From the Laboratory for Molecular Oncology, Department of Human Genetics, University of Leuven (K. U. Leuven) & Flanders Interuniversity Institute for Biotechnology (VIB), Herestraat 49, B-3000 Leuven, Belgium
Received for publication, June 19, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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Targeting of proteins to a particular cellular
compartment is a critical determinant for proper functioning. LPP
(LIM-containing lipoma-preferred partner) is a LIM domain protein that
is localized at sites of cell adhesion and transiently in the nucleus.
In various benign and malignant tumors, LPP is present in a mutant
form, which permanently localizes the LIM domains in the nucleus. Here, we have investigated which regions in LPP target the protein to its
subcellular locations. We found that the LIM domains are the main focal
adhesion targeting elements and that the proline-rich region of LPP,
which harbors binding sites for In recent years, it has become clear that compartmentalization
within mammalian cells is a key factor for the correct functioning of
the complex network of signaling pathways in these cells. Trafficking of signaling molecules between the cytoplasmic and nuclear
compartments, for instance, has important implications for the
magnitude and specificity of gene expression (1). An interesting recent
development is the realization that adhesion receptors and their
cytoskeletal partners can regulate this nucleocytoplasmic trafficking
of signaling proteins. Specialized cell adhesion sites not only play an
architectural role in organizing cell structure and polarity but also
are dynamic units directly involved in communication via the nuclear
trafficking capability of several adhesion site-associated proteins
(1). One such protein that may play a role in this process is the
LIM-containing lipoma-preferred partner
(LPP).1
LPP is a protein that is composed of an extensive proline-rich
N-terminal region and three C-terminal LIM domains (Fig.
1A) (2). LIM domains are cysteine- and histidine-rich double
zinc finger protein motifs that comprise ~55 residues, with the
primary sequence
CX2CX16-23HX2CX2CX2CX16-23CX2C
(where X is any amino acid) (3, 4) (Fig. 1C). The
LPP protein localizes in focal adhesions, which are membrane attachment
sites of cells to the extracellular matrix (5). In addition, LPP can be
transiently translocated to the nucleus (5). The nucleocytoplasmic
distribution of this protein involves a nuclear export signal (NES)
that resides in the proline-rich region (Fig. 1A). At cell
adhesions, LPP interacts with VASP (vasodilator-stimulated
phosphoprotein) via its proline-rich region that contains two
VASP-binding (FP4) motifs (Fig. 1A) (5). In
addition, LPP also interacts with Although the molecular function of LPP is not yet known in detail,
several characteristics of this protein suggest it has multiple
functions in different compartments of the cell. LPP binding to VASP
and In addition, LPP may play a role in the development of some benign and
malignant tumors. In a subgroup of lipomas, which are benign tumors of
adipose tissue, the LPP gene acts as the preferred translocation partner of HMGA2 and in these tumors,
HMGA2/LPP fusion transcripts are expressed (2, 15, 16). Identical fusion transcripts have also been found in a subgroup of pulmonary chondroid hamartomas (17) as well as in a parosteal lipoma (18). In a
case of acute monoblastic leukemia, the LPP gene acts as translocation partner of the MLL gene, and the tumor
expresses MLL/LPP fusion transcripts (19). All tumor-specific fusion
transcripts that are expressed in the above mentioned tumors encode
similar LPP fusion proteins containing AT-hooks (DNA binding domains) of the HMGA2 or MLL proteins followed by LIM domains of LPP. As we have
shown before, these fusion proteins are mainly expressed in the nucleus
(5).
As LPP appears to execute different functions depending on its
intracellular localization, in focal adhesions or in the nucleus, targeting of LPP to these intracellular compartments is expected to be
crucial for the differential functioning of this protein. It is
possible that LPP may contribute to the tumorigenic process when its
targeting is deregulated. To date however, little is known about the
parts of LPP that target the protein to focal adhesions or to the
nucleus. To obtain more insight into this matter, we made a variety of
GFP-LPP fusion constructs containing either full-length LPP molecules
or a number of mutated forms there off. We investigated the
intracellular distribution of these chimeras. In this way, we were able
to identify distinct regions in the LPP protein as key regulators of
the subcellular distribution of LPP.
Plasmids--
Plasmids for the expression of GFP (green
fluorescent protein)-tagged parts of the human LPP protein were made by
subcloning the appropriate PCR products in the EcoRI and
PstI sites of the pEGFP-C2 vector
(Clontech). The plasmid for the expression of GFP-tagged full-length LPP was described before (5). Plasmids for the
expression of GFP-tagged human zyxin, GFP-tagged human TRIP6, and
GFP-tagged mouse ajuba were made by cloning the appropriate cDNAs
into the EcoRI and BamHI sites of the pEGFP-C1
vector (for zyxin), in the SmaI site of the pEGFP-C2 vector
(for TRIP6), or in the BglII and EcoRI sites of
the pEGFP-C1 vector (for ajuba).
Plasmids for the expression of N-terminal GFP-tagged and C-terminal
Cell Culture and Transfections--
Cell lines used in this work
included CV-1 (African green monkey kidney fibroblast cells; ATCC
CCL-70), NIH/3T3 (mouse embryonal fibroblast cells; ATCC CRL-1658), and
293T (human embryonal kidney epithelial cells containing SV40
T-antigen). Cell lines were grown in Dulbecco's modified Eagle's
medium/F12 (1:1) (Invitrogen) supplemented with 10% fetal bovine serum
(Hyclone). All cells were cultured at 37 °C in a humidified
CO2 incubator.
Transient transfections were performed using FuGENETM 6 transfection reagent (Roche Molecular Biochemicals). Cells were grown on glass coverslides (coated with fibronectin in case of NIH/3T3 cells)
to 50-60% confluency in 24-well plates. For each transfection 1.5 µl of FuGENETM 6 transfection reagent in 50 µl of
serum-free Dulbecco's modified Eagle's medium (Invitrogen) was added
to 0.5 µg of DNA and incubated at room temperature for 20 min after
which the mixture was applied directly to the growth medium of the
cells. Cells were incubated further at 37 °C for 18-24 h before analysis.
SDS-PAGE and Western Blotting--
Expression of GFP-tagged and
GFP- Immunofluorescence--
Cells were fixed in 4% formaldehyde for
20 min followed by three washes in PBS containing 0.9 mM
CaCl2 and 0.5 mM MgCl2
(PBS2+). Quenching was performed by incubating the cells
for 10 min at room temperature in PBS2+ containing 50 mM NH4Cl. Cells were then permeabilized with
0.4% Triton X-100 for 10 min at room temperature. Subsequently, the slides were incubated with primary antibodies for 30 min at room temperature. After washing the cells three times in PBS2+,
bound primary antibodies were detected with fluorescently labeled secondary antibodies (Molecular Probes) for 30 min at room temperature. Following three washes in PBS2+, slides were mounted in
Vectashield mounting medium (Vector Laboratories). For detection of GFP
fluorescence, cells were fixed and thereafter directly mounted. Slides
were analyzed on a Zeiss Axiophot fluorescence microscope equipped with
a cooled digital CCD camera system (Photometrics) using
SmartCaptureTM software. Primary antibodies used included
rabbit polyclonal anti-LPP antibody MP2, dilution 1:200 (5) and a mouse
monoclonal anti-vinculin antibody hVIN-1, dilution 1:400 (Sigma).
The LPP LIM Domains Play an Important Role in Targeting the
Protein to Focal Adhesions--
As outlined above, certain benign
tumors express HMGA2/LPP fusion transcripts encoding HMGA2/LPP fusion
proteins. These proteins are mainly localized in the nucleus (5). In
lipomas, two different fusion transcripts are found encoding HMGA2/LPP
fusion proteins composed of the three DNA binding domains of HMGA2
followed by either the two most C-terminal LIM domains of LPP
(HMGA2/LPP-short) or a portion of the proline-rich region (amino acids
372-413) and all three LIM domains of LPP (HMGA2/LPP-long) (2). Our previous observations show that when GFP-tagged forms of these HMGA2/LPP fusion proteins are overexpressed in cells, both forms are
expressed only in the nucleus (5). However, in cells expressing very
high levels of HMGA2/LPP-long, this protein is also present in the
cytoplasm and in focal adhesions while in cells expressing similar
levels of HMGA2/LPP-short, staining in focal adhesions is not observed
(5). These observations were the first indication that the LIM domains
of LPP could play a role in targeting the LPP protein to focal adhesions.
To investigate the role of the LIM domains in targeting the LPP protein
to focal adhesions, we made a number of GFP fusion proteins containing
full-length LPP molecules carrying mutations in one or two of its LIM
domains (Fig. 1B). The
mutations in the LIM domains were made in such a way that these domains
were completely destroyed: four of eight conserved zinc-binding
cysteine and histidine residues were mutated to alanine (Fig.
1C). We compared the intracellular distribution of these
mutant LPP molecules to that of the wild-type protein also expressed as
a GFP fusion protein (Fig. 1B). The distribution of the
GFP-tagged wild-type LPP protein is indistinguishable from that of the
endogenous protein: GFP-LPP is highly concentrated in focal adhesions
and, at steady state, only very low levels of the protein can be
detected in the nucleus (Fig. 1, D and D', and
our previous observations, Ref. 5).
Mutations in any of the LPP LIM domains resulted in a reduction of the
focal adhesion targeting capacity of the LPP protein (Fig. 1,
E-G and E'-G'). The amount of reduction was
different depending on which of the LIM domains was targeted by
mutations. While mutations in the third LIM domain caused a minor
reduction in focal adhesion targeting capacity (Fig. 1, G
and G'), mutations in the first LIM domain caused a more severe
reduction (Fig. 1, E and E'), and mutations in
the second LIM domain caused the most severe reduction (Fig. 1,
F and F'). When two of the LPP LIM domains were
targeted by mutations at the same time, a severe reduction in focal
adhesion targeting capacity was observed in all possible cases (Fig. 1,
H-J and H'-J'). Mutations in the second and
third LIM domain (Fig. 1, I and I'), or in the
first and the third LIM domain (Fig. 1, J and J')
reduced the level of LPP in focal adhesions in a similar way as when
the second LIM domain was mutated. The most severe phenotype was
observed when the first and second LIM domains were mutated at the same
time. In this case, focal adhesion targeting of the LPP protein was
almost completely abolished (Fig. 1, H and H').
In conclusion, our results suggest that the LIM domains of LPP play an
important role in targeting the LPP protein to focal adhesions.
Role of the Zyxin, TRIP6, and
Zyxin/TRIP6/LIMD1 Similar Regions in Targeting
LPP to Focal Adhesions--
LPP is a member of a family of proteins,
which are all proline-rich in their N-terminal region and have three
LIM domains in their C-terminal region. LPP family members include
zyxin (10), TRIP6 (21), ajuba (22), and LIMD1 (23). While all family members are quite similar in their C-terminal LIM domains, there is
only limited similarity in their proline-rich regions. The LPP protein
contains regions similar to zyxin near the N terminus (the
Recently, it was shown that when human or chicken zyxin lacking their
We also investigated whether deletion of the TRIP6 similar region or
the zyxin/TRIP6/LIMD1 similar region in LPP has an influence on the
focal adhesion targeting capacity of the LPP protein. The function of
the TRIP6 similar region is not known, either in LPP or in TRIP6. The
zyxin/TRIP6/LIMD1 similar region contains a nuclear export signal (NES)
in zyxin (26). In LPP, the function of this region is not known.
According to our previous results (5) and Fig. 2, G and
G', it does not function as a NES in LPP. No difference in
focal adhesion targeting could be detected between the wild-type
protein and mutant GFP-LPP proteins containing a deletion of the TRIP6
similar region (GFP-LPP The Three LIM Domains of LPP Cooperate for Robust Targeting to
Focal Adhesions--
To obtain more insight into how the LIM domains
of LPP function to target LPP to focal adhesions, we deleted the entire
proline-rich region of the protein. In this way, we were able to
investigate the focal adhesion targeting capacity of the LIM domains as
a separate entity. We made a construct expressing a GFP fusion protein containing all three LIM domains of LPP (Fig.
3A). In CV-1 cells, this
GFP-LPP-(412-612) protein displayed robust targeting to focal adhesions (Fig. 3, B and B'). However, staining
in focal adhesions was not as strong as the staining obtained with the
full-length wild-type LPP protein. These observations indicate that
targeting of the LIM domains to focal adhesions is not as powerful as
for the full-length protein. This suggests that also the proline-rich region of LPP might have a function in targeting the protein to focal
adhesions.
To further analyze the focal adhesion targeting capacity of the LPP LIM
domains, we investigated the targeting capacity of paired LIM domains
and individual LIM domains of LPP. To do so, we made a number of
constructs expressing GFP fusions containing LIM domains 1 and 2 (GFP-LPP-(412-531)), LIM domains 2 and 3 (GFP-LPP-(471-612)), LIM
domain 1 (GFP-LPP-(412-473)), LIM domain 2 (GFP-LPP-(471-531)), or
LIM domain 3 (GFP-LPP-(531-612)) (Fig. 3A). Upon expression in CV-1 cells of these GFP-LPP fusion proteins, important observations could be made (Fig. 3, C-G and C'-G'). In
contrast to the GFP-LPP protein containing all three LIM domains of
LPP, which had strong focal adhesion targeting, paired LIM domains,
either LIM 1 and 2, or LIM 2 and 3, as well as each individual LIM
domain showed a drastic reduction in their focal adhesion targeting
capacity. These results suggest that the three LIM domains of LPP
cooperate to provide robust targeting of LPP to focal adhesions.
The LPP Proline-rich Region Harbors Targeting Capacity for Focal
Adhesions and Stress Fibers--
The above-mentioned results on the
focal adhesion targeting capacity of the LIM domains of LPP, as
compared with the complete LPP protein, suggested that the proline-rich
region of LPP also has a function in targeting the protein to focal
adhesions. To confirm these results, we made a construct expressing a
GFP-LPP protein containing only the proline-rich region, lacking all
three LIM domains (GFP-LPP-(2-415)) (Fig.
4). CV-1 cells expressing this protein
presented staining in focal adhesions indicating that the proline-rich
region of LPP has focal adhesion targeting capacity (Fig.
5, A and A').
However, whereas the strength of focal adhesion targeting capacity of
the LIM domains was comparable to that of the full-length protein, the
targeting capacity of the proline-rich region, although clearly
detectable, was found to be much weaker.
To narrow down the area in the proline-rich region responsible for its
focal adhesion targeting capacity, we made several deletion constructs
of the proline-rich region of LPP as depicted in Fig. 4. In this way,
we were able to study the effect of deletions in the proline-rich
region as a separate entity, isolated from the strong focal adhesion
targeting effect of the LIM domains. We first deleted the
In order to discriminate between these two possibilities, we made
constructs expressing GFP-LPP proteins containing either of these
segments of the proline-rich region with the TRIP6 similar region
(GFP-LPP-(94-258), GFP-LPP-(179-415)) or without the TRIP6 similar
region (GFP-LPP-(94-258) The LPP LIM Domains Can Target the The LIM Domains of LPP Can Deplete Endogenous LPP and Vinculin from
Focal Adhesions--
As our results indicated that the LIM domains of
LPP showed robust targeting to focal adhesions, we investigated whether
these domains could compete for the subcellular localization sites of endogenous LPP and as such could interfere with the function of this
protein. For this purpose, we expressed the GFP-LPP-(412-612) molecule
containing all three LIM domains of LPP in CV-1 cells and examined the
intracellular distribution of the endogenous LPP protein with our MP2
antibody. This antibody recognizes an epitope in the proline-rich
region of LPP (5). As a consequence of this, the antibody can
discriminate between the endogenous LPP protein and the ectopically
expressed GFP-LPP-(412-612) protein that lacks the proline-rich
region. The MP2 antibody also does not cross-react with LPP family
members zyxin, TRIP6, and ajuba (Fig.
7A). Our results showed that
high levels of the GFP-LPP-(412-612) protein could displace the
endogenous LPP protein from focal adhesions and could induce an
accumulation of endogenous LPP in the cytoplasm of cells (Fig. 7,
B and B'). This result suggests that the LIM region of LPP can indeed compete for the subcellular localization sites
of endogenous LPP, interfering with the normal function of the LPP
protein.
The GFP-LPP-(412-612) molecule is not only targeted to focal adhesions
but was also found to accumulate in the nucleus of cells, most likely
via passive diffusion (Fig. 7B). In this context, we
wondered whether the nuclear accumulation of the LPP LIM domains could
play a role in the observed reorganization of LPP's intracellular distribution. To investigate this, instead of GFP-LPP-(412-612), we
expressed the GFP-LPP-(412-612)-
To further investigate the consequences of overexpression of the LIM
domains of LPP, we looked into the distribution of vinculin in
CV1-cells that overexpressed the GFP-LPP-(412-612) protein. As shown
in Fig. 7, D and D', also vinculin was depleted
from focal adhesions in cells expressing high levels of the
GFP-LPP-(412-612) protein.
The Linker between LIM Domains 1 and 2 in LPP Is Important for Its
Focal Adhesion Targeting--
If LPP family members are compared with
respect to their focal adhesion targeting capacity, a striking
difference arises between LPP, TRIP6, and zyxin on the one hand, and
ajuba on the other hand (at this moment no data are available regarding
the focal adhesion targeting capacity of LIMD1). While LPP, TRIP6, and
zyxin were all reported to localize in focal adhesions (5, 10, 14, 27),
ajuba was reported not to be targeted to focal adhesions in NIH/3T3
cells (22). We used this information to investigate further the focal
adhesion targeting capacity of the LPP protein. As our results suggest
that the LIM domains of LPP are the main focal adhesion targeting
determinants of this protein and as the zyxin LIM domains were reported
to have a similar function (14), we compared the ajuba LIM domains to
the LIM domains of the other LPP family members on the amino acid
level. This shows a significant difference: in ajuba, the linker
between LIM domain 1 and LIM domain 2 is 5 amino acids longer as
compared with the linker in LPP, TRIP6, and zyxin (Fig.
8A).
To investigate whether in LPP the linker between the first and the
second LIM domain played a role in the focal adhesion targeting capacity of this protein, we replaced this linker in LPP with the
linker of ajuba as depicted in Fig. 8A. A GFP-LPP protein containing this mutation (GFP-LPPmut_ajuba) was introduced in NIH/3T3
cells plated on fibronectin and its focal adhesion targeting capacity
was compared with the targeting capacity of the wild-type GFP-LPP
protein. Our results showed that, while GFP-LPP was prominent in focal
adhesions (Fig. 8B), focal adhesion staining of
GFP-LPPmut_ajuba was significantly reduced (Fig. 8C).
Similar findings were obtained in CV-1 cells (results not shown). These
results indicate that the linker between LIM domains 1 and 2 in LPP
plays a role in focal adhesion targeting of this protein.
We next investigated whether reciprocal substitution in ajuba (Fig.
8A) could generate focal adhesion targeting capacity for ajuba in NIH/3T3 cells plated on fibronectin. A GFP-ajuba protein containing this mutation (GFP-ajubamut_LPP) was expressed in these cells; however, no focal adhesion staining could be observed (results not shown).
Interestingly, while no focal adhesion staining could be detected in
NIH/3T3 cells when introducing a wild-type GFP-ajuba protein in cells
that were already spread for 18-24 h on fibronectin (Fig.
8D), we did detect GFP-ajuba in focal adhesions in these cells when the transfected cells were trypsinized and allowed to spread
for only 1 h on fibronectin (Fig. 8E). However, focal adhesion staining of GFP-ajuba in these cells was not as strong as the
staining observed with GFP-LPP expressing NIH/3T3 cells that were
treated in the same way (Fig. 8F).
The Nuclear Targeting Capacity of the LPP Protein--
We have
shown before that LPP has the ability to shuttle to the nucleus and
that its nucleocytoplasmic distribution is regulated by an NES (5).
This NES prevents accumulation of LPP in the nucleus in steady state
cells. However, a very low percentage of cells show accumulation of LPP
in the nucleus, suggesting that LPP shuttling to the nucleus is tightly
regulated (5). To further study the nuclear targeting capacity of the
LPP protein, we investigated the influence of mutations in the LPP
protein on its capacity to shuttle to the nucleus. Previously (5), we
have shown that the LPP nuclear export can be blocked by leptomycin B
(LMB), a drug that blocks nuclear export by preventing the formation of the NES/CRM1/Ran-GTP complex (28-31). Therefore, we incubated cells expressing mutant LPP molecules with LMB and determined whether these
molecules could still accumulate in the nucleus as an indication that
these molecules could still shuttle to the nucleus.
In this way, we investigated the nuclear targeting capacity of LPP
molecules containing mutations in the first, second, or third LIM
domain (GFP-LPPmutLIM1, GFP-LPPmutLIM2, GFP-LPPmutLIM3), or in every
combination of two LIM domains (GFP-LPPmutLIM1/2, GFP-LPPmutLIM2/3,
GFP-LPPmutLIM1/3) (Fig. 1, B and C). All of these
molecules accumulated in the nucleus of cells upon incubation with LMB,
indicating that these mutations had no influence on the ability of LPP
to shuttle to the nucleus (Fig. 9,
A-F). These results suggest that any LIM domain of the LPP
protein is dispensable for nuclear targeting. We investigated further
this result by incubating cells expressing the GFP-LPP-(2-415)
molecule with LMB. We noticed that this molecule, which contains only
the proline-rich region of LPP lacking all three LIM domains (Fig. 4),
also accumulated in the nucleus under these conditions (Fig.
9G). These results suggest that the LIM domains of LPP are
dispensable for its nuclear targeting.
To get more insight into the LPP nuclear targeting capacity, we
investigated whether small regions of the protein could transport the
GFP-
As far as the proline-rich region is concerned, we made six constructs
expressing small parts of LPP as GFP-LPP-
Concerning the LIM domains of LPP, we investigated the capability of
each of its LIM domains individually, paired LIM domains (LIM 1 and 2 or LIM 2 and 3), or all of its LIM domains to target From our previous observations (5), it is suggested that the LPP
protein has different functions depending on its localization in focal
adhesions or in the nucleus. Therefore, it is expected that the
intracellular localization of LPP is crucial for its differential
functioning. We have used several strategies in order to map specific
regions in the LPP protein that are responsible for targeting to focal
adhesions and to the nucleus.
The LIM Domains Are the Main Focal Adhesion Targeting Elements in
the LPP Protein--
Our results showed that each of the LPP LIM
domains had the capacity to be targeted to focal adhesions. However,
while GFP fusion proteins containing individual or paired LIM domains
only possessed a rudimentary capacity to localize to these sites,
fusion proteins containing all of the LIM domains of LPP showed robust targeting to focal adhesions. These results indicate that the LIM
domains of LPP cooperate to target the protein to these sites of close
cellular contact with the extracellular matrix. These findings are an
example of the fact that, although individual LIM domains can operate
as protein binding units as demonstrated by structural analysis,
protein binding is often enhanced or dependent on the presence of more
than one LIM domain (32).
Our findings on the focal adhesion targeting of LPP are somewhat
different to the recently published findings on the focal adhesion
targeting of its family member zyxin (14). Like LPP, the three LIM
domains of zyxin show robust targeting and paired LIM domains show very
weak targeting to focal adhesions. However, in contrast to LPP,
individual LIM domains of zyxin do not target to focal adhesions
(14).
The finding that the LIM domains in LPP and zyxin cooperate to target
the proteins to focal adhesions is quite different from what has been
found for the protein paxillin. Paxillin is a focal adhesion-associated
adapter protein that contains four LIM domains in its C-terminal region
(33). Brown et al. (34) showed that the third LIM domain
plays a major role in targeting paxillin to focal adhesions, whereas
the second LIM domain plays a minor role.
Our analysis of the focal adhesion targeting capacity of the LPP LIM
domains opens a number of possibilities on the mechanism by which these
LIM domains incorporate into focal adhesions. As LIM domains are known
to function as protein-protein interaction units and as each individual
LIM domain still has the capacity to localize in focal adhesions, it
can be suggested that each LIM domain binds to a protein that resides
in focal adhesions. In this way, each LIM domain can interact with a
different protein, meaning that there could be more than one protein in
focal adhesions that binds to the LIM domains of LPP. However, it could
also be that only one protein contains three docking sites for each of the LIM domains of the LPP protein. Alternatively, it is also possible
that the three LIM domains of LPP form one large binding interface. It
would be very interesting to further investigate these possibilities.
To date, no binding partners of the LPP LIM domains have been
identified that have a robust localization to focal adhesions and
therefore could be focal adhesion targeting modules for LPP.
By replacing the linker between LIM domains 1 and 2 in LPP by the one
from ajuba, we showed that the level of this mutant LPP protein in
focal adhesions was lower as compared with the wild-type protein. These
results indicate that not only the LIM domains per se but
also the linker between the LIM domains, at least the one between LIM 1 and 2, has a role in the focal adhesion targeting of LPP. Our results
suggest that the three LIM domains of LPP form a specific structure,
and that it is this structure that is important for robust focal
adhesion targeting of LPP rather than the presence of the three
individual LIM domains as separate entities.
With regard to the functional relevance of focal adhesion targeting of
LPP through its LIM domains, we have identified in yeast two-hybrid
experiments proteins that interact with the LIM domains of LPP and have
preliminary confirmed this interaction by alternative approaches. One
of these proteins is Raly,4
an RNA-binding protein, pointing toward the possible involvement of
LPP, as a component of focal adhesion complexes, in rapid
post-transcriptional changes in gene expression mediated by
repositioning of translational components to sites of signal reception,
as has been suggested for zyxin, a family member of LPP (35). In that
report, the suggestion was based on the potential nucleic acid binding
capacity of zyxin. We cannot exclude the possibility that LPP also
possesses direct nucleic acid binding capacity. However, our data
indicate that, in case LPP is mechanistically involved in such a
post-transcriptional change in gene expression at focal adhesion
complexes, mRNA binding to LPP is probably not direct, as suggested
for zyxin, but is mediated via a protein-protein complex involving LPP.
Such an indirect mechanism has recently been described for another LIM domain protein, i.e. paxillin (36). The results of these
studies suggest a new mechanism whereby a paxillin-poly(A)-binding
protein 1 complex facilitates transport of mRNA from the nucleus to
sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.
The LPP Proline-rich Region Harbors Targeting Capacity for Focal
Adhesions and Stress Fibers--
In addition to the LIM domains, the
proline-rich region of LPP also contains focal adhesion targeting
capacity. However, while the LIM domains showed robust targeting to
focal adhesions, the targeting capacity of the proline-rich region was
much weaker. These results are similar to those reported for zyxin (14)
and paxillin (37). In both of these proteins, the LIM domains are the
main targeting units for focal adhesions; however, the non-LIM region
also has some weak targeting activity.
Near its N terminus, the LPP proline-rich region contains binding sites
for two proteins, VASP (5) and
Additional evidence for the fact that
By deletion analysis, we mapped the focal adhesion targeting unit of
the proline-rich region of LPP to the C-terminal half of this region.
This result suggested that this part of the proline-rich region
contains an interaction site for a protein that is localized in focal
adhesions. Further studies will be needed to test this hypothesis.
Furthermore, our studies indicate that the functionality of this focal
adhesion-targeting unit is influenced by the presence of mutated LIM
domains. Indeed, the focal adhesion targeting capacity of the
proline-rich region as a separate entity (this is without any LIM
domains present) is stronger than that of most of the full-length
proteins (that is with all LIM domains present) containing mutations in
the LIM domains. Mutation of the second LIM domain severely reduces the
focal adhesion targeting capacity of the LPP protein especially in
combination with mutations in the first LIM domain where focal adhesion
localization is hardly detectable. Mutations in the third LIM domain
have a minor effect except when in combination with mutations in the
first LIM domain.
Our deletion analysis also uncovered a targeting site for stress fibers
residing in the N-terminal half of the proline-rich region in the area
between the VASP binding sites and the TRIP6 similar region. By
expressing this area as a GFP fusion protein, it was found to have a
rather strong targeting capacity for stress fibers. However, stress
fiber localization was not observed in all cells, the majority of cells
showed a diffuse cytoplasmic distribution of the fusion protein, but no
apparent stress fibers. These results suggest that the targeting
capacity to stress fibers of this area is regulated. The presence of a
targeting site for stress fibers in the proline-rich region of LPP
suggests that the wild-type protein should also have the capability to
localize to stress fibers. Indeed, these findings confirm our
observations regarding the intracellular distribution of the LPP
protein. Examining cells that expressed the LPP protein, very
occasionally, we could indeed see wild-type LPP protein on stress
fibers.3 In this regard, the
LPP targeting capacity to stress fibers is very different from the
targeting capacity of zyxin. In contrast to LPP, zyxin is often
observed in a periodic distribution along stress fibers particularly in
cell types that have well developed bundles of actin filaments
(14).
The LPP Nuclear Targeting Capacity--
LPP shuttles between the
cytoplasm and the nucleus and its nucleocytoplasmic distribution is in
part regulated by a nuclear export signal (5). According to recent
literature, the regulation of the nucleocytoplasmic distribution of LPP
appears to be different from that of zyxin. During treatment of HeLa
cells with the nuclear export inhibitor leptomycin B, accumulation of
GFP-zyxin within cell nuclei occurs with kinetics that vary from cell
to cell (14) while the kinetics of the nuclear accumulation of GFP-LPP
in these cells is similar in every cell (5). Accumulation of TRIP6 in the nucleus of chicken embryo fibroblasts upon treatment with leptomycin B also occurs with similar kinetics in most cells (40).
Since LPP and its family members shuttle between the cytoplasm and the
nucleus, the question arises as to how these molecules are imported
into the nucleus. For ajuba, it was shown that induction of endodermal
differentiation of P19 embryonal carcinoma cells by means of
all-trans retinoic acid resulted in nuclear accumulation of
ajuba in these cells (41). Efforts to induce nuclear accumulation in
fibroblasts of zyxin (14) or TRIP6 (40) by manipulation of tissue
culture conditions have been unsuccessful, however. Similar
experiments, performed by us, with LPP also gave no results. In our
studies, we showed that the LIM domains of LPP are dispensable for its
nuclear targeting. These results are similar to those found for zyxin
(14) and TRIP6 (40) but are unlike those observed for ajuba for which
it was shown that the LIM domains are necessary for nuclear
accumulation (41).
The amino acid sequence of the LPP protein does not contain any
consensus nuclear localization signals suggesting that it may be
imported into the nucleus via an interaction with a nuclear localization signal containing transport protein (5). In an effort to
map the interaction site with this transport protein or to map any
unconventional nuclear localization sequences in the LPP protein, the
protein sequence was divided into shorter sequences, attempting to
conserve known functional domains, and investigated the nuclear
targeting capacity of these portions by expressing these as
GFP-LPP- The LIM Domains of LPP Can Deplete Endogenous LPP and Vinculin from
Focal Adhesions--
We have shown that the LIM domains of LPP were
able to compete for the subcellular localization sites of LPP thereby
interfering with the subcellular distribution of endogenous LPP. We
also showed that overexpression of these LIM domains could deplete
endogenous vinculin from focal adhesions. Our results are similar but
not identical to those obtained for zyxin (14). High level expression of the zyxin LIM domains was shown to deplete endogenous zyxin from
focal adhesions; however, vinculin retained a normal focal adhesion
distribution in the majority of cells (14).
There are several possibilities why the LIM domains of LPP are able to
deplete the endogenous protein from focal adhesions. From our results
we know that the LPP LIM domains have a strong focal adhesion targeting
capacity that is comparable to that of the wild-type protein. This
suggests that the endogenous LPP protein and the ectopic LIM domains
compete for the same binding site(s) in the focal adhesions. However,
because of their overexpression, the LIM domains apparently win this
competition thereby depleting the endogenous LPP from these sites and
as such populating the focal adhesions with LPP LIM domains uncoupled
from the proline-rich region. In this regard, it is noteworthy that
only in cells expressing high levels of the LPP LIM domains an obvious
redistribution of endogenous LPP molecules was observed whereas in
cells expressing lower levels of the LIM domains such a redistribution
of LPP molecules was not detectable. On the other hand, it is possible
that the LIM domains of LPP, when highly overexpressed in cells,
recruit binding partners of these LIM domains, and as such of LPP, to the cytoplasm. In this way, focal adhesions would be depleted from the
LPP LIM domain binding partner repertoire, and, because these LIM
domains are LPP's main focal adhesion targeting units, this would mean
that LPP's focal adhesion targeting is disturbed. In this regard, it
is interesting to note that in cells that express high levels of the
LIM domains of LPP, focal adhesion targeting of these LIM domains is
diminished as compared with cells with lower expression levels.
In conclusion, by using different strategies, we have identified
several regions in the LPP protein that have a function in targeting
this protein to focal adhesions, stress fibers, and the nucleus. In
addition, we show that the LIM domains of LPP can compete for the
subcellular localization sites of LPP interfering with the molecular
composition of focal adhesions.
-actinin and vasodilator-stimulated phosphoprotein (VASP), has a weak targeting capacity. All of the LIM
domains of LPP cooperate in order to provide robust targeting to focal
adhesions, and the linker between LIM domains 1 and 2 plays a pivotal
role in this targeting. When overexpressed in the cytoplasm of cells,
the LIM domains of LPP can deplete endogenous LPP and vinculin from
focal adhesions. The proline-rich region of LPP contains targeting
sites for focal adhesions and stress fibers that are distinct from the
-actinin and VASP binding sites, and the LPP LIM domains are
dispensable for targeting LPP to the nucleus. Our studies have
defined novel functional domains in the LPP protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin at these sites via its
-actinin binding site located near its N terminus in the
proline-rich region (Fig.
1A).2
-actinin suggests that it has a role in certain aspects of cell
motility and actin dynamics. VASP appears to have a universal role in
the control of these processes (6, 7).
-Actinin is a cross-linker of
filamentous actin and a dynamic constituent of focal adhesions (8, 9).
In this regard, the cytoskeletal role of LPP may be quite similar to
that of zyxin, which is a family member of LPP that also localizes to
focal adhesions and binds to VASP and
-actinin (10-13). Several
lines of evidence implicate zyxin in actin assembly and organization,
and in cell movements that are known to depend on actin (14). In the
nucleus, LPP harbors a significant transcriptional activation capacity residing in the proline-rich region as well as in the LIM domains suggesting that LPP is directly involved in the regulation of gene
transcription (5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (
GAL)-tagged parts of LPP were made by subcloning
the appropriate PCR products in the NheI and XbaI
(or SacII) sites of the pHM830 vector (20). The pHM840
construct has been described before (20). LPP constructs containing
mutations or small internal deletions were made by site-directed
mutagenesis using the QuikChangeTM site-directed
mutagenesis kit (Stratagene) according to the supplier's instructions.
All PCR amplifications were done with the Pwo DNA Polymerase
(Roche Molecular Biochemicals). All synthetic mutations and
PCR-amplified regions were verified by sequencing.
-galactosidase-tagged proteins was verified by Western blotting
using a polyclonal rabbit anti-GFP antibody, dilution 1:5000 (Santa
Cruz Biotechnology). Cell extracts from transfected cells in 24-well
plates were prepared by washing the cells three times in
phosphate-buffered saline (PBS) followed by direct lysis in 100 µl of
SDS-PAGE sample buffer (60 mM Tris-HCl, pH 6.8, 12%
glycerol, 4% SDS, 5%
-mercaptoethanol). 25 µl of each cell
extract were heated at 95 °C for 5 min and were loaded onto a 7.5%
SDS-polyacrylamide gel. After size-separation, proteins were
electrophoretically transferred to PROTEAN nitrocellulose membranes
(Schleicher and Schuell). ECL Western blotting was performed using
Renaissance Western blotting detection reagents (PerkinElmer Life
Sciences) according to the supplier's instructions. In Fig. 7A, a rabbit polyclonal anti-LPP antibody MP2 was used for
ECL Western blotting at a dilution of 1:3000.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Influence of mutations in the LPP LIM domains
on the focal adhesion targeting capacity of LPP. A,
schematic representation of the subdomain structure of the LPP protein.
B, table, representing the focal adhesion targeting capacity
of GFP-LPP molecules containing mutations in their LIM domains. The
focal adhesion targeting capacity was determined by transfecting
constructs expressing the various GFP-proteins in CV-1 cells. Strength
in targeting capacity is indicated and varies from strong (++++++) to
very weak or no detection in focal adhesions (±). C,
schematic structure of a LIM domain showing the conserved zinc binding
residues. The zinc binding residues that are indicated by a
circle are the ones that were mutated into alanine in the
LIM domains of the LPP molecules containing mutated LIM domains.
D-J, CV-1 cells transiently transfected with
constructs expressing GFP-LPP (D, D'),
GFP-LPPmutLIM1 (E, E'), GFP-LPPmutLIM2
(F, F'), GFP-LPPmutLIM3 (G,
G'), GFP-LPPmutLIM1/2 (H, H'),
GFP-LPPmutLIM2/3 (I, I'), GFP-LPPmutLIM1/3
(J, J'). Cells were fixed and labeled for
vinculin. Cells were visualized either for GFP
(D-J) or vinculin (D'-J').
-actinin
binding site) (Fig. 2, A and
B), TRIP6 in the center of the proline-rich region (Fig. 2,
A and C), and a region similar to zyxin, TRIP6,
and LIMD1 at the C terminus of the proline-rich region (Fig. 2,
A and D).
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Fig. 2.
Influence of mutations in the LPP
proline-rich region on its focal adhesion targeting capacity.
A, schematic representation of GFP-LPP molecules containing
mutations in their proline-rich region, and their focal adhesion
targeting capacity as compared with the GFP-LPP wild-type protein.
B-D, alignments of portions of the LPP amino acid sequence
with zyxin and/or TRIP6 showing the surroundings of the -actinin
binding site (zyxin similar region) (B), the TRIP6 similar
region (C), and the zyxin/TRIP6/LIMD1 similar region
(D). The sequences indicated in bold are the similar regions
and were deleted from the GFP-LPP molecules in A. E-G, CV-1 cells transiently transfected with constructs
expressing GFP-LPP
41-57 (E, E'),
GFP-LPP
205-230 (F, F'), GFP-LPP
387-408
(G, G'). Cells were fixed and labeled for
vinculin. Cells were visualized either for GFP (E-G) or
vinculin (E'-G').
-actinin binding site are expressed as fusion proteins with GFP,
targeting to focal adhesions is grossly impaired (14, 24, 25). We
investigated whether this region has the same function in targeting LPP
to focal adhesions as it has in zyxin. For this purpose, we made a
construct expressing a GFP-LPP protein containing a deletion of its
-actinin binding site (GFP-LPP
41-57) (Fig. 2, A and
B). However, no difference in focal adhesion targeting could
be detected between the mutant LPP protein and the wild-type protein
(Fig. 2, E and E').
205-230) (Fig. 2, A,
C, F, and F') or a deletion of the
zyxin/TRIP6/LIMD1 similar region (GFP-LPP
387-408) (Fig. 2,
A, D, G, and G'). In
conclusion, none of the similar regions in the proline-rich region of
LPP are important for the focal adhesion targeting of LPP.
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Fig. 3.
The three LIM domains of LPP cooperate for
robust targeting to focal adhesions. A, schematic
representation of GFP molecules containing LIM domains of LPP in
different combinations, and their focal adhesion targeting capacity as
compared with the wild-type protein. B-G, CV-1 cells
transiently transfected with constructs expressing GFP-LPP-(412-612)
(B, B'), GFP-LPP-(412-531) (C,
C'), GFP-LPP-(471-612) (D, D'),
GFP-LPP-(412-473) (E, E'), GFP-LPP-(471-531)
(F, F'), GFP-LPP-(531-612) (G,
G'). Cells were fixed and labeled for vinculin. Cells were
visualized either for GFP (B-G) or vinculin
(B'-G').
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Fig. 4.
The focal adhesion targeting capacity of the
LPP proline-rich region. Schematic representation of GFP molecules
containing the proline-rich region of LPP or portions of it, and their
focal adhesion targeting capacity as compared with the wild-type
protein.
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Fig. 5.
The LPP proline-rich region harbors targeting
capacity for focal adhesions and stress fibers. CV-1 cells
transiently transfected with constructs expressing GFP-LPP-(2-415)
(A, A'), GFP-LPP-(62-415) (B,
B'), GFP-LPP-(94-415) (C, C'),
GFP-LPP-(94-258) (D, D', E,
E'), GFP-LPP-(94-258) (205-230) (F,
F', G, G'), GFP-LPP-(179-415)
(H, H'), GFP-LPP-(179-415)
(205-230)
(I, I'). Cells were fixed and labeled for
vinculin. Cells were visualized either for GFP (A-I) or
vinculin (A'-I').
-actinin
binding site in LPP (GFP-LPP-(62-415)), because
-actinin is known
to be a component of focal adhesion sites and as such could provide a
docking site for the proline-rich region of the LPP protein. However,
deletion of the
-actinin binding site did not alter the targeting of
the proline-rich region to focal adhesions (Fig. 5, B and
B'). Deletion of the VASP binding sites in addition to the
-actinin binding site (GFP-LPP-(94-415)) had no effect on targeting
to focal adhesions (Fig. 5, C and C'). Further
deletion of the TRIP6 similar region in addition to the
-actinin and
VASP binding sites (GFP-LPP-(94-415)
(205-230)) also had no effect
(results not shown). These results indicate that the focal adhesion
targeting capacity of the proline-rich region of LPP is located either
between the VASP binding sites and the TRIP6 similar region or between
the TRIP6 similar region and the C-terminal end of the proline-rich region.
(205-230),
GFP-LPP-(179-415)
(205-230)) (Fig. 4). GFP-LPP-(94-258) and
GFP-LPP-(94-258)
(205-230) almost entirely lost the ability to
target to focal adhesions (Fig. 5, D, D',
F, and F'). However, occasionally, targeting to
stress fibers was observed (Fig. 5, E, E',
G, and G'). In this regard, we noticed that
wild-type LPP protein is also occasionally observed along stress
fibers.3 On the other hand,
GFP-LPP-(179-415) and GFP-LPP-(179-415)
(205-230) did show
targeting to focal adhesions in a way that was indistinguishable from
the targeting capacity of the entire proline-rich region (Fig. 5,
H, H', I, and I'). These
results indicate that the LPP proline-rich region harbors targeting
capacity for focal adhesions and stress fibers and that these
capacities are located between the TRIP6 similar region and the
C-terminal end of the proline-rich region, and between the VASP binding
sites and the TRIP6 similar region, respectively.
-Galactosidase Protein to
Focal Adhesions--
Our results suggest that the LIM domains are the
main focal adhesion targeting elements of the LPP protein. To further
investigate these results, we wanted to determine which parts of the
LPP protein were able to trans-port a non-related molecule, such as
-galactosidase to focal adhesions. For this purpose, we made a
number of constructs expressing GFP-LPP-
GAL proteins containing the
full-length or parts of the LPP protein fused to GFP and
GAL at the
N terminus and the C terminus, respectively (Fig.
6A). The GFP-
GAL protein itself is distributed throughout the cytoplasm in CV-1 cells, and no
staining in focal adhesions can be detected (results not shown). We
first investigated whether the full-length LPP protein (GFP-LPP-(2-612)-
GAL) could target
GAL to focal adhesions. As shown in Fig. 6, B and B', this was indeed the
case. Focal adhesion staining of the GFP-LPP-(2-612)-
GAL protein
was in fact as strong as the staining obtained with the GFP-LPP
protein. We next investigated whether the proline-rich region of LPP
(GFP-LPP-(2-415)-
GAL) or the LIM domains of LPP
(GFP-LPP-(412-612)-
GAL) were able to target
-galactosidase to
focal adhesions. While focal adhesion staining could hardly be detected
when the GFP-LPP-(2-415)-
GAL protein was expressed in CV-1 cells
(Fig. 6, C and C'), robust targeting to focal
adhesions was observed when the GFP-LPP-(412-612)-
GAL protein was
expressed in these cells (Fig. 6, D and D').
Staining in focal adhesions of the GFP-LPP-(412-612)-
GAL protein
was slightly diminished as compared with the GFP-LPP-(2-612)-
GAL
protein, and a higher level of diffuse cytoplasmic staining was
observed. We next investigated the ability of paired and individual LIM domains of LPP to target the
-galactosidase protein to focal adhesions. GFP-LPP-
GAL proteins containing LIM domains 1 and 2 (GFP-LPP-(412-531)-
GAL), LIM domains 2 and 3 (GFP-LPP-(471-612)-
GAL), LIM domain 1 (GFP-LPP-(412-473)-
GAL),
LIM domain 2 (GFP-LPP-(471-531)-
GAL), or LIM domain 3 (GFP-LPP-(531-612)-
GAL were expressed in CV-1 cells. However,
careful examination of these cells did not reveal any focal adhesion
staining (Fig. 6, E-I and E'-I'). In
conclusion, under these experimental conditions, only the full-length
protein or the three LIM domains of LPP were able to target the
-galactosidase protein to focal adhesions.
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Fig. 6.
The LPP LIM domains target
the -galactosidase protein to focal
adhesions. A, schematic representation of
GFP-LPP-
GAL molecules containing full-length or different portions
of LPP and their focal adhesion targeting capacity. B-I,
CV-1 cells transiently transfected with constructs expressing
GFP-LPP-(2-612)-
GAL (B, B'),
GFP-LPP-(2-415)-
GAL (C, C'),
GFP-LPP-(412-612)-
GAL (D, D'),
GFP-LPP-(412-531)-
GAL (E, E'),
GFP-LPP-(471-612)-
GAL (F, F'),
GFP-LPP-(412-473)-
GAL (G, G'),
GFP-LPP-(471-531)-
GAL (H, H'),
GFP-LPP-(531-612)-
GAL (I, I'). Cells were
fixed and labeled for vinculin. Cells were visualized either for GFP
(B-I) or vinculin (B'-I').
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Fig. 7.
The LIM domains of LPP can deplete endogenous
LPP and vinculin from focal adhesions. A, anti-LPP MP2
antibody does not cross-react with zyxin, TRIP6, or ajuba. Cell
extracts of 293T cells transiently transfected with constructs
expressing GFP-LPP (lanes 1 and 5), GFP-zyxin
(lanes 2 and 6), GFP-TRIP6 (lanes 3 and 7), or GFP-ajuba (lanes 4 and 8)
were analyzed by SDS-PAGE and Western blotting with an anti-GFP
antibody (lanes 1-4) or with the MP2 antibody (lanes
5-8). B-D, CV-1 cells were transiently transfected
with constructs expressing GFP-LPP-(412-612) (B,
B', D, D'), or
GFP-LPP-(412-612)- GAL (C, C') and stained
with the MP2 antibody (B', C'), or an
anti-vinculin antibody (D').
GAL protein in cells. Like GFP-LPP-(412-612), GFP-LPP-(412-612)-
GAL localizes to focal
adhesions but by fusing the LIM domains of LPP to
GAL, nuclear
accumulation of these LIM domains is prevented (Fig. 7C).
Our results showed that high levels of GFP-LPP-(412-612)-
GAL could
deplete endogenous LPP from focal adhesions and induce cytoplasmic
accumulation of LPP in a way that was indistinguishable from
GFP-LPP-(412-612) (Fig. 7, C and C'). These
results indicate that nuclear accumulation of the LPP LIM domains is
not necessary to deplete LPP from focal adhesions and cause its
cytoplasmic accumulation.
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Fig. 8.
The linker between LIM domains 1 and 2 in LPP
is important for its focal adhesion targeting. A,
comparison of the amino acid sequences of the linker between LIM
domains 1 and 2 in LPP, zyxin, TRIP6, and ajuba. The boxed
residues are the amino acids that were interchanged between LPP
and ajuba to produce GFP-LPPmut_ajuba and GFP-ajubamut_LPP.
B-D, NIH/3T3 cells plated on fibronectin, transiently
transfected with GFP-LPP wild-type (B), GFP-LPPmut_ajuba
(C), or GFP-ajuba wild-type (D). E and
F, NIH/3T3 cells were transiently transfected with GFP-ajuba
wild-type (E), or GFP-LPP wild-type (F). 24 h after transfection, the cells were trypsinized and allowed to spread
on fibronectin for 1 h. Detection of GFP fluorescence was done as
described under "Experimental Procedures."
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Fig. 9.
The LPP LIM domains are dispensable for
targeting LPP to the nucleus. A-G, CV-1 cells were
transiently transfected with constructs expressing, GFP-LPPmutLIM1
(A), GFP-LPPmutLIM2 (B), GFP-LPPmutLIM3
(C), GFP-LPPmutLIM1/2 (D), GFP-LPPmutLIM2/3
(E), GFP-LPPmutLIM1/3 (F), or GFP-LPP-(2-415)
(G). Cells were incubated with leptomycin B for 2 h.
Detection of GFP fluorescence was done as described under
"Experimental Procedures."
GAL protein into the nucleus. We used the
-galactosidase protein to increase the molecular weight of these short LPP peptides. In this way, we were able to discriminate between active targeting of
short fragments of the LPP protein to the nucleus and their accumulation in the nucleus caused by passive diffusion. When dividing
the LPP molecule into smaller parts, we took into consideration the
domain structure of LPP as presently known, to avoid affecting functional domains in the protein.
GAL fusions (Fig.
10A). These proteins
contained the N terminus of LPP (GFP-LPP-(2-43)-
GAL)), the
-actinin binding site (GFP-LPP-(36-65)-
GAL)), the VASP-binding sites (GFP-LPP-(62-101)-
GAL)), the region between the VASP binding sites and the TRIP6 similar region with a deletion of the NES (GFP-LPP-(94-208)
NES-
GAL)), the TRIP6 similar region
(GFP-LPP-(201-234)-
GAL)), or the remaining C-terminal part of the
proline-rich region (GFP-LPP-(227-413)-
GAL)). As a positive control
for nuclear accumulation, we used the plasmid pHM840, which expresses a
GFP-
GAL fusion protein containing the NLS of Simian Virus 40. This
NLS directs the GFP-
GAL fusion protein to the nucleus (20). We
expressed these proteins in CV-1 cells but none of the GFP-LPP-
GAL
fusions accumulated in the nucleus (Fig. 10, C-H) in
contrast to our positive control pHM840 (Fig. 10B). We also
investigated the nuclear targeting properties of the
GFP-LPP-(2-413)
NES-
GAL protein, which contains the entire proline-rich region with a deletion of the NES; however, we found that
it was not accumulating in the nucleus (Fig. 10I).
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Fig. 10.
GFP-LPP- GAL molecules are not targeted to
the nucleus. A, schematic representation of GFP-LPP-
GAL
molecules containing different portions of the proline-rich region of
LPP. B-I, CV-1 cells transiently transfected with
constructs expressing GFP-SV40NLS-
GAL (pHM840) (B),
GFP-LPP-(2-43)-
GAL (C), GFP-LPP-(36-65)-
GAL
(D), GFP-LPP-(62-101)-
GAL (E),
GFP-LPP-(94-208)
NES-
GAL (F), GFP-LPP(201-234)-
GAL
(G), GFP-LPP(227-413)
GAL (H),
GFP-LPP-(2-415)
NES-
GAL (I). Detection of GFP
fluorescence was done as described under "Experimental
Procedures."
GAL to the
nucleus by expressing GFP-LPP-
GAL proteins in CV-1 cells (Fig.
6A). However, these fusion proteins did not exhibit any
accumulation in the nucleus (Fig. 6, D-I).
Finally, we made a construct expressing full-length LPP containing a
deletion of the NES as a GFP-LPP-
GAL fusion protein and investigated
its intracellular distribution. Whereas the protein was expressed in
focal adhesions, it did not exhibit any nuclear staining (results not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin,2 which are both
localized in focal adhesions in addition to other sites in the cell
(38, 39). However, deletion of these binding sites from the
proline-rich region did not cause a reduction in the focal adhesion
targeting capacity of this region. These results suggest that VASP and
-actinin most likely do not play a role in the focal adhesion
targeting of LPP.
-actinin binding probably does
not play a significant role in targeting LPP to focal adhesions was
obtained from the fact that the focal adhesion targeting of an LPP
protein containing an internal deletion of the
-actinin binding site
was indistinguishable from that of the wild-type protein. These results
are quite different from those observed for zyxin. Recently, it was
shown that the
-actinin binding site in zyxin is essential for its
subcellular localization (24). It was shown that point mutations of
specific amino acids in the
-actinin binding site or deletion of the
entire
-actinin binding site grossly impaired zyxin targeting to
focal adhesions (24, 25). Our different results for LPP are probably
due to the fact that LPP has a lower affinity for
-actinin as
compared with zyxin.2
GAL fusion proteins. However, this approach did not give any
positive results. Our
-galactosidase fusion proteins were not
translocated into the nucleus, either by the LPP LIM domains or by its
proline-rich region containing a deletion of the NES. These findings
differ from those obtained for TRIP6 whose LIM domains and proline-rich
region are able to target
-galactosidase to the nucleus in chicken
embryo fibroblasts (40). It is possible that the nuclear targeting in
LPP is indeed different from that of TRIP6. However, another
explanation for these differences might be that in our case the
GFP-
GAL fusion proteins were made in such a way that GFP was fused
to the N terminus and
-galactosidase to the C terminus of the
LPP-portions (or vice versa3). In this way, both
LPP termini are blocked by external sequences and this may affect
functioning due to steric interference or incorrect folding. This might
also be the reason why our GFP-LPP-
GAL fusion protein containing
full-length LPP is able to localize at focal adhesions, indicating that
the LPP entity is functioning, but cannot be translocated to the
nucleus anymore. Our studies have provided us with interesting tools
for future experiments to obtain more insight in the physiological role
of LPP, e.g. we can express an LPP molecule in cells that is
still able to localize in focal adhesions (the full-length and the LIM
domain versions) but has lost its ability to be translocated into the nucleus.
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ACKNOWLEDGEMENTS |
---|
We thank Beat Trueb for sharing some of the
unpublished data from his laboratory with us. We are grateful to Greg
Longmore for fruitful discussions and for providing us with an ajuba
cDNA. We also thank Mary Beckerle for providing us with a zyxin and TRIP6 cDNA, and Thomas Stamminger for the GFP-GAL vectors.
Leptomycin B was kindly provided by Minoru Yoshida. We thank Koen
Crombez for interesting discussions and Nancy Weyns for excellent
technical assistance. We are also grateful to Neil Taylor for reading
the article.
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FOOTNOTES |
---|
* This work was supported in part by the Fonds voor Wetenschappelijk Onderzoek (F. W. O.) -Vlaanderen (Krediet aan Navorsers, nr. 1.5.108.02), and the Geconcerteerde Onderzoekacties (GOA) 2002-2006.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.
Postdoctoral Fellow of the Fund for Scientific Research, Flanders,
Belgium (F. W. O. -Vlaanderen). To whom correspondence should be
addressed. Tel.: 32-16-34-60-80 or 32-16-34-60-76; Fax: 32-16-34-60-73;
E-mail: marleen.petit@med.kuleuven.ac.be.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M206106200
2 B. Li, L. Zhuang, M. Reinhard, and B. Trueb, submitted for publication.
4 K. R. M. O. Crombez, manuscript in preparation.
3 M. Petit, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
LPP, LIM-containing
lipoma-preferred partner;
FA, focal adhesion;
NES, nuclear export
signal;
VASP, vasodilator-stimulated phosphoprotein;
HMGA2, high
mobility group AT-hook 2;
MLL, myeloid/lymphoid leukemia or mixed
lineage leukemia;
GFP, green fluorescent protein;
TRIP6, thyroid
hormone receptor-interacting protein 6;
GAL,
-galactosidase;
PBS, phosphate-buffered saline;
LMB, leptomycin B;
CRM1, chromosomal region
maintenance 1.
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