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Cell News 1/2015
Research news
Definitive proof for the lack of catalytic activity was provided
by the crystal structure of the ILK-KD bound to the CH2 do-
main of
α
-parvin, one of the 3 mammalian parvin isoforms. It
revealed that ILK-KD folds into a typical bilobial kinase structure
but it has a dramatically degenerated catalytic core compared
to known kinases. The P-loop structure that is essential for ATP-
binding contains a non-flexible motif in ILK, which is unable to
receive non-transferable phosphates of ATP. Due to the unusual
DVK motif the
γ
-phosphate is abnormally aligned and lies far
away from the putative catalytic site. Hence, bound ATP remains
in an unhydrolyzed state (Fukuda et al., 2009). It was further
demonstrated that kinase activity can be detected in impure
protein preparations of recombinant ILK, but this activity is lost
upon further purifications steps (Fukuda et al., 2009; Fukuda et
al., 2011).
Genetic studies provided further evidence for ILK being a pseu-
dokinase with adaptor function. Deletion of ILK in D. melano-
gaster leads to embryonic lethality with failure in muscle at-
tachment. Expression of ILK containing a mutation in the kinase
domain (E359K) (a reported kinase dead mutant ILK), in ILK-de-
ficient flies completely rescues the phenotype, indicating that
ILK fulfills its function independent of kinase activity (Zervas et
al., 2001). Similarly, the knockout of pat-4 (paralyzed, arrested
elongation at two-fold; C. elegans homolog of ILK) in C. elegans
impairs actin and myosin filament recruitment in embryonic mu-
scle, and this can be rescued by the expression of a kinase dead
ILK (Mackinnon et al., 2002).
The constitutive deletion of ILK in mice is embryonic lethal (Sakai
et al., 2003b). The embryos succumb during peri-implantation
due to a failure in epiblast polarization. This is caused by im-
paired F-actin rearrangement and basement membrane remo-
deling (Sakai et al., 2003b). In stark contrast, knock-in mice that
carry either a R211A mutation within the PH-domain leading
to a kinase-dead ILK, or specific mutations within the putative
autophosphorylation site leading to a kinase-dead (S343A) or
hyperactive (S343D) ILK are viable and healthy and show no dif-
ferences in phosphorylation of the reported ILK substrates AKT or
GSK-3
β
(Lange et al., 2009), supporting the kinase-independent
function of ILK. Knock-in mice with mutations in the ATP-bin-
ding site, K220A or K220M, die shortly after birth due to renal
dysgenesis (Lange et al., 2009). This mutation in ILK destabilizes
the KD and thereby interferes with its ability to bind to
α-
parvin,
demonstrating that ILK-
α
-parvin interaction is crucial for the
function of ILK.
α
-parvin knockout mice develop similar kidney
phenotype as that observed in ILK K220A/M mutants (Lange et
al., 2009). Together, all these studies confirm the non-catalytic,
adaptor function of ILK.
ILK regulates force generation, adhesion maturation,
and actin dynamics
The assembly and remodeling of integrin adhesion complexes is
a highly dynamic process that requires the recruitment of ad-
aptor proteins and myosin II-containing actin networks to ad-
hesion sites (Vicente-Manzanares and Horwitz, 2011). Upon cell
attachment, integrins bind to the underlying substrate and focal
complexes (FCs; also termed nascent adhesions) assemble at the
contact site of the cell with the ECM. The maturation of small
FCs (~100 nm in size) into large FAs (~1 µm) is driven by active
myosin II that enables further recruitment of adhesion-associa-
ted proteins with actin-binding or modulatory activity, such as
vinculin or paxillin, along polymerizing actin. The integrin-actin
connection is subsequently strengthened leading to formation of
stress fibers, antiparallel myosin II-containing actin bundles (Za-
mir and Geiger, 2001; Vicente-Manzanares and Horwitz, 2011).
A large number of studies implicate that the central cellular
function of ILK is to regulate adhesion maturation and to es-
tablish and maintain the integrin-actin linkage. Mammalian
cells lacking ILK display defects in actin reorganization and FA
maturation (Sakai et al., 2003b). ILK itself lacks actin-binding
properties and the precise molecular details of how ILK regulates
actin engagement at FAs are not clear. A possible adaptor linking
ILK to actin is parvin that was shown to bind actin through its
two in-tandem CH-domains (Olski et al., 2001) (Fig. 1). ILK has
also been reported to impact the activity of small GTPases such
as RhoA and Rac that modulate actin dynamics but the detailed
molecular mechanism of this regulation is not known (Boulter
et al., 2006; Kogata et al., 2009; Blumbach et al., 2010). Inte-
restingly, ILK also regulates the architecture and stability of the
microtubule network, which might have implications on intra-
cellular signaling as well as on the regulation of GTPase activity
(Wickström et al., 2010b).
During FA maturation, the connection between integrin ligands
and the actin cytoskeleton is strengthened and myosin II-de-
pendent actin stress fiber formation facilitates cell contraction
(Schiller and Fässler, 2013). ILK-deficient fibroblasts display large
FAs at the cell edges but absence of nascent FCs and fibrillar
adhesions (FBs), a specialized type of adhesion involved in ECM
remodeling (Stanchi et al., 2009; Radovanac et al., 2013) (Fig.
2A). It has been proposed that ILK collaborates with
α
-parvin to
segregate
α5β1
integrins from FAs, thus allowing the recruit-
ment of tensin and maturation of FBs (Stanchi et al., 2009). Fur-
thermore, the actin cytoskeleton of ILK-deficient fibroblasts is
disorganized and poorly linked to the abnormal FAs (Sakai et al.,
2003b). As a consequence, these cells are severely compromised
in their ability to generate traction forces and to exert force on
the underlying ECM (Radovanac et al., 2013) (Fig. 2B, C). Coll-
ectively these data indicate that ILK is important for adhesion
maturation by acting as an adaptor to establish and maintain
the integrin-actin linkage.
ILK is essential for ECM remodeling
During FA maturation fibronectin (FN)-bound integrins such as
α5β1
are segregated along the actin cytoskeleton. The subse-
quent generation of cellular forces and recruitment of additional
adaptor proteins such as tensin induces FB formation (Pankov
et al., 2000). The cellular force that is applied on FN leads to its
conformational changes and self-assembly, resulting in FN fibril-
logenesis (Zamir et al., 2000; Ohashi et al., 2002). FB maturation
is a prerequisite for subsequent FN fibrillogenesis. Although the
expression of FN in ILK-deficient fibroblasts is unaltered com-
pared to controls, FN fibrillogenesis is absent (Radovanac et al.,
2013) (Fig. 2D). As the affinity of integrin
α5β1
to its ligand FN
