1 ITI Research Institute, University of Bern, PO Box 54, CH-3010 Bern,
Switzerland
2 Institute for Clinical Biochemistry and Pathobiochemistry, University of
Würzburg, Versbacher Strasse 5, D-97078 Würzburg, Germany
* Author for correspondence (e-mail: beat.trueb{at}iti.unibe.ch)
Accepted 4 December 2002
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Summary |
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Key words: -Actinin, Cytoskeleton, Focal adhesion, Lipoma preferred partner, LPP, Zyxin
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Introduction |
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Zyxin has a modular structure with a proline-rich N-terminus that harbors a
nuclear export signal and three C-terminal LIM domains
(Fig. 1). It serves as a
versatile adapter protein that brings together various regulatory molecules
and cytoskeletal proteins. Although the precise function of zyxin in the
organization of the actin cytoskeleton is not yet clear, the identification of
its binding partners may shed some light onto its role. Zyxin has been
demonstrated to interact, via its C-terminal LIM domains, with members of the
cysteine-rich protein family CRP (Sadler
et al., 1992; Schmeichel et al., 1998). The LIM domains have also
been demonstrated to bind to LATS1, a tumor suppressor that appears to be
involved in the regulation of mitosis
(Hirota et al., 2000
).
Furthermore, zyxin binds to proteins of the Ena/VASP family, which control the
organization of the actin cytoskeleton
(Reinhard et al., 2001
). For
this interaction, the proline clusters within the N-terminal domain of zyxin
are responsible (Niebuhr et al.,
1997
; Drees et al.,
2000
). The proline clusters also appear to serve as binding sites
for the oncoprotein Vav, which is a guanosine exchange factor for the small
GTP-binding protein Rho (Hobert et al.,
1996
). Moreover, zyxin interacts with
-actinin, an
actin-crosslinking protein enriched at focal adhesion sites and along stress
fibers (Crawford et al., 1992
).
The exact binding site has been mapped to the extreme N-terminus of zyxin
(Reinhard et al., 1999
;
Drees et al., 1999
). We have
recently demonstrated that a linear motif of six amino acids (26-FGPVVA-31)
plays a critical role in this interaction. When a single amino acid within
this motif is replaced by using in vitro mutagenesis, binding of zyxin to
-actinin is abolished, and the subcellular distribution of zyxin is
significantly altered (Li and Trueb,
2001
).
|
Zyxin belongs to a small family of several related focal adhesion proteins.
Another member of this family is the lipoma preferred partner LPP
(Petit et al., 1996). LPP also
possesses a modular structure with a proline-rich N-terminus, including a
nuclear export signal and three C-terminal LIM domains
(Fig. 1). Similar to zyxin, LPP
has been localized to focal adhesions and to cell-cell adherence junctions
(Petit et al., 2000
). The gene
for LPP was originally discovered during the analysis of chromosomal
rearrangements in lipomas. Chromosomal translocations involving human
chromosomes 3 and 12 are found with high frequency in these benign tumors of
adipose tissues. The translocations often result in the fusion of the
HMGA2 gene on chromosome 12 with the LPP gene on chromosome
3. HMGA2 is known to code for a transcription factor of the high mobility
group of proteins (Ashar et al.,
1995
). The generated fusion proteins contain the N-terminal
sequence of HMGA2, including three DNA binding domains followed by the
C-terminal sequence of LPP with two or three LIM domains. Although a direct
relationship between tumorigenesis and the expression of these fusion proteins
has not been demonstrated in detail, it is likely that the LIM domains of LPP
contribute to the altered gene expression observed in lipomas.
A third member of the zyxin family is Trip6. This protein was originally
identified in a yeast two-hybrid screen as a protein that interacted with the
thyroid hormone receptor in a hormone-dependent manner
(Lee et al., 1995;
Yi and Beckerle, 1998
).
Similar to zyxin and LPP, Trip6 contains three LIM domains at the C-terminus
and a proline-rich N-terminus with a nuclear export signal. Furthermore, Trip6
exhibits a subcellular distribution at focal adhesion plaques quite similar to
zyxin and LPP (Wang and Gilmore,
2001
). Two additional proteins, LIMD1
(Kiss et al., 1999
) and Ajuba
(Goyal et al., 1999
), may also
be regarded as members of the zyxin protein family because they possess
similar domain structures. At the level of the amino-acid sequences, however,
these proteins are not as closely related as zyxin, LPP and Trip6.
Trip6 and LPP exhibit the highest sequence identity (53%) among all of the
zyxin family members. Their domain structures, however, are not strictly
conserved, as the proline-rich N-terminus of Trip6 is considerably shorter
than that of LPP (Fig. 1). On
the other hand, LPP and zyxin reveal a lower sequence identity (41%), but
exhibit a highly conserved domain structure. Interestingly, the sequence motif
that has previously been found in zyxin to be both necessary and sufficient
for -actinin binding is conserved in LPP
(Fig. 1). This motif, on the
other hand, is missing in the Trip6 sequence. The aim of the present study was
therefore to investigate whether LPP interacts with
-actinin in a way
similar to zyxin.
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Materials and Methods |
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For the expression of glutathione S-transferase (GST)-tagged LPP in bacteria, the cDNA sequence for LPP was amplified from a full-length human cDNA (Marathon-Ready cDNA, Invitrogen) by the polymerase chain reaction (PCR) utilizing two synthetic primers. The resulting full-length cDNA as well as a fragment derived thereof (encoding amino acid residues 1-109) were subcloned into the expression vector pGEX-4T2 (Amersham Pharmacia Biotech) downstream of the gst gene.
For expression in yeast, constructs were prepared by PCR with the help of
various synthetic oligonucleotide primers (Microsynth GmbH, Switzerland).
Selected cDNA fragments for -actinin were subcloned into the two-hybrid
prey vector pACT2 (Li and Trueb,
2001
). Two cDNA fragments for LPP that corresponded to amino-acid
residues 1-40 (LPP40) and 1-61 (LPP61), respectively, were subcloned into the
NcoI/PstI restriction site of the bait vector pGBKT7. For
competition experiments, a cDNA fragment coding for amino-acid residues 1-42
of zyxin was inserted into multiple cloning site I of the three-hybrid vector
pBridge (Clontech Laboratories, Palo Alto, CA) downstream of the sequence for
the GAL4 DNA-binding domain. As competitors, the sequences for LPP40, LPP61 or
zyxin (1-42) were ligated into multiple cloning site II of the same vector
downstream of the MET25 promoter. This promoter exhibits conditional
activity depending on the presence or absence of methionine in the culture
medium (Tirode et al.,
1997
).
Fusion constructs of LPP and green fluorescent protein (GFP) were prepared
by ligating the full-length LPP sequence or fragments LPP40 and LPP61,
respectively, into the EcoRI/SalI restriction site of the
expression vector pEGFP-C3 (Clontech) downstream of the GFP reporter
gene. A short deletion spanning nucleotides 367-417 (amino acids 41-57,
accession number U49957) was introduced into the full-length LPP construct by
the ExSite PCR-based mutagenesis method
(Costa et al., 1996;
Li and Trueb, 2001
), resulting
in the construct LPP
. For mitochondrial targeting experiments, a
synthetic oligonucleotide coding for the membrane anchor of ActA (amino-acid
residues 628-LILAMLAIGVFS LGAFIKIIQLRKNN-653) was purchased from Microsynth
GmbH (Switzerland). This oligonucleotide was inserted into the
ApaI/BamHI site of the pEGFP vector, downstream of the
sequences for LPP and GFP. Authenticity and reading frame of all constructs
were verified by DNA sequencing.
Blot overlays
GST-tagged LPP was expressed in E. coli BL21 after induction with
0.1 mM isopropylthio-ß-galactoside as suggested by the supplier (Amersham
Pharmacia Biotech). The bacteria were collected by centrifugation and lysed by
sonication. The fusion proteins were purified from the lysate by affinity
chromatography on glutathione Sepharose and analyzed on SDS polyacrylamide
gels. After transfer to nitrocellulose by electroblotting, the polypeptides
were detected with the GST detection module (Amersham) using goat anti-GST
antibodies, followed by alkaline-phosphatase-conjugated secondary antibodies
(Sigma). Similar blots that had been prepared in parallel were blocked with
bovine serum albumin and subsequently incubated with radiolabeled
-actinin in 10 mM NaCl, 1 mM Nonidet P-40, 0.1% 2-mercaptoethanol, 20
mM HEPES, pH 7.5 as previously described
(Crawford et al., 1992
;
Reinhard et al., 1999
). After
4 hours at room temperature, the blots were washed twice with the same buffer
and exposed to BioMax MS film (Eastman Kodak Co.).
Yeast two-hybrid and three-hybrid system
Yeast two-hybrid and three-hybrid experiments were carried out essentially
as described in the manuals provided by the supplier (Clontech). Yeast
reporter strain Y190 was cotransfected with the appropriate bait and prey
plasmids by the lithium acetate method. Selection for HIS3 reporter
gene activation was performed on agar plates lacking histidine, tryptophan and
leucine. Colonies that appeared after incubation for 5-10 days at 30°C
were assayed for activation of the lacZ reporter gene utilizing the
colony filter lift assay. For quantitative data, the colonies were grown in
liquid media and analyzed for ß-galactosidase activity using
O-nitrophenyl ß-D-galactopyranoside as a substrate.
Cell culture and GFP fusion protein expression
Cell lines were obtained from the American Type Culture Collection
(Manassas, VA) and kept in the laboratory at 37°C under an atmosphere of
5% CO2. PtK2 cells (CCL-56) were cultivated in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml
penicillin and 100 µg/ml streptomycin. H9C2 myoblasts (CRL-1466) were grown
in RPMI-1640 medium containing the same supplements. GFP plasmids (1
µg/well) were mixed with 100 µl Opti-MEM 1 (Life Technologies)
containing 3 µl of FuGENE-6 reagent (Roche) and added to the cells that had
grown to 60% confluence in six-well plates. Two days after transfection, the
cells were fixed with formaldehyde and prepared for indirect
immunofluorescence as described previously
(Reinhard et al., 1999). A
monoclonal antibody against human
-actinin (Sigma) was used at a
dilution of 1:400. A polyclonal antiserum that had previously been prepared in
our laboratory against human zyxin (amino acid residues 134-147) was used at a
1:50 dilution (Reinhard et al.,
1999
). After incubation with rhodamine-labeled secondary
antibodies, the slides were inspected under a Zeiss Axiovert microscope
equipped with epifluorescence optics. Electronic pictures were taken with
filter settings optimized for green (515-565 nm) and red (>590 nm) light
emission, respectively, and merged with the help of a computer software
program.
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Results |
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The analogous result was obtained with a full-length GST-LPP construct,
which migrated on a gel with a relative mobility of 100 kDa
(Fig. 2, right). In this case,
our probe reacted with the full-length polypeptide as well as with several
shorter, minor polypeptides that had probably been created from the
full-length construct by unspecific degradation. However, our probe did not
react with GST alone or with any of the polypeptide markers included on the
blot. Thus, -actinin interacts specifically with LPP in vitro.
Mapping of binding sites in -actinin and in LPP
To map the binding sites in detail, we employed the yeast two-hybrid system
(Fig. 3). The cDNA sequence for
human LPP was ligated into the bait vector pAS2-1 downstream of the sequence
for the DNA-binding domain of GAL4. By contrast, various fragments derived
from an -actinin cDNA were cloned into the prey vector pACT2 downstream
of the sequence for the transactivation domain of GAL4. A potential
interaction of the resulting fusion proteins was analyzed by growth of
transfected yeast on histidine-deficient agar plates and by transcription of
the reporter gene lacZ. All results were compared to interactions
observed between
-actinin and zyxin.
|
Initial studies demonstrated that the full-length LPP construct possessed
autonomous transactivating properties. Yeast transfected with the bait plasmid
alone grew on selective agar plates and expressed the lacZ reporter
gene. We therefore restricted our studies to the N-terminal sequence of LPP
(amino acids 1-61), which did not show autonomous transactivation as
demonstrated in a control experiment. When the corresponding GAL4-LPP
construct was transfected into yeast together with various fusion constructs
coding for -actinin, transcription of the reporter genes HIS3
and lacZ was observed. Specific interactions were noted with those
fragments that comprised the central SPEC domains of
-actinin
(Fig. 3). No interaction was
detected with the N-terminal calponin homology domains or with the C-terminal
EF hands. The minimal fragment of
-actinin that showed a positive
interaction with LPP consisted of SPEC domain 2-3. No interaction was observed
with a tandem array spanning SPEC domains 1-2 or 3-4. Furthermore, no
interaction was observed with SPEC domain 2 or SPEC domain 3 alone. In a
parallel experiment, the tandem array of SPEC domains 2-3 was also found to be
the minimal fragment interacting with the N-terminus of zyxin (residues 1-42)
(Fig. 3). In this context it
should be noted that the tandem array of SPEC domains 2-3 is the shortest
fragment of
-actinin that forms dimers in vitro (Djinovic-Carago et
al., 1999; Li and Trueb,
2001
). Taken together, our results suggest that
-actinin
binds via the same site to both zyxin and LPP.
For a quantitative comparison, we utilized a colorimetric assay and
determined the relative expression of the reporter gene lacZ. We
found that the N-terminus of LPP (residues 1-61) interacted with
-actinin (residues 264-725) with a relative affinity of
1.46±0.24 (n=3), whereas the N-terminus of zyxin (residues
1-42) interacted with the same fragment with a relative affinity of
10.28±3.1. Thus, the interaction of
-actinin with zyxin is
considerably stronger than that with LPP.
To map the binding site in LPP in more detail, we prepared a shorter
construct corresponding to amino acids 1-40 of human LPP. We found that LPP
(1-61) did interact with -actinin, whereas LPP (1-40) did not (data not
shown). The sequence deleted in the shorter LPP construct corresponded to the
-actinin-binding motif that was conserved in zyxin and LPP (see
Fig. 1). Thus, the conserved
motif appears to be responsible for
-actinin binding.
LPP competes with zyxin for -actinin binding
Since both, LPP and zyxin bind to -actinin, they may either bind
simultaneously or compete for binding to the same site. To distinguish between
these two possibilities, we employed the yeast three-hybrid system
(Fig. 4). This system makes use
of the pBridge vector, which contains two multiple cloning sites, MCSI and
MCSII. Sequences cloned into the first site are expressed as fusion proteins
with the DNA-binding domain of GAL4 similar to the situation in the normal
two-hybrid system. Sequences cloned into the second site, however, are
expressed as individual proteins (without GAL4 domain) from a conditional
promoter. This promoter is active in the absence of methionine, but repressed
in the presence of methionine. With the pBridge vector, it is therefore
possible to investigate stimulating or inhibiting effects of a third protein
onto a regular two-hybrid interaction.
|
The N-terminal sequences for LPP (residues 1-61 or 1-40) or zyxin (residues
1-42) were ligated into MCSII of the pBridge vector. The other cloning site
harbored the N-terminal sequence of zyxin (residues 1-42). These constructs
were transfected into yeast together with the bait vector pACT2 that contained
the sequence for the central -actinin rod (residues 264-725) as
outlined above.
A control experiment with a pBridge construct lacking any insert in MCSII
showed transcription of the reporter gene lacZ in the presence as
well as absence of methionine, which is indicative of a positive interaction
of zyxin with -actinin (Fig.
4). When MCSII contained the sequence for zyxin (1-42), this
interaction was strongly reduced in the absence of methionine because the
additionally expressed zyxin (without GAL4 DNA-binding domain) competed for
-actinin binding with the GAL4-zyxin fusion protein expressed from
MCSI. In the presence of methionine, no competition was observed since
transcription from MCSII was repressed. When LPP (1-61) rather than zyxin
(1-42) was expressed from MCSII, the zyxin
-actinin interaction
was also substantially reduced (Fig.
4). However, the colorimetric assay showed that zyxin (1-42)
inhibited the zyxin
-actinin interaction to a larger extent than
LPP (1-61). This result is consistent with the observation made above that
zyxin shows stronger affinity for
-actinin than LPP. No significant
inhibition was observed with LPP (1-40), which lacked the conserved
-actinin-binding site.
Our results were confirmed by the converse experiment. The sequence for LPP
(1-61) was cloned into MCSI of the pBridge vector and expressed as a fusion
protein with the DNA-binding domain of GAL4. When zyxin (1-42) was expressed
from MCSII, it strongly inhibited the LPP-actinin interaction
(data not shown). In this case, competition was nearly complete, suggesting
once more that zyxin possessed higher affinity for
-actinin than
LPP.
Taken together, our results clearly demonstrate that LPP and zyxin bind to
the same site of -actinin in a mutually exclusive manner.
Comparative subcellular distribution of LPP and zyxin
To compare the subcellular distribution of LPP and zyxin, the cDNA sequence
of LPP was ligated into a GFP expression vector. H9C2 cells were transfected
with this construct and inspected by epifluorescence microscopy
(Fig. 5). The GFP-fusion
protein was found to be distributed specifically at focal adhesion plaques as
previously demonstrated (Petit et al.,
2000) with specific antibodies. When the transfected cells were
labeled with antibodies against
-actinin and inspected by double label
fluorescence, a punctate staining was observed at focal contacts and along
stress fibers. Labeling of the transfected cells with antibodies against zyxin
specifically marked the focal adhesion plaques. The staining pattern was very
similar to that observed above with GFP-LPP
(Fig. 5). No significant
differences in the distribution of zyxin were detected between
GFP-LPP-transfected and non-transfected cells. Thus, LPP and zyxin exhibit a
very similar subcellular distribution, but GFP-LPP is not able to displace
zyxin from its normal subcellular sites, at least not to an extent detectable
under the conditions used.
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LPP interacts with -actinin in vivo
The interaction of LPP with -actinin was also verified in living
cells. An experiment was designed to investigate whether LPP, which has been
artificially targeted to mitochondria, recruits
-actinin to these
ectopic sites (Fig. 6). As a
sorting signal we used the membrane anchor of the protein ActA from
Listeria monocytogenes, which is able to direct a fusion protein to
the surface of mitochondria. Several constructs were prepared that encoded GFP
fusion proteins with the membrane anchor (M) of ActA and full-length LPP or
selected fragments derived thereof. PtK2 cells were transfected with these
constructs and analyzed under epifluorescence. The full-length construct of
LPP (GFP-LPP-M) was found to be distributed specifically at the mitochondrial
surfaces (Fig. 6).
Interestingly, this construct appeared to have the ability to induce
aggregation of the mitochondria into a few prominent clusters. When the
transfected cells were stained with antibodies against
-actinin and
inspected by double label fluorescence, a strict codistribution of
-actinin and the full-length construct was observed. This phenomenon
was particularly evident at larger clusters of mitochondria
(Fig. 6, arrows). When the
conserved
-actinin-binding motif was removed from the full-length
construct by deleting residues 41-57 (construct GFP-LPP
-M), no
codistribution of the fluorescence signal from
-actinin and GFP was
observed. Analogous results were obtained with shorter fragments derived from
the full-length LPP construct. A fragment spanning only the N-terminal amino
acids 1-61 of LPP (GFP-LPP61-M) codistributed with
-actinin and induced
clustering of the mitochondria that was very similar to the full-length
construct (Fig. 6, arrows). In
contrast, a shorter construct that lacked the conserved
-actinin-binding site (GFP-LPP40-M spanning residues 1-40) did not show
any codistribution and did not induce mitochondrial clustering. Likewise, no
codistribution of
-actinin and GFP and no clustering of mitochondria
was observed in cells transfected with GFP-M alone.
|
These results demonstrate that LPP interacts with -actinin in vitro
as well as in vivo. Furthermore, the conserved
-actinin binding site is
essential for recruitment of
-actinin to an ectopic site.
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Discussion |
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The functional similarities between zyxin and LPP also extend to their
binding partners. Both zyxin and LPP bind to VASP, a protein involved in the
control of actin polymerization (Petit et
al., 2000; Reinhard et al.,
2001
). In the present publication we showed that LPP, similar to
zyxin, also interacts with
-actinin. This interaction could be
demonstrated in vitro with the isolated proteins (blot overlay) as well as in
yeast cells by the two-hybrid system. The interaction was also verified in
mammalian cells in an experiment where LPP was able to recruit
-actinin
to the surface of mitochondria when artificially targeted to these ectopic
sites.
Truncation analyses allowed us to localize the binding site to the central
rod of -actinin, which contains the spectrin-like repeats SPEC 2 and 3.
Only fragments that could dimerize in vitro were able to interact with LPP,
suggesting that the dimeric conformation of
-actinin is required for
binding. A similar conclusion has previously been reached with zyxin
(Li and Trueb, 2001
). The
formation of such dimers offers a plausible explanation why the recruitment of
-actinin to LPP, which has been expressed on mitochondrial surfaces,
may lead to the striking clustering of the mitochondria:
-actinin in
its dimeric form might function as a divalent crosslinker and connect two LPP
molecules expressed on two different mitochondria. It is possible that this
crosslinking property of the
-actinin/LPP complex has functional
implications in the formation of focal adhesions in living cells.
The binding site of -actinin in LPP, by contrast, was mapped to a
motif present at the extreme N-terminus of LPP that is fully conserved in
zyxin. When this motif was deleted, LPP did not interact with
-actinin
in the two-hybrid system and lost its ability to recruit
-actinin to an
ectopic site in mammalian cells. The conserved motif contains hydrophobic and
basic amino acids (KKFXPVVAPKPK) and does not occur in any other protein
except zyxin and LPP.
In this publication we made extensive use of the three-hybrid system to
tackle the question of whether LPP and zyxin bind to -actinin in a
mutually exclusive manner. Our results demonstrate that the two proteins
compete for the same binding site in
-actinin. Since LPP and zyxin
coexist in most fibroblastic and epithelial cells, the question about the
biological significance for this functional redundancy arises. The answer
might be found in the existence of subtle differences between the two
proteins. Petit et al. described minor differences in the intracellular
distribution (Petit et al.,
2000
). Although both proteins were found at focal adhesions and
cell-cell contacts, zyxin was more prominently distributed than LPP along
stress fibers. Furthermore, there was a difference in the relative abundance
of the two proteins. In fibroblasts, the level of zyxin was about five times
higher than that of LPP. However, no significant difference in the relative
abundance was observed in epithelial cells
(Petit et al., 2000
). In this
publication we demonstrated that there is also a manifest difference between
the two proteins in their relative affinity for
-actinin. Utilizing a
quantitative colorimetric assay, we found that zyxin bound to
-actinin
with much higher affinity than LPP. A similar colorimetric approach was
originally used to determine the dissociation constants of the retinoblastoma
protein and its binding partners (Yang et
al., 1995
). The authors found that the binding affinities
determined by surface plasmon resonance correlated well with the results
obtained by the two-hybrid assay. We could confirm the difference between
zyxin and LPP in the affinity for
-actinin by direct competition
experiments. Zyxin completely abolished binding of LPP to
-actinin,
whereas LPP just reduced binding of zyxin to
-actinin in the converse
experiment.
In spite of the fact that LPP and zyxin compete for the same binding site
in -actinin, LPP was not able to displace zyxin from its normal
subcellular site when overexpressed in mammalian cells. Likewise, our
preliminary experiments (B. Li, unpublished) suggest that zyxin is not able to
displace LPP from focal adhesions, at least not to an extent detectable under
our experimental conditions. We interpret these findings as indicating the
existence of a multitude of additional binding partners for zyxin and LPP. It
might therefore not be possible to disturb a pre-existing focal adhesion
complex by the mere overexpression of a single component.
We assume that the subtle differences between LPP and zyxin, as outlined
above, play a decisive role in the differential function of the two proteins.
Zyxin is actively involved in the organization of focal adhesions, complexes
that are composed of more than 50 different proteins
(Zamir and Geiger, 2001). When
the expression of zyxin is inhibited by interfering RNAs, focal adhesions are
diminished and stress fibers are greatly reduced
(Harborth et al., 2001
). The
assembly and disassembly of these complexes is critical for cell motility and
may represent the limiting step for maximal speed of migration
(Palecek et al., 1998
).
Disassembly at the distal edge of the focal adhesions and assembly at the
proximal edge is controlled by dynamic protein-protein interactions. Zyxin is
one of the first molecules that dissociates from dissolving focal adhesions
(Rottner et al., 2001
). Our
previous studies showed that the interaction with
-actinin is necessary
for zyxin to localize to focal adhesions
(Reinhard et al., 1999
). Thus,
the competition of LPP (and other competitors) with zyxin for
-actinin
could provide one of the mechanisms for the dissociation of zyxin from
dissolving focal adhesions. It would therefore be interesting to investigate
whether zyxin and LPP show differences in their relative distribution at the
distal and the proximal end of focal adhesions.
So far, all our studies have been limited to bilateral protein-protein interactions. It is obvious, however, that a comprehensive understanding of the function of focal adhesions cannot be gained without extending our analyses to multiple protein interactions as they exist under physiological conditions. It is possible that the new technology of proteomics will be instrumental in tackling this challenge.
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Acknowledgments |
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