1 Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261,
USA
2 Structural Biology Program, Lerner Research Institute, The Cleveland Clinic
Foundation, Cleveland, OH 44195, USA
* Author for correspondence (e-mail: carywu{at}pitt.edu)
Accepted 11 September 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Integrin-linked kinase, PINCH, CH-ILKBP, Focal adhesions, Protein kinase C
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrin-linked kinase (ILK) is a cytoplasmic component of cell-ECM
adhesion structures that plays critical roles in the coupling of ECM to actin
cytoskeleton and signaling complexes
(Dedhar et al., 1999;
Wu, 1999
;
Wu and Dedhar, 2001
).
Structurally, ILK comprises an N-terminal ankyrin (ANK) repeat domain, a
C-terminal kinase domain that exhibits significant homology to other protein
kinase catalytic domains and a pleckstrin homology (PH)-like motif that
partially overlaps with the N-terminal region of the kinase domain. ILK is
capable of interacting with several other focal adhesion proteins including
ß1 integrins (Hannigan et al.,
1996
), LIM-domain containing protein PINCH
(Tu et al., 1999
), calponin
homology-containing protein CH-ILKBP (Tu
et al., 2001
) [also known as actopaxin
(Nikolopoulos and Turner,
2000
) or
-parvin (Olski
et al., 2001
)], affixin
(Yamaji et al., 2001
) [also
known as ß-parvin (Olski et al.,
2001
)] and paxillin
(Nikolopoulos and Turner,
2001
). We have previously shown that ILK binds to PINCH and
CH-ILKBP simultaneously, resulting in the formation of a PINCH-ILK-CH-ILKBP
ternary complex in mammalian cells (Tu et
al., 2001
). In mammalian systems, inhibition of the
PINCH-ILK-CH-ILKBP complex formation with dominant negative inhibitors results
in defects in cell shape change, migration, proliferation and ECM deposition
(Guo and Wu, 2002
;
Zhang et al., 2002
),
indicating a critical role of the PINCH-ILK-CH-ILKBP complex in these
processes. The interactions of ILK with PINCH and CH-ILKBP are well conserved
during evolution (Mackinnon et al.,
2002
; Wu and Dedhar,
2001
; Zervas and Brown,
2002
). In genetic model systems such as C. elegans,
mutations in any one of the components [pinch-1/unc-97
(Hobert et al., 1999
),
ilk/pat-4 (Mackinnon et al.,
2002
) or ch-ilkbp/pat-6 (Lin and Williams, 40th ASCB
Annual Meeting, 2000, Abstract 2666)] all resulted in a Pat phenotype similar
to that of ß-integrin/pat-3 or
-integrin/pat-2,
which is characterized by defects in the assembly of muscle cell attachment
structures (Gettner et al.,
1995
; Williams and Waterston,
1994
). Thus, the PINCH-ILK-CH-ILKBP complex appears to represent a
functionally important and evolutionally conserved cell-ECM adhesion complex
that is indispensable for the proper assembly of the actin-integrin adhesion
structures.
To better understand the molecular and cellular mechanisms underlying the assembly and regulation of cell-ECM adhesion structures, we have defined the PINCH and ILK sites that mediate the assembly of the PINCH-ILK-CH-ILKBP complex, identified upstream signaling proteins that are involved in the regulation of the ILK ternary complex and investigated the mechanism underlying the localization of PINCH and ILK to cell-ECM adhesion sites.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Site-directed mutagenesis
A QuickChangeTM site-directed mutagenesis system (Stratagene)
was used to introduce substitution mutations at positions as specified in each
experiment. All point mutations were confirmed by DNA sequencing.
Construction of GFP- or FLAG-tagged PINCH and ILK expression vectors
and transfection
DNA fragments encoding wild-type or mutant forms of PINCH or ILK were
cloned into the EcoRI/SalI sites of the pEGFP-C2 vector
(Clontech) or the EcoRI/XbaI sites of the pFLAG-CMV-2 vector
(Sigma). Mouse C2C12 cells or human 293 cells were transfected with the
expression vectors using LipofectAmine PLUS (Life Technologies)
(Huang et al., 2000). The
expression of GFP- or FLAG-tagged wild-type or mutant forms of PINCH or ILK
was confirmed by western blotting.
Immunofluorescence staining
Immunofluorescence staining was performed as described
(Zhang et al., 2002). Briefly,
cells were plated on fibronectin-coated coverslips, fixed, and stained with
mouse monoclonal antibodies as specified in each experiment. The primary mouse
monoclonal antibodies were detected with a Rhodamine
RedTX-conjugated anti-mouse IgG antibody and observed under a
fluorescence microscopy.
Cell preparation
To prepare cells that lacked ECM adhesion structures, cells were harvested
by trypsinization and washed twice with normal medium. Cells were then
resuspended in normal culture medium and maintained in suspension on a rocking
platform for 60 minutes and then harvested by centrifugation. The cells were
washed once with PBS and the pellets were lysed with 1% Triton X-100 in 50 mM
Tris-HCl (pH 7.4) containing 150 mM NaCl, 10 mM
Na4P2O7, 2 mM Na3 VO4,
100 mM NaF and protease inhibitors (lysis buffer). To prepare cells that had
adhered and spread on fibronectin, cells were plated in fibronectin (10
µg/ml) coated tissue culture dishes for different periods of time. Under
the condition used, cells began to spread within 30 minutes and were fully
spread after two hours. Cells that adhered and spread on fibronectin were
rinsed once with PBS and then lysed on the plates with the lysis buffer. In
some experiments, cell monolayers were incubated in medium containing
cytochalasin D or protein kinase C inhibitors calphostin C (under light) or
chelerylthrine chloride for 50 to 60 minutes (as specified in each experiment)
prior to lysis with the lysis buffer.
Immunoprecipitation assays
Immunoprecipitation of the PINCH-ILK-CH-ILKBP complex was performed as
previously described (Tu et al.,
2001; Zhang et al.,
2002
). Briefly, lysates (500 µg) of cells that were prepared as
described above were mixed with 500 µl of hybridoma culture supernatant
containing monoclonal anti-CH-ILKBP antibody 1D4. The samples were incubated
for 3 hours, mixed with 40 µl of UltraLink Immobilized Protein G (Pierce)
and then incubated for an additional 1.5 hours. The beads were washed four
times and the proteins bound were released from the beads by boiling in
SDS-PAGE sample buffer for 5 minutes. The samples were analyzed by western
blotting with anti-CH-ILKBP antibody 3B5, anti-ILK antibody 65.1, or rabbit
polyclonal anti-PINCH antibodies as specified in each experiment.
For immunoprecipitation of GFP-tagged or FLAG-tagged wild-type or mutant forms of ILK or PINCH, cells expressing the GFP- or FLAG-tagged proteins were lysed as described above. To immunoprecipitate GFP-tagged proteins, cell lysates were mixed with rabbit polyclonal anti-GFP antibodies (Clontech) and the immune complexes were precipitated with UltraLink Immobilized Protein G beads. To immunoprecipitate FLAG-tagged proteins, cell lysates were mixed with agarose beads conjugated with anti-FLAG antibody M2 (Sigma). The immunoprecipitated proteins were released from the beads by boiling in SDS-PAGE sample buffer for 5 min and analyzed by western blotting with antibodies as specified in each experiment.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The Q40A point mutation that ablates the ILK binding abolishes
the localization of PINCH to focal adhesions
We next tested whether the interaction with ILK is required for the
localization of PINCH to focal adhesions. To do this, we stained cells
expressing the GFP-tagged ILK-binding defective PINCH point mutant, and those
expressing GFP-PINCH as a control, with a monoclonal anti-paxillin antibody
(as a marker of focal adhesions). The results showed that, as expected,
GFP-PINCH readily localized to focal adhesions
(Fig. 1D,E). By contrast, the
ILK-binding defective Q40A point mutant failed to localize to focal
adhesions where abundant paxillin was detected
(Fig. 1F,G), suggesting that
the ILK-binding is required for the localization of PINCH to focal
adhesions.
Defining the ILK site that mediates the interaction with PINCH
We have previously shown that the PINCH-binding site is located within the
ILK N-terminal ankyrin (ANK) repeat domain
(Li et al., 1999;
Tu et al., 1999
). Although the
details of the three-dimensional structure of the ILK ANK domain remain to be
determined, sequence homology indicates that it likely adapts a fold similar
to other known ANK repeat protein structures, which are cupped-handed
consisting of an antiparallel beta-hairpin (fingers) and alpha-helix bundles
(palm) that are perpendicular to the plane of the beta-hairpin
(Venkataramani et al., 1998
).
Our previous deletion data has shown that the first ANK repeat is important
for ILK-PINCH interaction (Li et al.,
1999
). Because previous structural studies of ANK repeat protein
complexes suggested that ANK repeats may use their hairpin regions to interact
with the target proteins (Batchelor et al.,
1998
; Gorina and Pavletich,
1996
; Huxford et al.,
1998
; Jacobs and Harrison,
1998
), we investigated if this occurs to ILK by substituting
Asp31, which is located at the perspective hairpin region of the first ANK
repeat, with Ala. We expressed a GFP-fusion protein containing the ILK point
mutant (GFP-D31A), and a GFP-fusion protein containing the wild-type ILK as a
control, in mammalian cells. The expression of GFP-D31A and GFP-ILK was
confirmed by western blotting analyses of the cells
(Fig. 2A, lanes 1,2). To test
the PINCH-binding, we immunoprecipitated GFP-D31A
(Fig. 2A, lane 3) and GFP-ILK
(Fig. 2A, lane 4),
respectively, from the cell lysates. Western blotting analyses of the
immunoprecipitates with anti-PINCH antibodies showed that PINCH was
co-immunoprecipitated with GFP-ILK (Fig.
2B, lane 4) but not with GFP-D31A
(Fig. 2B, lane 3), suggesting
that residue D31 is required for the interaction with PINCH. Analyses of the
same immunoprecipitates with monoclonal anti-CH-ILKBP antibody 3B5 showed
that, as expected, CH-ILKBP was co-immunoprecipitated with GFP-D31A
(Fig. 2C, lane 3) as well as
with GFP-ILK (Fig. 2C, lane
4).
|
The D31A point mutation that ablates the PINCH binding abolishes the
localization of ILK to focal adhesions
To test whether the PINCH binding is required for the localization of ILK
to cell-ECM adhesion sites, we stained the cells expressing GFP-D31A and those
expressing GFP-ILK with a monoclonal anti-paxillin antibody. The GFP-tagged
PINCH-binding defective ILK point mutant
(Fig. 2D,E), unlike GFP-ILK
(Fig. 2F,G), was unable to
localize to focal adhesions. To further analyze this, we substituted Lys43,
which is located twelve residues C-terminal of D31 and is predicted to be in
the helix region, with Ala. The K43A point mutant, unlike D31A point mutant,
readily interacted with PINCH and localized to focal adhesions (data not
shown). Taken together, these results suggest that the interaction with PINCH,
which involves D31, is required for the localization of ILK to cell-ECM
adhesion sites.
The PH-like motif is not required for the localization of ILK to
focal adhesions
ILK contains a PH-like motif (residues 180-212) that is C-terminal to the
ANK domain and partially overlaps with the N-terminal region of the C-terminal
kinase domain. To test whether the PH-like motif is required for the
localization of ILK to cell-ECM adhesion sites, we deleted GTLNKHSGIDFK
(residues 180-190) from the PH-like motif. Residues 180-190 were chosen
because (1) they contain the consensus sequence GXLXK-GXXXK and therefore are
an essential part of the PH-like motif and (2) they do not overlap with the
C-terminal kinase domain and therefore unlikely interfere with the binding
activities mediated by the C-terminal domain. We expressed a GFP-fusion
protein containing the ILK deletion mutant (GFP-dPH)
(Fig. 3, lane 2), GFP-ILK
(Fig. 3, lane 1) as a positive
control and GFP (Fig. 3, lane
3) as a negative control in mammalian cells. Co-immunoprecipitation
experiments showed that GFP-dPH (lane 5), like GFP-ILK (lane 4) but unlike GFP
(lane 6), bound to both PINCH (Fig.
3B) and CH-ILKBP (Fig.
3C). Consistent with the binding activities towards PINCH and
CH-ILKBP, analyses of cells expressing GFP-dPH shows that it was recruited to
focal adhesions (Fig. 3D,E).
Thus, the PH-like motif is neither required for the assembly of the
PINCH-ILK-CH-ILKBP complex nor required for the localization of ILK to focal
adhesions.
|
Disruption of the interaction with CH-ILKBP impairs the localization
of ILK to focal adhesions
To assess whether the interaction with CH-ILKBP is required for the
localization of ILK to focal adhesions, we extended the region of deletion
mutation into the CH-ILKBP-binding C-terminal domain of ILK. To do this, we
expressed in mammalian cells a GFP fusion protein containing an ILK mutant
(D215) in which residues 180-215, which include residues within the N-terminal
region of the C-terminal domain, were deleted. Co-immunoprecipitation analyses
showed that deletion of residues 180-215, unlike deletion of residues within
the PH-like motif only (Fig.
3C, lane 5), disrupted the interaction with CH-ILKBP
(Fig. 4A,B, lane 2). GFP-D215
(Fig. 4C, lane 2), like GFP-dPH
(Fig. 3B, lane 5), bound to
PINCH. As expected, GFP-ILK (lane 1), but not GFP (lane 3), bound to both
CH-ILKBP (Fig. 4B) and PINCH
(Fig. 4C). Importantly, the
CH-ILKBP binding defective GFP-D215 (Fig.
4D,E), unlike the CH-ILKBP binding GFP-dPH
(Fig. 3D,E), failed to
localized to focal adhesions, suggesting that the localization of ILK to focal
adhesions requires not only the interaction with PINCH but also the
interaction with CH-ILKBP.
|
The formation of the PINCH-ILK-CH-ILKBP complex precedes
integrin-mediated cell adhesion and spreading
The foregoing experiments, together with our previous observation that the
interaction with ILK is required for the localization of CH-ILKBP to cell-ECM
adhesion sites (Tu et al.,
2001), suggest that PINCH, ILK and CH-ILKBP are interdependent in
their localization to cell-ECM adhesion sites. This predicts that the
formation of the PINCH-ILK-CH-ILKBP complex does not require the presence of
cell-ECM contacts. To test this, we immunoprecipitated CH-ILKBP from cells
that were maintained in suspension (so that they lacked any cell-ECM contacts)
with monoclonal anti-CH-ILKBP antibody 1D4
(Fig. 5A, lane 1). In parallel
experiments, CH-ILKBP was immunoprecipitated from cells that had adhered and
spread on fibronectin-coated surface for different periods of time
(Fig. 5A, lanes 2-4). Analyses
of the CH-ILKBP immunoprecipitates with anti-ILK
(Fig. 5B) and anti-PINCH
(Fig. 5C) antibodies showed
that PINCH, ILK and CH-ILKBP formed a complex in cells that lacked any
cell-ECM contacts (lane 1) as well as in cells that were plated for a
relatively short period of time (and therefore contained nascent focal
complexes) (lane 2) or well-spread (and therefore contained mature focal
adhesions and ECM contacts) (lane 4). Thus, consistent with the
interdependence between PINCH, ILK and CH-ILKBP in their localization to
cell-ECM adhesion sites, the formation of the PINCH-ILK-CH-ILKBP complex
precedes integrin-mediated cell adhesion and spreading.
|
Inhibition of protein kinase C, but not that of actin polymerization,
inhibits the PINCH-ILK-CH-ILKBP complex formation
It has been well described that protein kinase C plays an important role in
the cellular control of assembly of cell-ECM adhesion structures
(Berrier et al., 2000;
Chun and Jacobson, 1993
;
Defilippi et al., 1997
;
Lewis et al., 1996
;
Woods and Couchman, 1992
) and
the protein kinase C mediated-regulation precedes integrin-mediated cell
spreading (Vuori and Ruoslahti,
1993
). Because the formation of the PINCH-ILK-CH-ILKBP complex is
critical to cell spreading (Zhang et al.,
2002
) and it occurs prior to the formation of cell-ECM adhesion
structures (Fig. 5), we tested
whether protein kinase C is involved in the regulation of the
PINCH-ILK-CH-ILKBP complex formation. Cells were treated with calphostin C, a
specific protein kinase C inhibitor, and the formation of the
PINCH-ILK-CH-ILKBP complex was analyzed
(Fig. 6). The results showed
that inhibition of protein kinase C significantly reduced the amount of ILK
(Fig. 6B) and PINCH
(Fig. 6C) that were in complex
with CH-ILKBP (Fig. 6A). Treatment of cells with chelerylthrine chloride, another inhibitor of protein
kinase C, resulted in a similar reduction of the PINCH-ILK-CH-ILKBP complex
(data not shown). To determine whether protein kinase C regulates the ILK
ternary complex formation by regulating the ILK-CH-ILKBP interaction, the
PINCH-ILK interaction or both, we transfected human 293 cells with an
expression vector encoding FLAG-tagged ILK. FLAG-ILK was immunoprecipitated
with a monoclonal anti-FLAG antibody (Fig.
6D, lane 1). Analyses of the FLAG-ILK immunoprecipitates with
anti-CH-ILKBP and anti-PINCH antibodies showed that both CH-ILKBP
(Fig. 6E, lane 1) and PINCH
(Fig. 6F, lane 1) were
co-immunoprecipitated with FLAG-ILK. Treatment of the cells with calphostin C
significantly reduced the amount of CH-ILKBP
(Fig. 6E, lane 2) and PINCH
(Fig. 6F, lane 2) that were in
complex with FLAG-ILK (Fig. 6D,
lane 2), suggesting that protein kinase C is involved in the regulation of
both the ILK-CH-ILKBP and the PINCH-ILK interactions. In additional
experiments, we immunoprecipitated FLAG-PINCH form FLAG-PINCH transfectants
that were treated with or without calphostin C and found that much less ILK
and CH-ILKBP were co-immunoprecipitated with FLAG-PINCH in the presence of the
protein kinase C inhibitor (data not shown). In contrast to the effect of the
protein kinase C inhibitors, inhibition of actin polymerization with
cytochalasin D did not inhibit the complex formation
(Fig. 6D-F, lane 3), indicating
that actin polymerization is not required for the formation of the ILK
complex.
|
ILK interactions with PINCH and CH-ILKBP, although necessary, are not
sufficient for mediating ILK localization to focal adhesions
To test whether ILK interactions with PINCH and CH-ILKBP are sufficient for
mediating ILK localization to focal adhesions or there exist other sites that
are critical to this process, we generated an ILK point mutant in which
residue Phe438 at ILK C-terminus was substituted with Ala. We expressed a
GFP-fusion protein containing the ILK F438A mutant in mammalian cells.
GFP-F438A (Fig. 7A, lane 6),
like GFP-ILK (Fig. 7A, lane 5),
formed a complex with both CH-ILKBP (Fig.
7B, lanes 5,6) and PINCH (Fig.
7C, lanes 5,6). However, although the F438A point mutation
did not inhibit the ILK complex formation with PINCH and CH-ILKBP, it impaired
the ability of ILK to localize to focal adhesions
(Fig. 7D,E). These results
indicate that the interactions of ILK with PINCH and CH-ILKBP are necessary
but not sufficient for mediating ILK localization to focal adhesions.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
What are the other interactions that are involved in the localization of
the PINCH-ILK-CH-ILKBP complex to cell-ECM adhesion sites? Our finding that a
point mutation at the ILK C-terminus (F438A) does not disrupt the ILK
complex formation with PINCH and CH-ILKBP but impairs ILK localization to
cell-ECM adhesion sites suggests that this site likely mediates one of the
other interactions that are involved in this process. In addition to
interacting with PINCH and CH-ILKBP, ILK is capable of interacting with
several other focal adhesion proteins including the ß1 integrins
(Hannigan et al., 1996
),
affixin/ß-parvin (Olski et al.,
2001
; Yamaji et al.,
2001
) and paxillin
(Nikolopoulos and Turner,
2001
). These proteins are strong candidates for recruiting the
PINCH-ILK-CH-ILKBP complex to cell-ECM adhesion sites. In addition, there
likely exist other proteins that participate in this process. Mackinnon et
al., recently showed that in C. elegans, PAT-4/ILK requires UNC-112,
a newly identified FERM domain containing ILK-binding protein
(Rogalski et al., 2000
), to be
properly recruited to muscle attachments
(Mackinnon et al., 2002
). It
will be interesting to test in future studies whether ILK interacts with
Mig-2, a mammalian homologue of C. elegans UNC-112
(Rogalski et al., 2000
), and
if it does, whether it involves the F438 site.
It has been well established that protein kinase C plays important roles in
the regulation of cell spreading, migration, proliferation and fibronectin
matrix assembly (Berrier et al.,
2000; Chun and Jacobson,
1993
; Defilippi et al.,
1997
; Huang et al.,
1998
; Lewis et al.,
1996
; Miranti et al.,
1999
; Schlaepfer and Hunter,
1998
; Schwartz et al.,
1995
; Somers and Mosher,
1993
; Woods and Couchman,
1992
). The finding that inhibition of protein kinase C
down-regulates the PINCH-ILK-CH-ILKBP complex formation
(Fig. 6), together with our
previous findings that disruption of the PINCH-ILK-CH-ILKBP complex formation
inhibits cell spreading, migration, proliferation and fibronectin matrix
assembly (Guo and Wu, 2002
;
Zhang et al., 2002
), suggest
that the PINCH-ILK-CH-ILKBP complex likely serves as an important down stream
effector in protein kinase C-mediated regulation of cell spreading, migration,
proliferation and fibronectin matrix assembly. It is interesting to note that
cytochalasin D, which inhibits cell spreading, migration and fibronectin
matrix assembly by inhibition of actin polymerization, does not inhibit the
PINCH-ILK-CH-ILKBP complex formation (Fig.
6). Thus, the formation of the PINCH-ILK-CH-ILKBP complex does not
require actin polymerization, which is consistent with our observation that
the formation of the PINCH-ILK-CH-ILKBP complex precedes cell adhesion and
spreading. These results, together with the well documented effects of protein
kinase C on actin cytoskeleton organization and recent findings that the
PINCH-ILK-CH-ILKBP complex couples integrins to the actin cytoskeleton
(Mackinnon et al., 2002
;
Tu et al., 2001
;
Wu, 2001
;
Wu and Dedhar, 2001
;
Zervas and Brown, 2002
;
Zervas et al., 2001
), suggest
a new pathway (Fig. 8) in which
protein kinase C regulates cell adhesion and actin cytoskeleton organization
by, at least in part, modulating the assembly of the PINCH-ILK-CH-ILKBP
complex.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Batchelor, A. H., Piper, D. E., de la Brousse, F. C., McKnight,
S. L. and Wolberger, C. (1998). The structure of
GABPalpha/beta: an ETS domain-ankyrin repeat heterodimer bound to DNA.
Science 279,1037
-1041.
Berrier, A. L., Mastrangelo, A. M., Downward, J., Ginsberg, M.
and LaFlamme, S. E. (2000). Activated R-ras, Rac1, PI
3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated
integrin beta1 cytoplasmic domains. J. Cell Biol.
151,1549
-1560.
Burridge, K. and Chrzanowska-Wodnicka, M. (1996). Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12,463 -518.[CrossRef][Medline]
Calderwood, D. A., Shattil, S. J. and Ginsberg, M. H.
(2000). Integrins and actin filaments: reciprocal regulation of
cell adhesion and signaling. J. Biol. Chem.
275,22607
-22610.
Chun, J. S. and Jacobson, B. S. (1993). Requirement for diacylglycerol and protein kinase C in HeLa cell- substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading. Mol. Biol. Cell 4, 271-281.[Abstract]
Clark, E. A. and Brugge, J. S. (1995). Integrins and signal transduction pathways: the road taken. Science 268,233 -239.[Medline]
Dedhar, S., Williams, B. and Hannigan, G. (1999). Integrin linked kinase (ILK): a regulator of integrin and growth-factor signaling. Trends Cell Biol. 9, 319-323.[CrossRef][Medline]
Defilippi, P., Venturino, M., Gulino, D., Duperray, A., Boquet,
P., Fiorentini, C., Volpe, G., Palmieri, M., Silengo, L. and Tarone, G.
(1997). Dissection of pathways implicated in integrin-mediated
actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and
tyrosine phosphorylation. J. Biol. Chem.
272,21726
-21734.
Geiger, B., Bershadsky, A., Pankov, R. and Yamada, K. M. (2001). Transmembrane crosstalk between the extracellular matrixcytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2,793 -805.[CrossRef][Medline]
Gettner, S. N., Kenyon, C. and Reichardt, L. F. (1995). Characterization of beta pat-3 heterodimers, a family of essential integrin receptors in C. elegans. J. Cell Biol. 129,1127 -1141.[Abstract]
Giancotti, F. G. and Ruoslahti, E. (1999).
Integrin signaling. Science
285,1028
-1032.
Gorina, S. and Pavletich, N. P. (1996).
Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of
53BP2. Science 274,1001
-1005.
Guo, L. and Wu, C. (2002). Regulation of
fibronectin matrix deposition and cell proliferation by the PINCH-ILK-CH-ILKBP
complex. FASEB J. 16,1298
-1300.
Guo, L., Sanders, P. W., Woods, A. and Wu, C.
(2001). The distribution and regulation of integrin-linked kinase
in normal and diabetic kidneys. Am. J. Pathol.
159,1735
-1742.
Hannigan, G. E., Leung-Hagesteijn, C., Fitz-Gibbon, L., Coppolino, M. G., Radeva, G., Filmus, J., Bell, J. C. and Dedhar, S. (1996). Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase. Nature 379,91 -96.[CrossRef][Medline]
Hobert, O., Moerman, D. G., Clark, K. A., Beckerle, M. C. and
Ruvkun, G. (1999). A conserved LIM protein that affects
muscular adherens junction integrity and mechanosensory function in
Caenorhabditis elegans. J. Cell Biol.
144, 45-57.
Howe, A., Aplin, A. E., Alahari, S. K. and Juliano, R. L. (1998). Integrin signaling and cell growth control. Curr. Opin. Cell Biol. 10,220 -231.[CrossRef][Medline]
Huang, X., Wu, J., Spong, S. and Sheppard, D.
(1998). The integrin alphavbeta6 is critical for keratinocyte
migration on both its known ligand, fibronectin, and on vitronectin.
J. Cell Sci. 111,2189
-2195.
Huang, Y., Li, J., Zhang, Z. and Wu, C. (2000).
The roles of integrin-linked kinase in the regulation of myogenic
differentiation. J. Cell Biol.
150,861
-871.
Huxford, T., Huang, D. B., Malek, S. and Ghosh, G. (1998). The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation. Cell 95,759 -770.[Medline]
Jacobs, M. D. and Harrison, S. C. (1998). Structure of an IkappaBalpha/NF-kappaB complex. Cell 95,749 -758.[Medline]
Jockusch, B. M., Bubeck, P., Giehl, K., Kroemker, M., Moschner, J., Rothkegel, M., Rudiger, M., Schluter, K., Stanke, G. and Winkler, J. (1995). The molecular architecture of focal adhesions. Annu. Rev. Cell Dev. Biol. 11,379 -416.[CrossRef][Medline]
Lewis, J. M., Cheresh, D. A. and Schwartz, M. A. (1996). Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation. J. Cell Biol. 134,1323 -1332.[Abstract]
Li, F., Zhang, Y. and Wu, C. (1999).
Integrin-linked kinase is localized to cell-matrix focal adhesions but not
cell-cell adhesion sites and the focal adhesion localization of
integrin-linked kinase is regulated by the PINCH-binding ANK repeats.
J. Cell Sci. 112,4589
-4599.
Mackinnon, A. C., Qadota, H., Norman, K. R., Moerman, D. G. and Williams, B. D. (2002). C. elegans PAT-4/ILK Functions as an Adaptor Protein within Integrin Adhesion Complexes. Curr. Biol. 12,787 -797.[CrossRef][Medline]
Miranti, C. K., Ohno, S. and Brugge, J. S.
(1999). Protein kinase C regulates integrin-induced activation of
the extracellular regulated kinase pathway upstream of Shc. J.
Biol. Chem. 274,10571
-10581.
Nikolopoulos, S. N. and Turner, C. E. (2000).
Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and
actin and regulates cell adhesion. J. Cell Biol.
151,1435
-1448.
Nikolopoulos, S. N. and Turner, C. E. (2001).
Integrin-linked kinase (ILK) binding to paxillin LD1 motif regulates ILK
localization to focal adhesions. J. Biol. Chem.
276,23499
-23505.
Olski, T. M., Noegel, A. A. and Korenbaum, E.
(2001). Parvin, a 42 kDa focal adhesion protein, related to the
alpha-actinin superfamily. J. Cell. Sci.
114,525
-538.
Rogalski, T. M., Mullen, G. P., Gilbert, M. M., Williams, B. D.
and Moerman, D. G. (2000). The UNC-112 gene in Caenorhabditis
elegans encodes a novel component of cell-matrix adhesion structures required
for integrin localization in the muscle cell membrane. J. Cell
Biol. 150,253
-264.
Schlaepfer, D. D. and Hunter, T. (1998). Integrin signalling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol. 8,151 -157.[CrossRef][Medline]
Schwartz, M. A., Schaller, M. D. and Ginsberg, M. H. (1995). Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11,549 -599.[CrossRef][Medline]
Somers, C. E. and Mosher, D. F. (1993). Protein
kinase C modulation of fibronectin matrix assembly. J. Biol.
Chem. 268,22277
-22280.
Tu, Y., Li, F., Goicoechea, S. and Wu, C.
(1999). The LIM-only protein PINCH directly interacts with
integrin-linked kinase and is recruited to integrin-rich sites in spreading
cells. Mol. Cell. Biol.
19,2425
-2434.
Tu, Y., Huang, Y., Zhang, Z., Hua, Y. and Wu, C.
(2001). A new focal adhesion protein that interacts with
integrin-linked kinase and regulates cell adhesion and spreading.
J. Cell Biol. 153,585
-598.
Velyvis, A., Yang, Y., Wu, C. and Qin, J.
(2001). Solution structure of the focal adhesion adaptor PINCH
LIM1 domain and characterization of its interaction with integrin linked
kinase ankyrin repeat domain. J. Biol. Chem.
276,4932
-4939.
Venkataramani, R., Swaminathan, K. and Marmorstein, R. (1998). Crystal structure of the CDK4/6 inhibitory protein p18INK4c provides insights into ankyrin-like repeat structure/function and tumor-derived p16INK4 mutations. Nat. Struct. Biol. 5, 74-81.[Medline]
Vuori, K. and Ruoslahti, E. (1993). Activation
of protein kinase C precedes alpha 5 beta 1 integrin- mediated cell spreading
on fibronectin. J. Biol. Chem.
268,21459
-21462.
Williams, B. D. and Waterston, R. H. (1994). Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations. J. Cell Biol. 124,475 -490.[Abstract]
Woods, A. and Couchman, J. R. (1992). Protein kinase C involvement in focal adhesion formation. J. Cell Sci. 101,277 -290.[Abstract]
Wu, C. (1999). Integrin-linked kinase and
PINCH: partners in regulation of cell-extracellular matrix interaction and
signal transduction. J. Cell Sci.
112,4485
-4489.
Wu, C. (2001). ILK interactions. J. Cell Sci. 114,2549 -2550.[Medline]
Wu, C. and Dedhar, S. (2001). Integrin linked
kinase (ILK) and its interactors: a new paradigm for the coupling of
extracellular matrix to actin cytoskeleton and signaling complexes.
J. Cell Biol. 155,505
-510.
Yamaji, S., Suzuki, A., Sugiyama, Y., Koide, Y., Yoshida, M.,
Kanamori, H., Mohri, H., Ohno, S. and Ishigatsubo, Y. (2001).
A novel integrin-linked kinase-binding protein, affixin, is involved in the
early stage of cell-substrate interaction. J. Cell
Biol. 153,1251
-1264.
Zamir, E. and Geiger, B. (2001). Molecular
complexity and dynamics of cell-matrix adhesions. J. Cell
Sci. 114,3583
-3590.
Zervas, C. G. and Brown, N. H. (2002). Integrin adhesion: when is a kinase a kinase? Curr. Biol. 12,R350 -R351.[CrossRef][Medline]
Zervas, C. G., Gregory, S. L. and Brown, N. H.
(2001). Drosophila integrin-linked kinase is required at sites of
integrin adhesion to link the cytoskeleton to the plasma membrane.
J. Cell Biol. 152,1007
-1018.
Zhang, Y., Guo, L., Chen, K. and Wu, C. (2002).
A critical role of the PINCH-integrin-linked kinase interaction in the
regulation of cell shape change and migration. J. Biol.
Chem. 277,318
-326.