16mackinnon

16mackinnon
Current Biology, Vol. 12, 787–797, May 14, 2002, 2002 Elsevier Science Ltd. All rights reserved.
PII S0960-9822(02)00810-2
C. elegans PAT-4/ILK Functions as an Adaptor
Protein within Integrin Adhesion Complexes
A. Craig Mackinnon,1 Hiroshi Qadota,2
Kenneth R. Norman,2 Donald G. Moerman,2
and Benjamin D. Williams1,3
1
Department of Cell and Structural Biology
University of Illinois, Urbana-Champaign
601 South Goodwin Avenue
Urbana, Illinois 61801
2
Department of Zoology
University of British Columbia
Vancouver, British Columbia V6T 1Z4
Canada
Summary
Background: Mammalian integrin-linked kinase (ILK)
was identified in a yeast two-hybrid screen for proteins
binding the integrin ␤1 subunit cytoplasmic domain. ILK
has been implicated in integrin-mediated signaling and
is also an adaptor within integrin-associated cytoskeletal complexes.
Results: We identified the C. elegans pat-4 gene in previous genetic screens for mutants unable to assemble
integrin-mediated muscle cell attachments. Here, we
report that pat-4 encodes the sole C. elegans homolog
of ILK. In pat-4 null mutants, embryonic muscle cells
form integrin foci, but the subsequent recruitment of
vinculin and UNC-89 as well as actin and myosin filaments to these in vivo focal adhesion analogs is blocked.
Conversely, PAT-4/ILK requires the ECM component
UNC-52/perlecan, the transmembrane protein integrin,
and the novel cytoplasmic attachment protein UNC-112
to be properly recruited to nascent attachments. Transgenically expressed “kinase-dead” ILK fully rescues
pat-4 loss-of-function mutants. We also identify UNC112 as a new binding partner for ILK.
Conclusions: Our data strengthens the emerging view
that ILK functions primarily as an adaptor protein within
integrin adhesion complexes and identifies UNC-112 as
a new ILK binding partner.
Introduction
Caenorhabditis elegans has proven to be an advantageous system for the genetic analysis of muscle development [1, 2]. Myoblasts are born near the end of gastrulation and soon begin to express muscle-specific
structural proteins [3, 4]. Subsequently, they migrate to
their final positions in the body wall and flatten against
the hypodermis, or “skin” of the embryo [5, 6]. Structural
proteins, such as integrin and vinculin, then polarize and
accumulate at the basal sarcolemma adjacent to the
hypodermis where they assemble into a highly ordered
set of related muscle attachment structures [4] called
dense bodies and M-lines, which attach actin thin filaments and myosin thick filaments, respectively, to the
basal sarcolemma (Figure 1). Based upon their function
3
Correspondence: [email protected]
and polypeptide composition [7–14], dense bodies and
M-lines are analogs and indeed homologs of vertebrate
adhesion plaques (Figure 1C).
We are focusing upon a subset of C. elegans Pat
mutants (Paralyzed, Arrested elongation at Two-fold)
[15] that have deficits in dense body and M-line assembly. The corresponding genes investigated to date include unc-52 [7], pat-3 [8], unc-112 [11], unc-97 [10],
and deb-1 [13, 16], each encoding a muscle attachment
protein (Figure 1C). Here, we report our investigation of
pat-4, which we show codes for the nematode homolog
of integrin-linked kinase (ILK), a vertebrate focal adhesion protein implicated in integrin signaling [17, 18].
ILK was first identified in a yeast two-hybrid screen
using the integrin ␤1 subunit cytoplasmic domain as bait
[19]. ILK has four N-terminal ankyrin repeats, followed
by a plecstrin homology domain that partially overlaps
a C-terminal kinase domain [20]. The ankyrin repeats
bind to the first LIM domain of UNC-97/PINCH [10, 21],
a LIM-only focal adhesion adaptor protein, while the
plecstrin homology domain has been proposed to bind
phosphoinositides, which can activate the kinase activity of ILK in vitro [20]. The ILK kinase domain, which is
most similar to the serine/threonine kinase Raf, binds
the integrin ␤1 subunit cytoplasmic tail [19]. Functional
analysis of ILK has relied heavily upon the expression
of high levels of wild-type and “kinase-dead” forms of
the protein in cultured cells. A wide range of effects
has been reported in these experiments, and significant
attention has focused upon the Wnt signaling pathway
[20, 22] and alterations in the protein kinase B (PKB/
AKT) pathway regulating apoptosis [20]. It is of particular
interest, therefore, that a recently reported reverse genetic analysis of ILK function in Drosophila failed to find
evidence for ILK function in well-characterized PKB or
Wnt signaling pathways [23]. In fact, these authors
showed that a kinase-dead ILK construct rescues ILK
mutants as well as the wild-type gene. They did find that
ILK colocalizes with integrin and is needed to stabilize
muscle attachments, leading them to propose that ILK
functions primarily as an adaptor molecule.
Here, we show that PAT-4/ILK is a component of C.
elegans muscle dense bodies and M-lines and is needed
for their proper assembly. In reciprocal investigations,
we show that PAT-4/ILK is dependent upon integrin
for its assembly into muscle attachments. Kinase-dead
forms of PAT-4/ILK rescue the mutant pat-4 phenotype,
and we also have failed to find any evidence for PAT4/ILK function in Wnt signaling cascades. Our results
therefore concur with the findings from the Drosophila
ILK analysis, suggesting that ILK functions primarily as
an adaptor molecule during muscle assembly. We extend these results by identifying the ERM adhesion complex protein UNC-112 [11] as a new ILK-interacting
partner.
Results
pat-4 Encodes Integrin-Linked Kinase
We used a combination of positional cloning and candidate gene approaches to molecularly isolate the pat-4
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Figure 1. Schematic Diagram of the C. elegans Body-Wall Muscle Structure
(A) An adult worm with body-wall muscle
quadrants visible (orange).
(B) A body-wall cross-section with cuticle, hypodermis, and basal lamina peeled away to
reveal the basal membrane of two body-wall
muscle cells.
(C) A longitudinal section through a body-wall
muscle cell. Dense bodies and M-lines attach
actin thin filaments and myosin thick filaments, respectively, to the basal sarcolemma.
(D) Locations of several different muscle attachment proteins. ␣-actinin and vinculin are
present in dense bodies but not M-lines. Conversely, UNC-89 is present only in M-lines.
Localization of the ␣PAT-2 integrin subunit is
unpublished data from B.D.W. and R. Waterston.
gene (Figure 2A). RNA interference (RNAi) experiments
performed on predicted gene C29F9.7 produced 98%
Pat F1 progeny (n ⬎ 4000). Subsequent transformation
experiments confirmed that a genomic DNA fragment
containing C29F9.7 completely rescues the phenotype
of pat-4 homozygotes. Two of the four existing pat-4
Figure 2. Molecular Isolation of pat-4
(A) A genetic map of the left arm of LGIII. The genomic cosmid clone
C29F9 rescues pat-4 mutant embryos.
(B) A genomic structure of the pat-4 gene. Exons (gray boxes) and
the methionine start codon (small arrow) are indicated.
(C) pat-4 point mutations st551 and st579.
(D) A structure of the predicted PAT-4/ILK protein. This diagram
has been simplified. The C-terminal half of the PH motif is actually
contained within the kinase domain.
alleles are point mutations (Figure 2C) in C29F9.7, including a nonsense mutation within the N-terminal part
of the protein-coding region, providing unambiguous
confirmation of the pat-4 molecular identity. The remaining two pat-4 alleles are deletions that completely
remove C29F9.7 (see the Experimental Procedures).
Embryos homozygous for the nonsense allele pat4(st551) fail to stain with PAT-4 polyclonal antiserum
and are therefore likely to be protein null. The predicted
primary peptide structure of PAT-4 is similar (56% identity) to the human molecule integrin-linked kinase (ILK)
(Figure 2D). Searches of the essentially complete C.
elegans genome sequence show that PAT-4 is the sole
C. elegans ILK homolog. We refer to the PAT-4 protein
as PAT-4/ILK.
PAT-4/ILK Colocalizes with Integrin
Transgenic minigenes encoding translational fusions
between PAT-4/ILK and either green or yellow fluorescent protein (GFP, YFP) fully rescue pat-4(st551) mutants, providing strong evidence that the fusion proteins
are functional in vivo and are likely to have a functionally
relevant subcellular localization. Immunostaining experiments using anti-PAT-4/ILK show an identical localization pattern in wild-type embryos and adults (see below,
and data not shown).
The pat-4::gfp transgene is initially expressed weakly
throughout postgastrulation embryos (data not shown).
By 420 min postfertilization, PAT-4/ILK::GFP is primarily
pat-4 and Muscle Assembly
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Figure 3. PAT-4/ILK and ␤PAT-3 Integrin
Colocalize at Muscle Attachments
(A) A wild-type, ⵑ420 min embryo. PAT4::GFP localizes to body-wall muscle attachments (arrow). The scale bar represents 2 ␮m.
(B) The same embryo as that shown in (A)
double stained with monoclonal antibodies
MH25, recognizing ␤PAT-3 integrin, and
MH27, recognizing hypodermal-hypodermal
cell junctions (included for developmental
staging and orientation purposes).
(C) An overlay of (A) and (B). Areas of PAT4::GFP and integrin colocalization appear
yellow.
(D–F) Detail of body-wall muscle from a rescued pat-4(st551) adult hermaphrodite coexpressing (D) pat-4::yfp and (E) pat-3::cfp.
Dense body (arrows) and M-line (arrowheads)
attachment structures are indicated. (F) An
overlay of panels (D) and (E). Regions in which
␤PAT-3::CFP and PAT-4::YFP colocalize appear white. The scale bar in (D) represents
5 ␮m.
observed in the body-wall muscle cells (Figure 3A),
where it colocalizes with the integrin ␤PAT-3 subunit
(Figures 3A–3C). pat-4::gfp is also transiently expressed
in the pharynx during embryogenesis (data not shown).
In adult hermaphrodites, pat-4::gfp expression is observed in body-wall muscle cells (Figure 3D), the spermatheca, the vulva muscles, a subset of the mechanosensory neurons (ALM, AVM, PLM, and PVM), the distal
tip cells, the uterine muscles, and the anal depressor
and anal sphincter muscles (data not shown). In adult
body-wall muscle cells, PAT-4/ILK::YFP colocalizes with
a functional ␤PAT-3::CFP integrin subunit in M-lines and
dense bodies (Figures 3D–3F) and at muscle-muscle
adherent junctions (data not shown). PAT-4/ILK::GFP is
located near the cell membrane and does not extend
very deeply into body-wall muscle cells. An identical
localization has been reported previously for UNC-97/
PINCH [10] and UNC-112 [11]. The fact that PAT-4/ILK
is only observed at sites reported to contain the ␤PAT-3
integrin subunit in body-wall muscle cells (see below)
is an expected result based on the studies of mammalian
and Drosophila ILK [23, 24].
PAT-4/ILK Is Required for Dense Body
and M-Line Assembly
Previous analysis of actin and myosin organization in the
embryonic muscles of pat-4 mutants revealed defects in
sarcomere assembly quite similar to those in unc-52
perlecan and pat-3 integrin loss-of-function mutants
[15]. In both unc-52 and pat-3 mutants, structural proteins do not polarize to the muscle cell basal membrane,
and integrin fails to aggregate into nascent attachments
[4, 15]. To determine the extent of dense body and M-line
assembly in pat-4 embryos, we investigated the localiza-
tion of UNC-52/perlecan and ␤PAT-3 integrin by antibody staining. The early localization of UNC-52/perlecan
into a longitudinal stripe adjacent to each muscle occurs
normally in pat-4 mutants (compare arrows in Figures
4A and 4B). The initial polarization and subsequent clustering of integrin into foci also occurs normally (compare
arrows in Figures 4C and 4D). There is, however, some
irregularity in the integrin pattern in the pat-4 mutant
embryos. Some of the integrin foci are not located within
the tight stripe of nascent attachments that normally
forms near the centerline of each quadrant. Instead, they
are in positions that are spread out laterally across the
entire width of each muscle cell (arrowheads, Figure
4C). Integrin organization never improves in the pat-4
embryos and, in fact, deteriorates in older animals (data
not shown). In contrast, in wild-type embryos, the integrin foci soon organize further, forming a highly ordered
striated array of morphologically distinguishable dense
bodies and M-lines [4].
Formation of integrin foci in pat-4 mutants suggests
that dense body and M-line assembly is initiated in the
absence of PAT-4/ILK protein. We next asked whether
several dense body- and M-line-specific proteins are
recruited to the integrin foci. In wild-type embryos 430
min postfertilization, the dense body protein DEB-1/vinculin has already polarized to the basal sarcolemma and
is organized into nascent attachments that appear as a
continuous stripe of staining along each muscle quadrant (Figure 4E). By 520 min, this pattern resolves into
a recognizable array of dense bodies (see Figure 6C in
[4]). In contrast, DEB-1/vinculin polarizes within the
pat-4 embryos but does not appear to fully assemble
into nascent attachments (Figure 4F), as indicated by
the different integrin and DEB-1/vinculin localization in
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Figure 5. Integrin and UNC-112 Do Not Colocalize Extensively in a
pat-4(st551) Mutant
(A) At ⵑ430 min postfertilization, ␤PAT-3 integrin, stained with antibody MH25, is located in nascent muscle attachments.
(B) A GFP channel in the same focal plane as (A). UNC-112::GFP is
observed in some foci (arrows), but, for the most part, does not
colocalize with integrin and instead is distributed diffusely within
the cytoplasm (small arrowheads).
Figure 4. Muscle Assembly in pat-4 Mutant Embryos
(A–H) Wild-type (left column) and pat-4(st551) mutant (right column)
embryos 430 min postfertilization stained for (A and B) perlecan
(antibody MH2), (C and D) integrin (antibody MH25), (E and F) vinculin
(antibody MH24), and (G and H) UNC-89 (antibody MH42). All animals
were also stained with antibody MH27 to show hypodermis-hypodermis junctions (arrowhead, all panels). The circular hypodermal
cell junction formed at the derid sensillum is seen in all panels and
can be used for orientation. All panels show a dorsolateral view of
the embryo. One of the two dorsal body-wall muscle quadrants
(arrow in all panels) is in the plane of focus. We obtained identical
results for all four pat-4 mutants. The scale bar in (A) represents 2 ␮m.
pat-4 mutants (compare arrows in Figures 4D and 4F).
We also investigated the M-line protein UNC-89, which,
in wild-type embryos, becomes polarized and recruited
into an array of nascent M-lines [4]. In pat-4 mutants,
UNC-89 fails to polarize and remains in a large clump
within the cytoplasm of each muscle cell (compare
arrows in Figures 4G and 4H), indicating that UNC-89
also fails to be recruited to the nascent M-lines formed
in the absence of PAT-4/ILK.
Because our previous work has shown that UNC-112
and integrin colocalize at muscle attachment structures
[11], we were interested in determining if this colocalization requires PAT-4/ILK. Our efforts to generate antisera
directed against UNC-112 were unsuccessful. Therefore, to determine the localization of UNC-112 in pat4(st551) mutant embryos, we first crossed an unc112::gfp transgene [11] into pat-4 mutants and then
stained the mutant embryos with ␤PAT-3 integrin antisera. In pat-4 mutants, we observe that UNC-112 does
not appear to extensively colocalize with integrin in the
sarcolemmas (compare Figures 5A and 5B). Instead,
UNC-112 mainly appears to be distributed diffusely
throughout the cytoplasm (arrowheads, Figure 4B), suggesting that PAT-4/ILK is required for UNC-112 to properly assemble into nascent attachments. Identically
staged wild-type embryos expressing the same unc112::gfp transgene and stained with the same ␤PAT-3
monoclonal antibody show extensive colocalization of
UNC-112 and ␤PAT-3 (see Figures 5G–5F from [11]).
Perlecan, Integrin, and UNC-112, but Not Vinculin,
Are Necessary for PAT-4/ILK Assembly
In order to determine the requirements for PAT-4/ILK
recruitment to muscle attachments, we generated an
antibody to PAT-4/ILK and used it to localize this protein
in wild-type embryos and in mutants blocked at different
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791
Figure 6. PAT-4/ILK Assembly at the Muscle Cell Membrane Is Blocked in unc-52 Perlecan, pat-2 Integrin, and unc-112 Mutants, but Not in
deb-1 Vinculin Mutants
(A–F) Same view and developmental stage as in Figure 3. Embryos were double stained with anti-PAT-4 and with antibody MH27 to show
hypodermal junctions (arrowheads). See Results for details. The scale bar in (A) represents 2 ␮m.
stages of attachment assembly. This antiserum is specific to PAT-4/ILK, indicated by failure to detect any
PAT-4/ILK protein in pat-4(st551) mutants (Figure 6B).
This figure shows a pat-4(st551) embryo double stained
with anti-PAT-4/ILK and antibody MH27, the latter of
which stains hypodermal-hypodermal junctions and is
included for orientation purposes. Body-wall muscle
cells do not stain (arrow, Figure 6B), and only the MH27
staining pattern is observed (arrowhead, Figure 6B). In
wild-type embryos, PAT-4/ILK polarizes and is recruited
to nascent muscle attachments (Figure 6A). It has been
shown previously that, in protein null unc-52 perlecan
mutants, integrin and other structural proteins including
vinculin fail to polarize, and muscle attachments do not
form [7, 15]. We stained unc-52 perlecan mutants with
the anti-PAT-4/ILK antiserum and found that PAT-4/ILK
also fails to polarize to the membrane, accumulating
instead in the cytoplasm (Figure 6C, compare with wildtype in Figure 6A). pat-2 codes for the ␣PAT-2 integrin
subunit in dense bodies and M-lines (unpublished data),
and pat-3 codes for the ␤PAT-3 integrin subunit [8]. It
has been shown previously that muscle structural proteins fail to polarize in pat-3 integrin mutants, accumulating instead in the muscle cell cytoplasm [4]. We
stained both pat-2 integrin ␣ subunit (Figure 6D) and
pat-3 integrin ␤ subunit mutant embryos (data not
shown) and found that much of the PAT-4/ILK staining
is distributed throughout the muscle cell cytoplasm (Figure 6D, arrow). PAT-4/ILK has a similar mislocalization
in unc-112 mutants (Figure 6E, arrow). It has also been
shown that, in deb-1 vinculin protein null mutants, integrin forms recognizable arrays of dense bodies and
M-lines, but the recruitment of actin filaments to the
sarcolemma fails to occur, indicating that dense body
assembly is incomplete [4, 16]. We find that PAT-4/ILK
is able to polarize and localize into nascent attachments
in deb-1 vinculin mutants (arrow, Figure 6F), indicating
that vinculin is not required to recruit PAT-4/ILK to dense
bodies.
PAT-4/ILK Binds UNC-112 in Yeast
Two-Hydrid Assays
Work published by others has already demonstrated
that ILK can behave as an adaptor protein as well as a
kinase [19, 20, 22]. Besides integrin, ILK can bind paxillin, PINCH, and actopaxin/CHILKBP/affixin, all components of adhesion complexes [21, 25–27]. We have discovered through yeast two-hydrid assays that ILK can
also bind UNC-112, a recently described member of the
ERM family of proteins that localizes to myofilament
adhesion complexes in nematode body-wall muscle
[11]. Using UNC-112 as the bait protein in a yeast twohybrid screen of a C. elegans cDNA library, we identified
several positive clones encoding PAT-4/ILK.
Full-length UNC-112 and full-length PAT-4/ILK interact strongly in the yeast two-hybrid system (Figures 7A
and 7B). To map the regions of UNC-112 and PAT-4/
ILK that are required for this interaction, we made a
series of in-frame deletions and found that the amino
half of UNC-112, corresponding to amino acid residues
1–396, is critical for its ability to bind PAT-4/ILK, as
demonstrated by the significant decrease in the number
of viable yeast colonies when these residues are removed from UNC-112 (Figure 7A). Residues 213–474
of PAT-4/ILK, corresponding to the carboxy-terminal
kinase domain, were found to be required for PAT-4/
ILK to bind full-length UNC-112 (Figure 7B). This is the
same region identified as binding actopaxin [28]. Results
from in vitro binding assays performed with purified recombinant UNC-112 and PAT-4 are consistent with direct binding between UNC-112 and PAT-4/ILK (Figure
7C). PINCH binds to the amino ankyrin repeats of ILK
[21], and we observe that UNC-97/PINCH and PAT-4/ILK
interact in an analogous manner (Figure 7D). In addition,
using UNC-97/PINCH as the bait protein in a yeast twohybrid screen of a C. elegans library, we identified positive clones containing PAT-4/ILK. One surprise from the
Drosophila study of ILK was that no detectable interaction was found between dILK and the COOH tail of ␤PS
integrin [23]. We also fail to detect an interaction between PAT-4/ILK and the ␤PAT-3 integrin cytoplasmic
domain in a yeast two-hybrid assay (Figure 7D).
Previous work has shown that a point mutation in
human ILK corresponding to E359K confers dominantnegative activity to ILK, presumably by disrupting ILK
catalytic activity [22]. However, when the equivalent mutation was introduced into Drosophila ILK, it did not
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Figure 7. Yeast Two-Hybrid Analysis
UNC-112 and PAT-4/ILK Interactions
of
The plus and minus symbols provide an indication of how well the yeast grew, and the
strip on the right shows three replicate yeast
clones grown on selective media.
(A) Interactions between various regions of
UNC-112 (open boxes) and full-length PAT-4.
(B) Interactions between various regions of
the PAT-4 protein (open boxes) and fulllength UNC-112.
(C) We tested the ability of either a fusion
between UNC-112 and maltose binding protein (MBP), or MBP alone, to bind to purified
GST::PAT-4/ILK in vitro. As shown in Figure
6C, MBP::UNC-112 binds to GST::PAT-4/ILK
(lane 3), but MBP alone fails to bind to
GST::PAT-4/ILK (lane 4). Degradation products of GST::PAT-4/ILK are indicated by an
asterisk. MBP::UNC-112 fails to bind GST
only (data not shown).
(D) Interactions between various regions of
PAT-4/ILK (open boxes) and full-length UNC97/PINCH or the cytoplasmic domain of
␤PAT-3 integrin.
disrupt the in vivo function of this protein, and it rescued
the mutant phenotype [23], suggesting that dILK can
function independently of its kinase activity. We were
interested to see if disruption of the kinase would inhibit
protein-protein interactions in the two-hybrid assay.
When we introduced the equivalent mutation into PAT4/ILK, we noticed no decrease in PAT-4/ILK’s ability
to bind UNC-112 (Figure 7B). This result reinforces the
observations made in Drosophila and suggests that
PAT-4/ILK is primarily acting as an adaptor molecule at
this stage in development.
PAT-4 Functions as an Adaptor
during Attachment Assembly
The accumulating evidence that ILK is an adaptor molecule within adhesion complexes and the striking obser-
pat-4 and Muscle Assembly
793
Figure 8. Summary of Interactions between
PAT-4/ILK, UNC-112, and UNC-97/PINCH
The carboxy kinase domain of wild-type and
kinase-dead PAT-4/ILK interacts with the
amino-terminal half of UNC-112. PAT-4/ILK
interacts with UNC-97/PINCH in a manner
analogous to the interaction observed between the vertebrate homologs [21]. Previously, the integrin ␤1 subunit cytoplasmic
domain, PINCH, actopaxin, CH-ILKBP,
affixin, ILKAP, and paxillin [19, 21, 25, 28, 38,
46] have been reported to bind ILK. In this
report, we present UNC-112 as a new ILK
binding protein.
vation that kinase-dead ILK (E359K) can fully rescue
mutant Drosophila lacking functional ILK led us to perform a similar experiment. We were able to completely
rescue the pat-4 mutant phenotype when we introduced
a pat-4 transgene containing the E359K mutation into
mutant animals. A recent report shows that vertebrate
E359K ILK retains approximately 20% of the kinase activity, while the S343A mutant form has no detectable
activity [29]. We therefore made a second kinase-dead
construct, corresponding to S343A in vertebrate ILK,
and this construct also fully rescues the pat-4 mutant
phenotype. Curiously, these animals not only have normal muscle development, but they also appear wildtype in every other respect. This is not the expected
result if the kinases were important in other developmental pathways. To further explore this avenue, we
examined whether PAT-4/ILK has a role in a Wnt signaling pathway in C. elegans.
Biochemical studies have shown that overexpression
of ILK in epithelial cells stimulates the ␤-catenin/LEF
signaling pathway, leading to a downregulation in
E-cadherin expression and deactivation of GSK-3␤ by
both direct and PKB/AKT-mediated phosphorylation
[20, 22, 24, 30]. In light of these findings, we examined
whether PAT-4/ILK plays a role in a Wnt signaling pathway that occurs very early during C. elegans development and is responsible for inducing the asymmetric
division of the EMS blastomere [31, 32]. In the absence
of Wnt signaling and gsk-3␤ activity, EMS divides to
produce two MS-like blastomeres, and embryos produce extra mesodermal tissue. If PAT-4/ILK is indeed
required for this signaling process, then we predict that
pat-4 mutant animals should have a mutant phenotype
similar to Wnt and gsk-3␤ mutant animals. Our analysis
of pat-4 mutant embryos and the results from our pat4(RNAi) experiments, the latter likely to remove maternally contributed PAT-4/ILK from early embryos [33],
are both inconsistent with this predicted outcome. We
therefore can provide no evidence that PAT-4/ILK regulates GSK-3␤ during Wnt signaling between P2 and
EMS.
Discussion
We have shown that the pat-4 gene encodes the sole
ILK homolog in C. elegans and that PAT-4/ILK not only
colocalizes with integrin at muscle attachments but is
essential for proper attachment assembly. During at-
tachment assembly, ILK functions downstream of perlecan and integrin and upstream of vinculin, UNC-89,
and the connection of thin and thick filaments to the
sarcolemma. Conversely, proper PAT-4/ILK assembly
into muscle attachments requires perlecan and integrin,
but not vinculin. Interestingly, PAT-4/ILK and UNC-112
exhibit a mutual requirement for their proper recruitment
to nascent attachments. The essential function of
PAT-4/ILK in C. elegans is independent of its kinase
activity, consistent with the emerging view that ILK acts
primarily as an adaptor protein (Figure 8). Supporting
this interpretation, we have identified a binding interaction between PAT-4/ILK and the novel attachment protein UNC-112. Surprisingly, PAT-4/ILK does not bind
to the cytoplasmic tail of integrin in yeast two-hybrid
assays. This seems peculiar, since it was this interaction
with the cytoplasmic tail of integrin that led to the discovery of vertebrate ILK [19], and yet a result similar to ours
was recently reported for Drosophila ILK and integrin [23].
The colocalization of PAT-4/ILK with integrin in C.
elegans muscle attachments is consistent with the previously reported colocalization of ILK and integrin in
vertebrate focal adhesions [24] and within Drosophila
muscle attachments [23]. Our analysis of pat-4 mutants
provides direct evidence that ILK is needed for the
proper assembly of muscle attachments. Early events
during assembly, including the deposition of UNC-52/
perlecan and the initial polarization and clustering of
integrin into foci, occur normally. Subsequent events,
including the patterning of the foci within the plane of
the membrane and the linkage of myofilaments to the
nascent attachments, do not occur. This is shown by
the failure of integrin foci to enter into the highly ordered
array that is characteristic of dense bodies and M-lines,
the compromised recruitment of vinculin and UNC-89
to dense bodies and M-lines, respectively, and the failure of actin and myosin filaments to associate normally
with the sarcolemma [15]. Mutations removing several
other attachment or sarcomere proteins do not cause
dramatic deficits in integrin patterning within the plane
of the membrane. Specifically, integrin organizes into
recognizable arrays of dense bodies and M-lines in the
absence of vinculin or myosin A, despite the fact that
sarcomere assembly is severely disrupted in either case
[4]. Together, these results indicate that a key set of
proteins including PAT-4/ILK and UNC-112 pattern the
nascent attachments within the plane of the membrane.
Other attachment and sarcomere proteins are subse-
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quently recruited, but they do not play an essential role
in setting up the initial pattern.
Our observations that PAT-4/ILK fails to properly organize in the absence of perlecan or integrin were predicted because perlecan and integrin are required to
form nascent dense bodies and M-lines [15, 34]. The
failure of PAT-4/ILK to assemble at the membrane in
the absence of integrin directly contrasts the results in
Drosophila in which dILK localizes to muscle attachment
sites in integrin mutants [23]. There is also a significant
difference between Drosophila and C. elegans regarding
the effects of ILK loss-of-function mutations on muscle
attachment assembly [23]. In Drosophila, loss of ILK
compromises the stability of attachments but does not
cause an obvious defect in their initial formation or in
the assembly of sarcomeres. This is in marked contrast
to the severe effects on attachment and sarcomere assembly that we observe in pat-4 mutants. Interestingly,
assembly defects observed in the muscle of ILK mutants
in Drosophila and C. elegans mirror those seen in integrin loss-of-function mutants in these organisms [35–37].
It is noteworthy that Drosophila muscle attachments
lacking integrin are able to form while C. elegans integrin
mutants never form attachments, suggesting that there
may be fundamental differences in integrin’s role during
muscle development in these two organisms.
The comparable rescue of pat-4 mutants by wild-type
and several different kinase-dead pat-4 transgenes
shows that the essential function of ILK is not dependent
upon its kinase activity. Experiments using kinase-dead
forms of the protein and investigation of early embryonic
Wnt signaling pathways both failed to provide any evidence that ILK functions as a kinase during signal transduction. Taken together, our results are consistent with
the idea originally proposed by Zervas [23] that the ILK
kinase domain is “vestigial”, working as a binding site
rather than as a physiologically relevant kinase. The
proposed role for vertebrate ILK in integrin signaling
may reflect a newly acquired function for this protein.
Alternatively, the effects on cellular signal transduction
that have been observed in vertebrate cells might be
due to nonspecific promiscuous kinase activity caused
by ectopic overexpression. Conservation of the ILK kinase domain across species may reflect a conserved
structural motif required for its interaction with various
pairing partners, including UNC-97/PINCH, UNC-112,
and actopaxin/CHILKBP/affixin.
It is important to note, however, that our results do
not rule out a role for ILK in signal transduction, since
other kinases with overlapping function may compensate for the ILK loss-of-function or kinase-dead mutations that we used in our experiments. We also may
have failed to identify subtle developmental deficits that
might yet provide evidence that PAT-4/ILK is involved
in signal transduction. Nevertheless, our rescue results
are remarkably similar to the kinase-dead rescue of Drosophila ILK mutants.
If ILK is not a signaling kinase, which is what our
experiments and those on Drosophila ILK suggest, then
its role in adhesion complexes is most likely as an adaptor protein bringing other components of the complex
together. The recent identification of additional binding
partners of ILK reinforces this conclusion [21, 25, 27,
28, 38]. Using the yeast two-hybrid approach, we have
reproduced the binding of ILK and PINCH homologs in
C. elegans. We have also identified a novel ILK binding
partner, the dense body and M-line protein UNC-112.
Several lines of evidence are consistent with a direct
binding interaction between PAT-4/ILK and UNC-112.
First, PAT-4/ILK and UNC-112 colocalize in vivo at dense
body and M-line muscle attachments. Second, we have
shown that PAT-4/ILK and UNC-112 are reciprocally
dependent upon each other for recruitment to the sarcolemma, raising the interesting possibility that they may
be recruited to nascent attachments as part of a multiprotein complex. Third, we have identified the subdomains of PAT-4/ILK and UNC-112 that are necessary
and sufficient for interaction in yeast two-hybrid assays.
Finally, we have obtained evidence for direct binding
between purified recombinant PAT-4/ILK and UNC-112
in vitro.
Conclusions
In conclusion, PAT-4/ILK mediates the formation of linkages between the nascent adhesion complexes and the
underlying cytoskeleton. Failure to complete these linkages in the absence of PAT-4/ILK blocks sarcomere
assembly, paralyzes the embryo, and ultimately results
in developmental arrest. On the cellular level, the nascent muscle attachments remain disorganized within
the plasma membrane. PAT-4/ILK function in vivo appears to be independent of its kinase activity. Our results
suggest that PAT-4/ILK is an adaptor protein. Consistent
with this view, we have identified a new ILK binding
partner, the novel protein UNC-112.
Experimental Procedures
Strains and Genetics Used in This Study
Worms were grown and genetic analysis was done as described
[39]. Wild-type worms are the N2 strain of the Bristol variety. The
following mutations and strains were used: deb-1(st555), pat2(st567), pat-3(st552), pat-4(st551), pat-4(st559), pat-4(st579), pat4(st580), unc-52(st549), unc-112(gk-1), unc-112(st561), WB72 (pat4(st551); zpEx55), WB125 (pat-4(st551); zpEx194), WB154 (unc44(e362) deb-1(st555)/unc-82(e1223) unc-24(e138); zpEx194),
WB156 (⫹/⫹; zpEx194), WB175 (pat-4(st551); zpEx209), WB201
(pat-4(st551);zpEx204), and WB204 (pat-4(st551); zpEx225). All extrachromosomal arrays contain the dominant transformation marker
pRF4 (rol-6) and the following: zpEx55 contains a PCR fragment,
generated using primers BW48/BW48 (all primer sequences are
available in Table S1 of the Supplementary Material available with
this article online, see below), corresponding to full-length pat-4;
zpEx194 contains plasmid pAT4.4; zpEx204 contains PCR fragments
(generated using primers T7/BW15, BW163/2051, BW70/2051, and
BW71/BW94), corresponding to pat-3::CFP and pat-4::YFP;
zpEx209 contains plasmid pAT4.6; and zpEx225 contains plasmids
pAT4.21. The molecular lesions corresponding to pat-4 alleles st551,
st559, st579, and st580 were identified by PCR amplification of the
C29F9.7 gene from homozygous Pat embryos and subsequent DNA
sequencing using the fmol kit (Promega). We used PCR to define
the right breakpoints of the st559 and st580 deletion alleles to an
interval defined by the pat-4 locus and K10F12.3, which is located
230 kbp to the right. The left breakpoints were defined using PCR
primers corresponding to the 5⬘ region of the predicted gene
F54C4.3, which is located 3.2 kbp to the left of pat-4. We are able
to detect the PCR product using pat-4(st580) embryos as templates,
but not when we use pat-4(st559) embryos as templates. cDNA
clone yk199a2 was sequenced and found to be a full-length pat-4
clone whose sequence matches the processed transcript predicted
by GENEFINDER. Seven additional full-length cDNA clones from Yuji
pat-4 and Muscle Assembly
795
Kohara’s cDNA collection (yk175b10, yk269g8, yk334c3, yk374e3,
yk415h4, yk455c5, and yk473d6) were also analyzed, and no alternatively spliced isoforms were detected. The pat-4 nucleotide and
protein sequence have been previously submitted to GenBank/
EMBL/DDBJ under the accession number T33574 [40]. PAT-4 homologs were identified using the BLAST algorithm to search the NCBI
database, and sequence alignments were performed using the
CLUSTAL V alignment method. Transformation rescue of pat4(st551) was done by injecting cosmid DE10 (canonical for C29F9)
[41]. To confirm that pat-4 corresponds to C29F9.7, we injected
pat-4(st551)/⫹ animals with a PCR product corresponding only to
C29F9.7. The promoter sequence used for this PCR product starts
1915 bp upstream from the predicted ATG start codon. Both injection mixes completely rescue the pat-4 phenotype.
Molecular Biology
Plasmids pAT4.4, pAT4.6, pAT4.16, and pAT4.21 are pat-4::gfp minigenes in which the pat-4-coding region is fused in frame to the
carboxy terminus of GFP. These minigenes either contain no pat-4
intronic sequence (pAT4.16) or contain the first intron of pat-4
(pAT4.4, pAT4.6, and pAT4.21). Plasmid pAT4.1 was constructed by
cloning a PCR fragment, generated using primers BW73 and BW59,
into the 4285-bp BamHI-NcoI fragment of plasmid yk199a2 (GenBank accession number C39322). This plasmid contains 1915 bp of
5⬘ UTR sequence and the first intron of the pat-4 gene. The plasmid
pAT4.3 was constructed by cloning a PCR product, generated using
primers BW99 and BW100 and corresponding to full-length pat-4
cDNA, into the vector pPD118.20 digested with EcoRI. The 2.13-kb
MluI-KpnI fragment of the resulting plasmid was then replaced with
a PCR product generated using BW-116 and BW-118 and digested
with MluI and KpnI. The plasmid pAT4.4 was constructed by replacing the 235-bp BstXI-EagI fragment of pAT4.3 with the 783-bp BstXIEagI fragment of pAT4.1. A mutagenic PCR fragment, generated
with primers BW190 and BW191 and corresponding to vertebrate
kinase-dead (E359K) ILK, was cloned into the 6.69-kb SacI-NheI
fragment of either pAT4.4 or pAT4.3 to generate pAT4.6 or pAT4.16,
respectively. The plasmid pAT4.21 was constructed by replacing
the 368-bp SacI-NheI wild-type-coding sequence of pAT4.4 with a
mutagenic PCR fragment generated with primers BW229 and
BW230, corresponding to vertebrate kinase-dead (S343A) ILK. All
plasmids were verified by sequencing.
A cDNA fragment of unc-112 was PCR amplified using primers
Bam-unc-112 and unc-112-NsiXho. This fragment was cloned into
pBluescript KS⫹ to make pDM#224. The NsiI-XhoI region of
pDM#224 was exchanged for the NsiI-XhoI region of pDM#205, a
cDNA clone of unc-112, see [11], to make pDM#225. A BamHIXhoI fragment of pDM#225 was cloned into the BamHI-SalI sites of
pGBDU-C1 or pGAD-C1, resulting in pDM#235 or pDM#236, respectively (amino acids [aa] 1–720). pDM#238 was made by inserting the
BamHI-BglII fragment of pDM#225 into pGAD-C1. To make
pDM#230 (aa 397–720), a BglII-XhoI fragment of pDM#205 was inserted into pGAD-C3. pDM#232 (aa 565–720) and pDM#234 (aa
32–720) were made by cloning a SnaBI (blunt ended with Klenow
fragment) and XhoI, or a StyI (blunt ended with Klenow fragment)
and XhoI fragment, of pDM#205 into the SmaI and XhoI sites of
pGAD-C1. pDM#264 (aa 32–396) was made by removing the BglIIXhoI region from pDM#234.
Among the pat-4 deletion plasmids, pDM#280, pDM#312, and
pDM#313 were originally isolated by two-hybrid screening with the
UNC-112 bait. To make the full-length PAT-4 plasmid pDM#292 (aa
1–474), the XhoI fragment of pDM#280 was inserted into the SalI
site of pGBDU-C2. pDM#301 (aa 213-349), pDM#327 (aa 4–212), and
pDM#328 were made by inserting SalI-SalI or SalI-BglII fragments of
pDM#292 into SalI sites of pGBDU-C1, pGBDU-C3, or pGAD-C3.
pDM#303 (aa 350–474) and pDM#304 were made by inserting a SalIBglII fragment of pDM#292 into SalI-BglII sites of pGBDU-C1 and
pGAD-C1. To make pDM#325 (aa 213–474) and pDM#326, a SalISalI fragment of pDM#302 was cloned into a SalI site of pDM#303
or pDM#304. pDM#323 (aa 1–349) was made by exchanging an
EcoRI-SacII region of pDM#301 for that of pDM#292. The EcoRISmaI fragment of pAT4.16.2 was cloned into the EcoRI and SmaI
sites of pDM#303 to make pDM#381 (E359K). pDM#321 (aa 46–474)
and pDM#322 (aa 104–474) were made by cloning an XhoI fragment
of pDM#312 and pDM#313 into the SalI site of pGBDU-C2.
A cDNA fragment of the pat-3 cytoplasmic region was PCR amplified using primers Bam-pat-3cyto and pat-3cyto-Xho. This fragment
was cloned into pBluescript KS⫹ to make pDM#226. The BamHIXhoI fragment of pDM#226 was cloned into pGBDU-C1 to make
pDM#227. A cDNA fragment of unc-97 was PCR amplified using
primers pU97-1 and pU97-2. This fragment was digested with BamHI
and Xho and was subsequently cloned into the BamHI and SalI sites
of pGBDU-C1 to make pDM#429.
A plasmid for bacterial expression of GST::PAT-4 (C terminus, aa
213–474) fusion (pDM#438) was made by inserting a BamHI-BglII
fragment of pDM#325 into pGEX-KK-1 (a gift from Dr. Kaibuchi). To
make pDM#367 for bacterial expression of the MBP::UNC-112 (N
terminus, aa 1–396) fusion, a BamHI-BglII fragment of pDM#225
was cloned into pMAL-KK-1 (a gift from Dr. Kaibuchi).
RNAi was performed according to [33]. dsRNA corresponding to
pat-4 was generated using the MegaScripe kit (Ambion) and the
cDNA clone yk199a2 as a template.
In Vitro Binding Assay
GST::PAT-4 and MBP::UNC-112 were prepared from E. coli harboring pDM#438 and pDM#367, respectively, and the interaction of
GST::PAT-4 with MBP::UNC-112 was examined as described [42].
Briefly, MBP alone or MBP::UNC-112 (25 ␮g) were mixed with glutathione affinity beads coated with GST::PAT-4 (10 ␮g). The beads
were then washed with buffer containing 100 mM NaCl and 0.1%
Triton X-100, and the bound proteins were extracted by the addition
of SDS-PAGE sample buffer. The extracts were subjected to SDSPAGE, followed by silver staining.
Antibody Staining
Populations of embryos were fixed and stained as previously described [16]. We used affinity-purified goat anti- mouse IgG conjugated to rhodamine (Chemicon International) diluted 1:100 as the
secondary antibody. For our analysis, we used the following monoclonal antibodies, diluted in PBS supplemented with 0.5% Tween20 and 30% normal goat serum: MH2 (1:100), MH24 (1:200), MH25
(1:250), MH27 (1:1500), and MH42 (1:50) [12, 43]. Polyclonal PAT-4
antisera was obtained from mice injected with a synthetic 31 amino
acid peptide (CKNTILEIAQEHGQSPNDRVPFKDTTWKGTK; single
letter amino acid notation).
Two-Hybrid Screening
Two-hybrid screening with the UNC-112 bait was performed using
the yeast strain PJ69-4A harboring pDM#235 as described [44].
Yeast cells were transformed by the lithium acetate method [45].
After transformation with the RB2 cDNA library (a gift from Dr. R.
Barstead), His⫹ colonies were selected. HIS3 expression was assayed using 2 mM 3-amino triazole. Two-hybrid interaction was
confirmed by ADE2 expression. Isolated library plasmids were confirmed. PCR-amplified cDNA fragments of library plasmids were
subjected to DNA sequencing. From 5 ⫻ 106 colonies, pat-4 cDNA
clones pDM#280, pDM#312, and pDM#313 were identified. To confirm two-hybrid interactions, three independent colonies were selected and assayed for colony formation.
Supplementary Material
Supplementary Material including Table S1, which lists primer
sequences, is available at http://images.cellpress.com/supmat/
supmatin.htm.
Acknowledgments
We are grateful to Dr. Andy Fire and his lab for plasmid vectors, Dr.
Alan Coulson for cosmids, Dr. Yuji Kohara for cDNA clones, Dr.
Robert Barstead for the RB2 cDNA library, Dr. Kozo Kaibuchi for
plasmids, Dr. Shinya Kuroda for advice on in vitro binding assays,
and Dr. John Speith and the C. elegans Genome Sequencing Consortium for pat-4 DNA sequence data. We wish to thank the lab of
Akira Chiba for allowing us the use of equipment and reagents. We
appreciate the technical assistance provided by Chris Zugates and
Patrick King. Some of the nematode strains used in this work were
provided by the Caenorhabditis Genetics Center, which is funded
by the National Institutes of Health National Center for Research
Current Biology
796
Resources (NCRR). This work was supported by a predoctoral fellowship from the American Heart Association, Midwest Affiliate to
A.C.M.; a Japan Society for the Promotion of Science Postdoctoral
Fellowship for Research Abroad (2000) to H.Q.; a grant from the
Canadian Institute for Health Research to D.G.M.; and National Institutes of Health grant R01 HD38464-01 and a Scientist Development
Grant from the American Heart Association, Midwest Affiliate to
B.D.W.
Received: January 17, 2002
Revised: March 4, 2002
Accepted: March 7, 2002
Published: May 14, 2002
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