From the Institute of Clinical Biochemistry and
Pathobiochemistry, University of Würzburg, Versbacher Strasse 5, D-97078 Würzburg, Germany and the ¶ M. E. Müller
Institute, University of Bern, P. O. Box 30, CH-3010 Bern, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The LIM domain protein zyxin is a component of
adherens type junctions, stress fibers, and highly dynamic membrane
areas and appears to be involved in microfilament organization. Chicken zyxin and its human counterpart display less than 60% sequence identity, raising concern about their functional identity. Here, we
demonstrate that human zyxin, like the avian protein, specifically interacts with A panoply of cellular processes, such as cell-cell and cell-matrix
adhesion, motility, morphogenesis, growth and differentiation, are
largely dependent on the structure and dynamics of the actin-based cytoskeleton and its associated signaling proteins. Elucidation of the
mutual interactions between the wide variety of known
microfilament-associated proteins has shed light on the underlying
organization of the multi-level protein network and also on its
linkages e.g. to transmembrane receptors (for a review see
Ref. 1). Due to their involvement in a variety of biological systems,
special interest has been devoted to zyxin and the F-actin-binding
protein Zyxin was originally identified in chicken fibroblasts as a protein
associated with focal adhesions, stress fibers, and cell-cell adherens
junctions (19). A human cDNA coding for a protein closely resembling chicken zyxin but with less than 60% sequence identity has
been isolated in a subtractive cDNA cloning approach by virtue of
its reduced expression in SV40 transformed fibroblasts (20). Independently, an equivalent cDNA has been cloned from an umbilical vein endothelial cell library (21). Zyxin is composed of an extended
N-terminal part, which is rich in prolines, and three C-terminal LIM
domains. In addition to its sequence similarity, the human protein
showed the same subcellular distribution (21, 23) and VASP binding
activity (23) as chicken zyxin and hence was proposed to represent the
mammalian homologue of avian zyxin (20, 21, 23).
There is a large body of evidence that zyxin plays a major role in the
organization of actin filaments in living cells. This evidence
originates from its structural and functional relationship with the
bacterial protein ActA found at the surface of the intracellular bacterial pathogen Listeria monocytogenes (23, 25, 27, 28). This microorganism invades eukaryotic cells and exploits the host actin-based cytoskeleton to promote its own intracellular motility and
cell to cell spread (for a review, see Ref. 29). Detailed studies have
revealed that the bacterial protein ActA is both necessary and
sufficient for the recruitment of actin filaments and actin-based
motility (29). ActA seems to accomplish this function by enhancing the
Arp2/3 complex driven nucleation of new actin filaments (30). The
N-terminal region of zyxin appears to share some structural and
functional similarity with the central and the C-terminal portion of
the ActA protein (23, 25, 28). In particular, both zyxin and ActA
harbor proline-rich binding sites for their common ligands VASP (23,
25, 27) and Mena (24, 25). The interactions with VASP and Mena are
thought to contribute to efficient actin polymerization by recruiting polymerization competent profilactin complexes (24, 31). Hence, zyxin
may likewise regulate actin filament organization and thus represent an
endogenous ActA analogue in eukaryotic cells (23, 25, 28).
By in vitro binding assays, chicken zyxin has been shown to
interact with Therefore, we set out to investigate a putative interaction between
human zyxin and GST Zyxin Fusion Proteins
Twelve cDNA clones for human zyxin had previously been
isolated from a placenta cDNA library (20). The inserts of seven clones with successively truncated 5'-ends (B91, B52, B4, 925, 835, 725, and 225) were selected and subcloned into the EcoRI restriction site of the expression vector pGEX-5X (Amersham Pharmacia Biotech) downstream of the GST gene. Orientation and reading frame of
the final constructs were verified by DNA sequencing. Shorter fusion
polypeptides were prepared by expression of different restriction fragments in E. coli (BL21). These fragments were prepared
from the 5'-end of the full-length zyxin cDNA by partial or
complete digestion with the enzymes EcoRI, SmaI,
NcoI, MspI, and HaeIII and
subsequently ligated in the desired reading frame into the expression
vector pGEX-5X.
Mitochondrial Targeting of Zyxin
An FspI fragment comprising bases 55-1318 of the
human zyxin cDNA (20) was inserted into the SmaI site of
the eukaryotic expression vector pSPL61 (33, 34). The
MscI-BamHI fragment of this construct was
replaced by sequential ligation with the zyxin
MscI-MaeII fragment and an
MaeII-BamHI adapter consisting of the
complementary oligonucleotides A1 and A2, resulting in the wild type
zyxin mitochondrial targeting construct (targZyxinWT).
Construction of Zyxin Deletion Mutants
Deletion of Zyxin Amino Acids 19-41--
Two PCR products were
amplified with the wild type mitochondrial targeting construct as a
template and the primer pairs U4589/S1L and S1U/L1708, introducing a
unique SacII restriction site into each PCR product. Both
products were digested with SacII and ligated. The ligation
product was then used as a template for PCR amplification with the
primer pair U159/L1423. The resulting PCR product was gel purified and
cloned into pCRII vector (Invitrogen) and a 267-base pair
NcoI-BglII fragment of the corresponding clone
was ligated into either NcoI-BglII digested
targZyxinWT or wild type GST-zyxin vector.
Deletion of Zyxin Amino Acids 261-283--
Two PCR products
amplified from wild type GST-zyxin vector with the primer pairs S1U/S2L
and S2U/L1868, respectively, were ligated after NgoMI
digestion. With the ligation product as a template and the primers S1U
and L1868, a PCR product was amplified, gel purified, and ligated into
pCR-Script Amp SK(+) vector (Stratagene). A 589-base pair fragment,
which comprises the deletion site corresponding to zyxin amino acids
261-283, was excised with BglII-DraIII and inserted into the BglII-DraIII sites of wild type
GST-zyxin, GST-zyxin Insertion of Zyxin Amino Acids 262-284 into Position
17-41--
Two complementary synthetic oligonucleotides, OU and OL,
were annealed, phosphorylated with T4 polynucleotide kinase, and inserted into the SacII-digested and dephosphorylated
targZyxin
All junctions of different sequence inserts as well as all
PCR-amplified regions were verified by DNA sequencing.
Oligonucleotides
The oligonucleotides used were as follows: A1,
5'-CGTGCTCTGTCGGAAGTGCCACACTGCTAGAGCCCAGACG-3'; A2,
5'-GATCCGTCTGGGCTCTAGCAGTGTGGCACTTCCGACAGAGCA-3'; L1423,
5'-ACTTAGGCGCTGGAGCCGGAGATGAG-3'; L1708,
5'-GCTCCTCCACCTCCTTCAGA-3'; L1868, 5'-GCAGGTGAAGCAGGCGATGTG-3'; OU,
5'-CCCTAAGTTTTCTCCAGTGACTCCTAAGTTTACTCCTGTGGCTTCCAAGTTCAGTCCTGGAGC-3'; OL,
5'-TCCAGGACTGAACTTGGAAGCCACAGGAGTAAACTTAGGAGTCACTGGAGAAAACTTAGGGGC-3'; S1L, 5'-TGCACCCCGCGGGAGCCGAGACCGAAACGGAG-3'; S1U,
5'-CGGCTCCCGCGGGTGACAGCGAGCCTCCCCCGGCAC-3'; S2L,
5'-GAAATCTTGCGGCCGCACGCCGGCGATGAGGCTGGGGGCCCTCGG-3'; S2U, 5'-GTTCAGTCCGCCGGCGCCAGGTGGATCTGGGTCACAA-3'; U159,
5'-GAATGAAAGACCCCACCTGTA-3'; U4589, 5'-CGACACGGAAATGTTGAATAC-3'.
Green Fluorescent Protein (GFP) Zyxin Fusion Constructs
The full-length cDNA sequence of human zyxin and three
deletion constructs (see above) were amplified by PCR and cloned into the EcoRI-BamHI site of the expression vector
pEGFP-C3 (CLONTECH, Palo Alto, CA) downstream of
the reporter gene for GFP. The final constructs harbored the zyxin
sequence for amino acid residues 1-570 (wild type) or the same
sequence with deletions corresponding to amino acid residues 19-41
(GFP-zyxin Transfection of Cells
pEGFP-C3 plasmids (1 µg/well) were mixed with 100 µl of
Opti-MEM 1 (Life Technologies) containing 3 µl of FuGENE-6 reagent (Roche Molecular Biochemicals) and were added to COS-1 cells (ATCC CRL-1650), which had grown to 60% confluence in 6 well plates. Chicken
tendon fibroblasts (36) were transfected in a similar way using
SuperFect reagent (Qiagen). One to 2 days after transfection, the cells
were washed with PBS and inspected at an excitation wave length of
450-490 nm.
PtK2 cells were grown on coverslips in minimum essential
medium with Eagle's salts, Glutamax ITM (Life
Technologies, Inc.), and 10% fetal calf serum. Cells were transfected
with the mitochondrial targeting constructs by the calcium phosphate
method, using an overnight incubation with 5 µg of DNA per 3-cm dish.
Two days posttransfection, cells were prepared for indirect
immunofluorescence microscopy.
Immunofluorescence Microscopy
Immunofluorescence microscopy of formaldehyde fixed cells was
done essentially as described (37). The following antibodies were used:
rabbit-anti zyxin serum AS83-1 (23), mouse anti- Immunoblots
Proteins were resolved on SDS-polyacrylamide gels and
transferred to nitrocellulose by electroblotting. The blots were
incubated with various antibodies or processed for blot overlays. Goat
anti-GST antibodies were purchased from Amersham Pharmacia Biotech. The preparation of the anti-human zyxin monoclonal antibody 164ID4 will be
described elsewhere.2 A
polyclonal antiserum against a synthetic peptide corresponding to human
zyxin residues 134-147 was raised in rabbits. Bound antibodies were
detected with 125I-labeled sheep anti-mouse antibody
(Amersham Pharmacia Biotech) or alkaline phosphatase-conjugated
anti-rabbit antibodies (Sigma) followed by development with
bromochloroindolyl phosphate and nitroblue tetrazolium substrate.
Blot Overlays
For radiolabeling, Blot overlays were performed essentially as described (18). Residual
protein-binding sites on the nitrocellulose blots were blocked
overnight with 0.75% bovine serum albumin in 10 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% 2-mercaptoethanol, 20 mM HEPES, pH 7.5. The blots were incubated at room
temperature on a rocking platform with the radiolabeled probe (5 × 105 cpm/ml). After 4 h, the blots were washed twice
with the same buffer and exposed to BioMax MS film (Eastman Kodak
Co.).
Solid Phase Binding Assay
For conjugation with peroxidase Peptides corresponding to human zyxin (20, 21) amino acids 21-42 plus
an N-terminal cysteine residue (zyxin21-42) and murine VASP
(EMBLTM accession number X98475) amino acids 313-333
including a terminal cysteine (control peptide) were synthesized with
acetylated Maleimide activated microtiter plates (Reacti-BindTM,
Pierce) and amine binding microtiter plates with an
N-oxysuccinimide surface (DNA-BINDTM, Costar)
were coated with 50 or 100 µg/ml peptide solution, respectively. After washing and blocking of remaining binding sites 0.5-50 µg/ml peroxidase-labeled Blot Overlays of N-terminal Zyxin Deletion Mutants Reveal a
Functional -actinin. Furthermore, we map the interaction site to
a motif of approximately 22 amino acids, present in the N-terminal
domain of human zyxin. This motif is both necessary and sufficient for
-actinin binding, whereas a downstream region, which is related in
sequence, appears to be dispensable. A synthetic peptide comprising
human zyxin residues 21-42 specifically binds to
-actinin in solid
phase binding assays. In contrast to full-length zyxin, constructs
lacking this motif do not interact with
-actinin in blot overlays
and fail to recruit
-actinin in living cells. When zyxin lacking the
-actinin binding site is expressed as a fusion protein with green
fluorescent protein, association of the recombinant protein with stress
fibers is abolished, and targeting to focal adhesions is grossly
impaired. Our results suggest a crucial role for the
-actinin-zyxin
interaction in subcellular zyxin localization and microfilament organization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin, two interacting constituents of these
supramolecular protein complexes.
-Actinin is a major constituent of diverse actin-based structures,
such as stress fibers, focal adhesions, and the peripheral belt of
epithelial cells (2, 3).
-Actinin is also found in the cortical
actin filament webs at the periphery of highly dynamic lamellae (2).
The protein forms a rod-shaped antiparallel dimer of approximately
100-kDa subunits, each subunit comprising an N-terminal actin binding
domain, four central spectrin-like repeats, and two C-terminal EF-hand
motifs (4). Whereas the spectrin-like repeats are thought to be
involved in dimerization, the C-terminal domain confers
Ca2+ sensitivity on actin binding of different nonmuscle
isoforms (5). Like few other cytoskeleton-associated proteins,
-actinin constitutes a direct link between the actin cytoskeleton
and the cytoplasmic domains of several cell surface receptors,
including integrins
1 (6),
2 (7), and
IIb
3 (6),
ICAM-11 (8), ICAM-2 (9),
L-selectin (10), and the
N-methyl-D-aspartate receptor (11). This
cytoskeletal connection has been implicated in receptor function and
anchorage, cytoskeletal reorganization, and concomitant signaling
events (1, 7, 11). Binding of
-actinin to PIP2 and
phosphatidylinositol 3-kinase, as well as a PIP2-regulated
interaction with the Rho effector kinase PKN (12) and vinculin (13) are
also consistent with functional roles of
-actinin in signal
transduction pathways and cytoskeletal organization (5).
-Actinin is
intimately linked with microfilamentous structures (5) not only by its
actin cross-linking activity but also by its interaction with the
cell-cell adherens junction protein
-catenin, different
cysteine-rich proteins (CRPs) (14-17) and zyxin (18). Due to the
substantial overlap in subcellular distribution and the emerging
functions of zyxin in actin filament regulation, the
-actinin-zyxin
interaction has gained special interest.
-actinin (18); the LIM-only proteins CRP1, CRP2, and
CRP3/MLP (14-17); and the Ena VASP homology 1 domain of the microfilament organizing proteins VASP and Mena (23-25). The
proto-oncogene product Vav binds zyxin in an SH3
domain-dependent manner (26). The in vitro
interaction between zyxin and
-actinin is of moderate affinity and
involves the N-terminal part of
-actinin (18), which includes the
actin binding domain (4) and also binds to PIP2 (5) and
chicken CRP1 (32). Vice versa, the
-actinin binding site has only
roughly been mapped to the N-terminal region of chicken zyxin (amino
acids 1-348; Ref. 15). A similar interaction with
-actinin has not
yet been established for human zyxin, and also, functional in
vivo evidence for a direct
-actinin-zyxin linkage is missing.
-actinin in more detail. In this study, we
demonstrate that the human protein binds to
-actinin as described for the avian protein. We further localize the interaction site to a
motif of about 22 amino acids, present in the zyxin N terminus. Using a
synthetic peptide and various deletion constructs, we finally show that
this motif, but not a related downstream region, is both necessary and
sufficient for zyxin interaction with
-actinin in vitro.
Furthermore, this
-actinin binding site is essential for
zyxin-dependent
-actinin recruitment in living cells as
well as for proper subcellular zyxin localization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
19-41, targZyxinWT, and targZyxin
19-41,
yielding the corresponding constructs GST-zyxin
261-283, GST-zyxin
19-41/
261-283, targZyxin
261-283, and targZyxin
19-41/
261-283, respectively.
19-41 construct. To reduce the background of parental
plasmids, the ligated product was digested with SacII prior
to transformation into competent DH5
cells.
19-41), 261-283 (GFP-zyxin
261-283) or 19-41 and
261-283 (GFP-zyxin
19-41/
261-283). The reading frame and the
authenticity of the final constructs were verified by DNA sequencing.
-actinin monoclonal
antibody BM 75.2 (Sigma), Oregon Green-labeled goat anti-rabbit
antibody (Molecular Probes, Eugene, OR, USA), and Cy3-labeled goat
anti-mouse IgG + IgM antibodies (Jackson ImmunoReasearch Laboratories,
West Grove, PA). Prior to use the latter antibodies were preadsorbed to
rabbit
globulins (Serva, Heidelberg, Germany) immobilized on a
Sepharose cartridge.
-actinin (Sigma A9776) was dialyzed
exhaustively against 500 mM NaCl, 50 mM
Tris-HCl, pH 7.6, and transferred to a test tube, which had been coated
with Iodogen according to the instructions of the manufacturer
(Pierce). Carrier-free Na125I (0.5 mCi/200 µg of protein;
final volume, 500 µl) was added, and the reaction was allowed to
proceed for 10 min on ice, followed by 5 min at room temperature.
Radiolabeled protein was separated from nonincorporated iodine by
chromatography on Sephadex G-50.
-actinin was purified from
chicken gizzard essentially as described (38), except that
phosphocellulose P11 was substituted by fibrous phosphocellulose
(Sigma, C-3145), and
-actinin was eluted with a linear gradient of
0-500 mM NaCl in equilibration buffer. The protein was
>95% pure as judged from SDS-polyacrylamide gel electrophoresis
analysis and Coomassie Blue staining. 300 µg of purified
-actinin
was coupled to 1 mg of preactivated horseradish peroxidase (Pierce)
according to the manufacturer's instructions.
-amino groups and amidated carboxyl termini. The peptides
were purified on a VYDAC RP C-8 column to >85% purity and were
verified by mass spectral analysis (Genosys Biotechnologies,
Pampisford, United Kingdom).
-actinin (protein concentration refers to
-actinin) was added (triplicates). Bound
-actinin-peroxidase conjugate was detected by incubation with o-phenylenediamine
peroxidase substrate solution (Sigma). The reaction was stopped by
addition of 1 volume of 750 mM HCl. For analysis,
background values obtained without peptide coating were subtracted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Actinin Binding Site
A potential interaction of
-actinin with human zyxin was investigated in a blot overlay assay.
To this end, several cDNA clones for human zyxin were expressed as GST fusion proteins in a prokaryotic expression system. These clones
coded for the full-length zyxin sequence and for fragments thereof that
were successively truncated from the N terminus. Detection of the
expressed fusion proteins with an anti-GST antibody showed that the
full-length protein and the truncated forms could successfully be
expressed in bacteria, but that a large percentage of the products was
degraded into a complex mixture of fragments (not shown). Changing the
conditions (i.e. lowering the temperature for expression or
utilizing a bacterial strain with minimal endogenous protease activity)
did not solve the problem. We therefore transferred the entire mixture
of GST fusion proteins to nitrocellulose membranes and probed this blot
with radioiodinated
-actinin (Fig. 1).
Our probe bound to bands of 80 and 36 kDa derived from the full-length construct (amino acids 1-572). A positive reaction with the
full-length fusion protein of 110 kDa was visible only when the gel was
overloaded and when the blot was exposed for a prolonged period of time
(not shown; see also Fig. 6). In contrast, no specific interaction was
observed with any of the successively truncated GST-zyxin fusion
proteins starting at zyxin amino acid 58 or C-terminal thereof. Some
unspecific binding was observed with two bacterial polypeptides of 28 and 33 kDa present in all samples, but these signals disappeared when
the fusion proteins were purified on glutathione-Sepharose before being
transferred to nitrocellulose. Thus, human zyxin contains at least one
major binding site for
-actinin, and this site must be located, at
least in part, within the first 57 amino acids of the polypeptide.
View larger version (86K):
[in a new window]
Fig. 1.
Interaction of
-actinin and human zyxin. Different zyxin
cDNA constructs were expressed in bacteria as GST fusion proteins,
and the proteins were resolved by 10% polyacrylamide gels. After
transfer to nitrocellulose, the proteins were probed with
radioiodinated
-actinin. The cDNA constructs coded for the
full-length zyxin sequence (amino acids 1-572) or for fragments that
were successively truncated from the N terminus as indicated at the
top.
To narrow down this site, we prepared several shorter constructs that
covered different regions within the first 51 amino acids of human
zyxin and expressed them in bacteria. These fusion peptides proved to
be fairly resistant against endogenous proteolysis and could be
immobilized on nitrocellulose in virtually intact form after
purification on glutathione-Sepharose (Fig.
2). Radiolabeled -actinin bound
specifically to the fusion peptides containing residues 1-51 and
1-41, but not to those containing residues 4-18 or 4-27. These
results suggest that amino acids 28-41 are part of a major binding
site for
-actinin.
|
In an effort to verify that the protein bound to the zyxin fusion
peptides was in fact -actinin, we excised one of the radiolabeled bands from the nitrocellulose membrane and eluted the protein with SDS
sample buffer. On a polyacrylamide gel the eluted protein migrated with
the same mobility as
-actinin, thus proving its identity (Fig.
3).
|
To investigate whether zyxin sequences that appear to be necessary for
the interaction with -actinin per se are capable of
-actinin binding, we performed the converse experiment. A peptide corresponding to residues 21-42, preceded by an N-terminal cysteine residue for coupling purposes, was synthesized and coupled to maleimide-activated microtiter plates. In a solid phase binding assay
with increasing concentrations of horseradish peroxidase-labeled
-actinin added as soluble ligand, this peptide showed a
concentration-dependent interaction with
-actinin,
whereas an unrelated control peptide was negative (Fig.
4). Similar results were also obtained
when the peptides were coupled to microtiter plates via their
-amino groups instead of terminal cysteine residues (not shown). These results
suggest that residues 21-42 of human zyxin represent a functional
-actinin binding site.
|
The -Actinin Binding Site of Human Zyxin Is Characterized by an
Evolutionary Conserved Internal Repeat Structure--
The
-actinin
binding site of human zyxin contains several prolines and lysines, with
the remainder being dominated by aliphatic and aromatic residues (Fig.
5a). It is also conserved in
mouse (21) and chicken zyxin (14). In addition, a similar sequence is
found in the N terminus of the human LIM domain protein lipoma preferred partner (LPP) (22), which shares limited similarity with
human zyxin over its entire sequence.
|
A closer analysis of the sequence comprising the -actinin binding
site of human zyxin showed that it consists of a 10 amino acids
submotif occurring twice in tandem (Fig. 5b). A more
detailed search for possible repeat structures revealed a second
sequence element within the N-terminal zyxin region that resembles the
-actinin binding site identified here. This sequence is located between residues 261 and 284 in human zyxin and is evolutionary conserved in mouse zyxin (21), but not in chicken zyxin (14) or LPP
(22). This sequence is characterized by a repeat structure similar to
that of the
-actinin binding site: it comprises one complete copy of
the 10 amino acids submotif (amino acids 261-270 in human zyxin),
followed by two truncated copies occurring twice in tandem (amino acids
271-277/278-284 in human zyxin). Each of the truncations lacks the
first three amino acids of the 10-amino acid submotif (Fig.
5b).
Our studies so far have identified an N-terminal -actinin binding
site in human zyxin and a downstream sequence with remarkable similarity. For a functional comparison of the two sites, three deletion constructs were prepared that lacked either the sequences corresponding to the first site (
19-41), the second site
(
261-283), or both sites (
19-41/
261-283). When these
constructs were expressed in bacteria as GST fusion proteins and tested
in a blot overlay (Fig. 6), an
interaction of radiolabeled
-actinin was observed with wild type
zyxin as well as with zyxin lacking the second site. No interaction was
detected with zyxin lacking either the first site or both sites. The
first site is therefore indispensable for
-actinin binding, whereas
there is no evidence for a functional significance of the downstream
sequence element.
|
The -Actinin Binding Site Is Essential for Proper Subcellular
Targeting of Zyxin--
To address possible functions of the
-actinin binding site in living cells, the wild type sequence and
the three deletion constructs, detailed above, were cloned into a GFP
expression vector and transfected into COS cells. With the GFP
fluorescence, the subcellular distribution of the GFP fusion proteins
was visualized in living cells. The fusion protein synthesized from the
wild type sequence was found to be localized at focal contacts and along stress fibers (Fig. 7a).
Its distribution was virtually identical to that observed in a parallel
experiment by indirect immunofluorescence with an anti-zyxin antibody
(not shown; see also Refs. 21 and 23). No difference was noticed with
the construct GFP-zyxin
261-283, which encoded GFP-zyxin lacking the downstream site (Fig. 7c). However, after expression of
GFP-zyxin lacking the
-actinin binding site (GFP-zyxin
19-41) or
the first and second site (GFP-zyxin
19-41/
261-283), the
majority of the label was diffusely distributed in the cytoplasm (Fig.
7 , b and d), in a way reminiscent of control
cells that had been transfected with a GFP plasmid alone. No signal was
observed along stress fibers, and only some focal contacts were stained
to a minor extent. Similar results were obtained when primary chicken
fibroblasts rather than COS cells were transfected with the GFP-zyxin
constructs, albeit the transfection efficiency was considerably lower
in this case (Fig. 7, e-h). Thus, the
-actinin binding
site is absolutely required for the localization of zyxin to stress
fibers (and to some extent also for focal contact localization),
whereas the downstream site does not play any obvious role in these
experiments.
|
The -Actinin Binding Site Is Required for
Zyxin-dependent
-Actinin Recruitment in Living
Cells--
To further analyze the
-actinin binding activity of
zyxin constructs in living cells, we sought to ectopically express
different zyxin constructs at discrete subcellular sites. For this, we
made use of a mitochondrial targeting system described by Pistor
et al. (33). Wild type zyxin and zyxin constructs bearing
deletions of either the
-actinin binding site (targZyxin
19-41),
the related sequence described above (targZyxin
261-283), or both
(targZyxin
19-41/
261-283) were cloned into the pSPL61 vector
(33, 34), a derivative of the eukaryotic expression vector pMPSVHE
(35), for expression as fusion proteins with a 29 amino acid C-terminal peptide. This peptide is derived from the membrane anchor of the L. monocytogenes ActA protein. After transfection into
eukaryotic cells, it directs the fusion protein to the mitochondrial
surface (33). A major benefit of the mitochondrial targeting system is
the absence of any endogenous zyxin and
-actinin at these ectopic
sites. Therefore, there is no need to discriminate against a background
of endogenous proteins. In addition, functionally related proteins are
also absent from mitochondria and hence will not interfere with the
binding assay.
Transfection of PtK2 cells with these vectors, encoding
zyxin fusion proteins under the control of the myeloproliferative sarcoma virus promoter, resulted in high level protein expression, regardless of the construct. Double staining of transfected cells with
an antiserum to zyxin and the mitochondrial dye CMTMRos demonstrated that the zyxin fusion proteins almost exclusively colocalized with
mitochondria (not shown). Also, independent of the construct used for
transfection, mitochondria of transfected cells tended to form
extensive clusters (Fig. 8,
a-d).
|
Double label indirect immunofluorescence microscopy with antibodies to
zyxin and -actinin revealed that wild type zyxin fusion protein was
capable of recruiting endogenous
-actinin to the mitochondria of
transfected cells, a phenomenon that was especially evident at
mitochondrial clusters (Fig. 8, a and a';
open arrows). In sharp contrast, a zyxin deletion construct
lacking the
-actinin binding site (targZyxin
19-41), completely
lost its
-actinin binding activity (Fig. 8, b and
b').
Several binding sites for the focal adhesion and
microfilament-associated protein VASP map to a zyxin region starting
from about amino acid 69 (25), i.e. in close proximity to
the -actinin binding site. Both wild type zyxin and targZyxin
19-41 recruited VASP to the mitochondrial surfaces (not shown). As
these closely adjacent VASP binding sites were still functionally
preserved, we conclude that the
19-41 deletion did not cause any
gross structural disturbance, although we cannot rule out a possible
conformational change.
Unlike targZyxin 19-41, deletion of the related sequence element
(targZyxin
261-283) did not abolish mitochondrial
-actinin recruitment (Fig. 8, c and c'). Finally,
constructs bearing deletions of both sites (targZyxin
19-41/
261-283) also failed to direct
-actinin to the
mitochondria of transfected cells (Fig. 8, d and
d'). Obviously, zyxin residues 19-41 are indispensable for
-actinin binding not only in blot overlays but also in transfected cells. Moreover,
-actinin binding via this site accounts for the
major part, if not all, of the
-actinin binding activity of zyxin in
living cells, as revealed in the mitochondrial targeting assay.
The striking sequence homology between the -actinin binding site and
its related downstream sequence raised the question of whether the
latter could be rendered a functional
-actinin binding site when
grafted into the position of the authentic binding site within the
polypeptide chain. Therefore, we made a construct, coding for peptide
sequence 262-284 inserted into the deletion site of targZyxin
19-41. After transfection of this construct into PtK2
cells, no colocalization of zyxin and
-actinin at mitochondria was
observed (not shown). This indicates that despite similarities in
sequence and charge, the downstream sequence cannot functionally rescue
deletion of the
-actinin binding site, even if an additional copy is
introduced at the normal binding site position.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using different in vitro and transfection experiments,
we have mapped an -actinin binding site in human zyxin. This binding site was found to be both necessary and sufficient for
-actinin binding in blot overlay and solid phase binding assays, respectively. The site is also involved in zyxin localization to stress fibers and
focal adhesions and is required for
-actinin binding to zyxin in a
mitochondrial targeting approach, i.e. in the physiological ambience of living cells. In addition, we have shown that the human
protein LPP shares a sequence element closely related to the zyxin
-actinin binding site, raising the possibility that LPP is also an
-actinin-binding protein.
The -Actinin Binding Site of Zyxin Shares Some Features with
-Actinin Binding Sites in Proteins That Bind to the
-Actinin
Central and C-Terminal Region--
Similar to the
-actinin binding
site of zyxin characterized here (Fig. 5), basic and hydrophobic
residues also prevail in several other
-actinin binding sites,
including those of integrin
1 (39, 40),
L-selectin (10), and the NR1 subunit of the N-methyl-D-aspartate receptor (11). Integrin
1 (6) and L-selectin (10) bind to the 53-kDa
thermolysin fragment of
-actinin (which comprises both the central
and C-terminal regions of the protein). In a yeast two-hybrid assay,
the NR1 subunit of the N-methyl-D-aspartate receptor interacts with the region of the central spectrin-like repeats
(11). This is remarkable, because zyxin binding has previously been
attributed to the N-terminal 27-kDa fragment, not to the 53-kDa
fragment of
-actinin (18). Apparently, different
-actinin domains
mediate binding to zyxin and to the other proteins mentioned above.
Therefore, we questioned whether the similarities between the
-actinin binding sites of zyxin and those other proteins are only fortuitous.
Is There More Than One -Actinin:Zyxin Interface?--
In
resolving this issue, it is important to note that the blotted
N-terminal 27-kDa proteolytic fragment of
-actinin was previously
shown to bind radiolabeled full-length zyxin (18). In sharp contrast to
these experiments, we used an overlay with radiolabeled
-actinin to
identify the zyxin region involved in the interaction. Generally, in
blot overlays, a labeled native protein is used as probe to detect an
interaction with denatured proteins separated by SDS-polyacrylamide gel
electrophoresis and blotted to a membrane. Renaturation on the blot
membrane should, for example, be easier for small linear peptide motifs
than for complex protein folds or complete domains. Therefore it may be unexpected that overlays probing the
-actinin-zyxin interaction work
in either direction. The existence of two independent binding interfaces may be an obvious explanation. This raises the attractive possibility that native zyxin interacts with a peptide motif in the
27-kDa fragment of
-actinin (18), whereas another interaction may be
due to binding of a zyxin peptide motif by a different
-actinin
region (possibly in the central or C-terminal region). In support of
this view, both the 27- and 53-kDa
-actinin fragments, but not the
full-length protein, failed to compete for
-actinin binding to zyxin
in solid phase binding assays (18). This hypothesis may also explain
the similarities between the
-actinin binding motifs of zyxin and
those of known ligands for the central/C-terminal part of
-actinin.
In fact, interaction with more than one -actinin region appears to
be quite common and has been suggested for several proteins, such as
ICAM-2 (9), actinin-associated LIM protein (41), titin (42), and PKN
(12). Vice versa,
-actinin ligands often harbor two
essentially nonoverlapping, apparently unrelated binding sites in close
proximity, as shown for integrin
1 (39, 40), Ep-CAM
(40), and ICAM-2 (9).
-Actinin Binding Sites: Specific Sequence Requirements or
Compositional Bias?--
Besides some bias in amino acid composition,
there is no obvious consensus motif for the
-actinin binding sites
of zyxin (this study), integrin
1 (39, 40),
L-selectin (10), the NR1 subunit of the
N-methyl-D-aspartate receptor (11), and some other
-actinin binding sites (8, 9, 40). Indeed, there are reports
that even certain scrambled or reverse sequence ICAM-1 and
1 integrin peptides show comparable binding activity
when tested in vitro (8, 39). Contrary to
-actinin
binding sites of ICAM-1 and integrin
1, our results
suggest that the
-actinin binding site of zyxin in blot overlays and
in transfection experiments is strictly dependent on the sequence of
the binding site. In particular, the related sequence element between
residues 261 and 283 of human zyxin (Fig. 5) was neither necessary nor
sufficient for
-actinin binding in blot overlays or in living cells.
The negative results obtained with this latter human zyxin peptide are
in contrast, however, with overlay experiments performed with synthetic
peptides (not shown). 9-21-mer peptides were synthesized in parallel
arrays on a solid paper support using the SPOTsTM
technology (43). When these peptides were incubated with
-actinin, we observed spurious binding to peptides corresponding either to the
actual binding site as characterized here or to the nonfunctional downstream element and even to a reverse sequence. This indicates that
in the case of
-actinin
binding data based primarily on this
type of assay should be interpreted with caution unless confirmed by
independent approaches. It will be interesting to investigate whether
or not this downstream motif is related to a putative second
-actinin binding site that is responsible for
-actinin binding in
the reciprocal overlay assay. However, the demonstration that chicken
zyxin interacts with
-actinin in 125I-labeled zyxin
overlays (18), although chicken zyxin lacks an element related in
sequence to residues 261-283 of human zyxin, argues against such a relationship.
Requirement of the -Actinin-Zyxin Interaction for Proper
Subcellular Targeting of Zyxin--
As judged from transfection
experiments with GFP-zyxin fusion constructs, the zyxin
-actinin
interaction appears to be essential for zyxin to adopt the typical
dotted pattern of stress fiber localization, because GFP-zyxin
19-41
failed to locate at stress fibers. Nevertheless, interactions of zyxin
with proteins other than
-actinin appear to recruit the same fusion
protein to focal adhesions, although not very efficiently. However, we
cannot distinguish whether these differences in subcellular targeting
to focal adhesions versus stress fibers reflect true
qualitative or only quantitative differences. Zyxin is predominantly
concentrated at focal adhesions and the terminal portions of stress
fibers, whereas its association with stress fibers proper is
characterized by a much finer and weaker staining pattern in indirect
immunofluorescence analysis (Fig. 7) (14, 18, 19, 21, 23). Thus, if the
deletion of the
-actinin binding site would equally impair
association with both types of structures, the weaker staining of the
fusion protein at stress fibers might be lost, whereas some residual staining of focal adhesions could persist.
GFP-zyxin 19-41 failed to associate with stress fibers. Obviously,
the known zyxin interactions with CRPs (14-17) and VASP (23) cannot
compensate for direct
-actinin binding, although both proteins
localize with zyxin along stress fibers and their terminal portions
(14, 17, 23, 37). This may be surprising in light of the observation
that VASP and zyxin display a virtually indistinguishable subcellular
distribution, including a dot-like stress fiber association of
identical spacing and periodicity (23) that (at stress fibers) also
coincides with
-actinin staining (37). In contrast, the indirect
immunofluorescence patterns of zyxin and
-actinin vastly overlap but
are also distinctly different. Specifically,
-actinin staining is
more pronounced along stress fibers, whereas zyxin is concentrated in
the focal adhesions proper (18). However, it has been pointed out that possible differences in antigen accessibility may contribute to these
immunofluorescence patterns (3, 18).
Zyxin-dependent -Actinin Recruitment in Living
Cells--
Chicken gizzard vinculin, the vinculin head, and the
vinculin hinge plus tail domains were very inefficient in recruiting
-actinin when tested in the same mitochondrial targeting system as
used in this report (34). This is despite the fact that an interaction
has been shown in vitro that involves an
-actinin binding
site in the vinculin head domain (13). These results obtained with
mitochondrial targeting of vinculin and vinculin fragments are in sharp
contrast to the strong
-actinin recruitment observed with a hybrid
construct replacing most of the vinculin head by the ActA N-terminal
part (amino acids 31-236) (34). Similar
-actinin recruitment has
been reported for wild type ActA (33, 44). It is still an unresolved
issue whether
-actinin recruitment by ActA or the ActA-vinculin
chimera is due to direct binding or whether
-actinin is recruited by
the considerable amounts of F-actin that accumulate at the
mitochondrial surfaces of cells transfected with these constructs. Like
ActA, zyxin is able to recruit F-actin to the mitochondria of
transfected cells.3
Nevertheless, in the case of zyxin, we can rule out the possibility that
-actinin recruitment is primarily due to an indirect
association mediated by F-actin (18) or some other protein(s): a
deletion, which is confined to a short zyxin peptide motif that has
been positively identified as an
-actinin binding site in a parallel in vitro assay, virtually abolishes
-actinin recruitment
in the targeting assay and
-actinin binding to GST-zyxin fusion
proteins in blot overlays in vitro.
Regardless of whether the -actinin recruitment by ActA (33, 44) or
ActA chimeras (34) turns out to be direct or indirect, the efficient
-actinin binding to zyxin and zyxin-dependent F-actin recruitment in living cells lend further support to the view that ActA
and zyxin share common functional features.
Similar to the failure of -actinin binding site deletion mutants to
localize to stress fibers, the equivalent fusion proteins assayed in
the mitochondrial targeting system demonstrated that there was no
efficient backup system for recruitment of
-actinin to zyxin. In
particular, at least in these cells, cysteine-rich protein family
members, which bind both zyxin and
-actinin (17, 32), do not
functionally substitute for the direct
-actinin-zyxin interaction.
In conclusion, we have identified and evaluated an -actinin binding
site in human zyxin that maps close to the zyxin N terminus and is
required for zyxin-dependent
-actinin recruitment and proper zyxin localization in living cells. The results presented here
have set the stage for investigating the contribution of the
-actinin-zyxin interaction in microfilament architecture and regulation.
![]() |
ACKNOWLEDGEMENTS |
---|
M. R. is grateful to Jürgen Otte and Dietmar von der Ahe (Bad Nauheim, Germany) for sharing human zyxin cDNA prior to publication. We thank Susanne Pistor and Jürgen Wehland (Braunschweig, Germany) for the kind gift of the mitochondrial targeting vector pSPL61 and our colleagues for valuable criticism and comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This study was supported by Grant 31-50571.97 from the Swiss National Science Foundation, the Bernese Cancer Liga, and the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 49-931-201-3144; Fax: 49-931-201-3153; E-mail: Matthias.Reinhard{at}mail.uniwuerzburg.de.
2 Krause et al., manuscript in preparation.
3 M. Reinhard and U. Walter, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ICAM, intercellular adhesion molecule; CRP, cysteine-rich protein; GFP, green fluorescent protein; GST, glutathione S-transferase; LPP, lipoma preferred partner; VASP, vasodilator-stimulated phosphoprotein; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|