forms of
p38. Anisomycin-treatment resulted in phosphorylation of p38, in both
suspended and adherent cells, over a similar concentration range used for the
JNK experiments (Fig. 5A).
Phosphorylation of p38 occurred within 15 minutes and was persistent through
the 60 minute time-point (Fig.
5B). Thus, similar to the JNK pathway, anisomycin treatment of NIH
3T3 cells activates the p38 pathway, irrespective of the state of
anchorage.
|
Active p38 accumulates in the nucleus of non-adherent and adherent
cells
We next examined the effects of adhesion on the accumulation of active p38
in the nucleus upon anisomycin-treatment. NIH 3T3 cells were placed in
suspension or re-adhered to fibronectin and treated with anisomycin for 30
minutes, and the localization of phospho-p38 analyzed by immunofluorescence.
Treatment of cells with anisomycin resulted in increased phospho-p38 staining
in both adherent and suspended cells (Fig.
6). In both states of adhesion, active p38 accumulated in the
nucleus. Since both Elk-1 and ATF-2 are phosphorylated by both JNK and p38, we
were unable to examine direct phosphorylation of these transcription factors
as a read-out of nuclear p38 activity in response to anisomycin treatment.
However, expression of an active form of a p38 upstream activator, MKK3,
initiated phosphorylation of Elk-1 in both suspended and adherent cells (data
not shown). Together, these data indicate that, similar to our findings with
JNK, the nucleocytoplasmic distribution of activated p38 is unaffected by
changes in cellular adhesion.
|
Expression of active nuclear-targeted form of ERK recovers Elk-1
phosphorylation in suspended cells
Our findings highlight differences between adhesion regulation of MAP
kinases, notably in their localization and ability to signal to nuclear
transcription factors. In these and previous studies
[Fig. 4C
(Aplin et al., 2001)], we have
shown that ERK activated by 22W Raf poorly signals to the nucleus in suspended
cells. Next, we decided to determine whether enhancing nuclear-targeting of
ERK could recover phosphorylation of Elk-1. To this end, we expressed a
construct (ERK2-MEK1-LA) encoding an ERK-MEK fusion protein that has the
leucine-based nuclear export signal in MEK rendered inactive by mutation to
alanine residues. In cells, ERK2-MEK1-LA accumulates in the nucleus and is
constitutively active (Robinson et al.,
1998
). When expressed in NIH 3T3 cells, western blot and in vitro
kinase analyses determined that the phosphorylation and kinase activity of
ERK2-MEK1-LA were anchorage-independent
(Fig. 7A). As before,
expression of active Raf resulted in efficient phosphorylation of Elk-1 in
adherent but not suspended cells. By contrast, Elk-1 phosphorylation at Ser383
in NIH 3T3 cells expressing the ERK2-MEK1-LA construct was
anchorage-independent (Fig.
7B). These data indicate that adhesion regulation of ERK
localization is critical for efficient phosphorylation of Elk-1; control that
is by-passed by targeting nuclear accumulation of ERK.
|
![]() |
Discussion |
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Here, we have expanded our studies on adhesion regulation of MAP kinase
compartmentalization to the JNK and p38 subfamilies. We find that in contrast
to the ERK pathway, in which accumulation in the nucleus is impaired in
non-adherent cells, activated JNK and p38 are able to efficiently localize
and, in the case of JNK, impact on nuclear events in the absence of
integrin-mediated engagement. Studies on the JNK pathway were of particular
interest since recent reports have demonstrated the presence of phosphorylated
forms of JNK both in focal adhesions and in the nucleus upon adhesion to
fibronectin (Almeida et al.,
2000). However, our observations argue against the possibility of
adhesion-dependent trafficking of JNK between focal adhesion sites and the
nucleus. Several reports have demonstrated that JNK is directly activated by
integrin-mediated adhesion in the absence of additional signals
(MacKenna et al., 1998
;
Mainiero et al., 1997
;
Miyamoto et al., 1995
;
Oktay et al., 1999
). In our
studies, it is important to emphasize that we examined changes in nuclear
activity in response to activation in pre-adherent cells, as opposed to JNK
activation directly in response to adhesion. Whether differences exist in the
ability of JNK to localize to the nucleus, when activated by adhesion versus
anisomycin treatment, remains to be determined.
Studies in COS cells show that over-expressed p38 is detected both in the
cytoplasm and the nucleus under basal conditions
(Raingeaud et al., 1995). In
contrast, p38 in 293T cells is primarily localized to the nucleus, a proposed
site of activation (Ben-Levy et al.,
1998
). In 293T cells, activation of p38 results in the
phosphorylation of MAPKAP kinase-2, which then promotes nuclear export of
active p38 via a leptomycin-B dependent mechanism
(Ben-Levy et al., 1998
;
Engel et al., 1998
). In NIH
3T3 cells, we clearly observed phosphorylated p38 co-localization with
staining for the nucleus, although it remains unresolved in our cell system
whether activation of p38 occurs in the cytoplasm or in the nucleus.
Finally, we have extended our studies on the ERK cascade. We have
previously shown that activation of ERK by expression of active versions of
upstream components in the pathway, namely Raf and MEK, renders ERK activation
anchorage-independent. However, subsequent accumulation of ERK within the
nucleus and phosphorylation of Elk-1 are anchorage-dependent
(Aplin et al., 2001). The
mechanism whereby adhesion regulates ERK nucleocytoplasmic trafficking remains
to be determined. At the level of ERK, adhesion may regulate the release from
cytoplasmic anchoring proteins, the rate of import and/or the rate of export
(Cyert, 2001
). Here, we have
used an ERK-MEK fusion, which is active and accumulates in the nucleus due to
mutation of the nuclear export sequence in MEK
(Robinson et al., 1998
). Our
findings with ERK-MEK-LA add to our understanding of adhesion regulation of
ERK. Notably, ERK-MEK-LA is phosphorylated within the activation loop and is
active in suspended cells (Fig.
7A) indicating that the activities of MKPs that downregulate ERK
are not dramatically increased in suspended cells. Additionally, expression of
ERK-MEK-LA leads to Elk-1 phosphorylation in suspended cells arguing against
the possibility that increased Elk-1 phosphatase activity underlies the
decreased Elk-1 phosphorylation in our 22W Raf experiments. Our findings with
ERK-MEK-LA do not distinguish between altered import and export because these
processes may be affected by the overexpression of ERK-MEK-LA, and the
comparable wild-type version of ERK-MEK does not possess a suitable specific
activity (Robinson et al.,
1998
). Future experiments involving temporal regulation of
activated endogenous ERK should shed light on this issue. However, these
findings underscore the importance of adhesion regulation of ERK trafficking
between the cytoplasm and the nucleus for effects on nuclear transcription
factors and possibly for adhesion-dependent cell cycle events.
Recently much attention has been paid to identifying protein-protein
interaction domains with the rationale that they will provide the molecular
basis for specificity of signaling cascades
(Hunter, 2000;
Pawson and Nash, 2000
). It has
been proposed that ERK, JNK and p38 interact with their respective cytoplasmic
anchoring proteins through a common docking (CD) domain in the C-terminus of
each molecule (Tanoue et al.,
2000
). The CD domain is characterized by several negatively
charged amino acids and is located on the opposite side to the active site.
Many of the cytoplasmic anchors that regulate localization of these MAP
kinases bind to the CD domain. For example, ERK is sequestered in the
cytoplasm through its association with partners, such as MEK and MAP kinase
phosphatase-3 (MKP-3), both of which bind to the CD domain
(Camps et al., 1998
;
Fukuda et al., 1997
;
Tanoue et al., 2000
). MKP1 and
2 also contain CD domain docking motifs and represent likely candidates for
nuclear ERK anchoring proteins (Lenormand
et al., 1998
; Tanoue et al.,
2000
; Volmat et al.,
2001
). Furthermore, it has been postulated that nuclear
translocation of ERK is dependent upon homodimerization
(Khokhlatchev et al., 1998
).
One attractive possibility from our studies on ERK is that adhesion regulates
the release of ERK from its cytoplasmic binding partners and/or
homodimerization possibly through an actin-organized scaffold
(Aplin et al., 2001
;
Leinweber et al., 1999
). CD
regions have also been identified within JNK and p38; however, the identities
of the cytoplasmic and nuclear anchors for these MAP kinases are less certain.
Nevertheless, adhesion may differentially modulate the manner by which
different members of the MAP kinase family interact with cytoplasmic anchor
proteins via the CD domain.
The role of cell adhesion molecules in the regulation of nucleocytoplasmic
trafficking of signaling molecules appears to be an emerging concept and
warrants further investigation. The ability of cadherins to inhibit
translocation of ß-catenin to the nucleus and subsequent activation of
LEF/TCF-1-mediated transcription events has been well-studied
(Gottardi et al., 2001;
Orsulic et al., 1999
;
Sadot et al., 1998
). More
recently, additional cases whereby cell adhesion receptors regulate the
nucleocytoplasmic trafficking of signaling molecules have been found
(Bianchi et al., 2000
;
Hsueh et al., 2000
;
Zimmermann et al., 2001
). In
summary, these and our studies highlight the fact that a multitude of
connections exist between cell adhesion receptors and key signaling molecules
that act as transcriptional regulators. Cell adhesion/actin cytoskeletal
regulation of nucleocytoplasmic trafficking may provide a direct channel for
communicating information from the extracellular environment to the
nucleus.
![]() |
Acknowledgments |
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