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ILK, PINCH and parvin: the tIPP of integrin signalling
Kyle R. Legate, Eloi Montañez, Oliver Kudlacek and Reinhard Fässler Abstract | The ternary complex of integrin-linked kinase (ILK), PINCH and parvin functions as a signalling platform for integrins by interfacing with the actin cytoskeleton and many diverse signalling pathways. All these proteins have synergistic functions at focal adhesions, but recent work has indicated that these proteins might also have separate roles within a cell. They function as regulators of gene transcription or cell–cell adhesion.
Extracellular matrix The extracellular matrix (ECM) provides the structural which indicates that certain intracellular proteins might (ECM). A network of secreted framework for the formation of tissues and organs. The have key roles in the regulation of the function of β1 proteins and polysaccharides ECM binds to substrate-adhesion molecules on the sur- integrins. that surrounds all the face of cells and influences various intracellular signalling Three proteins that have emerged from these connective tissues and underlines all the epithelial and pathways that regulate survival, proliferation, polarity and studies as important regulators of integrin-mediated the endothelial sheets. It differentiation. An important family of adhesion mol- signalling are the integrin-linked kinase (ILK), and provides a physical support for ecules that bind to the ECM are the integrins. Integrins the adaptor proteins PINCH (particularly interesting tissues, as well as a sink for the are heterodimeric transmembrane molecules that consist Cys-His-rich protein) and parvin. These molecules storage, release and of α and β subunits, and they are composed of large form a heterotrimeric complex we refer to as the IPP presentation of growth factors. extracellular domains and relatively small cytoplasmic complex, which is named after its components in order Focal adhesion domains1. Integrins can switch between active and inac- of their discovery. Recent reports have provided a wealth A highly specialized cell- tive conformations. In the inactive state, integrins have a of data to expand the known functions of the IPP com- adhesion structure that low affinity for ligands. Intracellular signalling events such plex into almost every aspect of cell behaviour and fate. connects actin filaments to the ECM through integrins. as protein-kinase-C stimulation can prime the integrins, This review will provide an overview of our current Immature focal adhesions are which results in a conformational change that exposes the knowledge regarding the function of the IPP complex. known as focal complexes, and ligand-binding site (FIG. 1). Ligand binding activates signal- Interactions between the IPP components and numerous those that are formed through ling cascades that lead to the assembly of a multiprotein binding partners will be discussed to explain how the IPP interactions with fibronectin complex at the site of cell adhesion to the ECM. These complex functions both as an adaptor between integrins mature into structures known as fibrillar adhesions. events have two important impacts on the cell: they forge a and the actin cytoskeleton, and as a hub that regulates connection between the ECM and the actin cytoskeleton, several signalling pathways. Furthermore, this review and they alter the fluxes of many intracellular signalling will address the latest results in the ongoing controversy pathways. regarding the function of the putative kinase activity of Among the integrins, β1 integrin contributes to a large ILK. We will conclude by describing the results of in vivo number of integrin heterodimers whereas β3 integrin has studies in model organisms, which provide insights into an important adhesion role in platelets — an excellent the role of the IPP-mediated integrin-signalling func- 1 β β Department of Molecular system in which to study cell–ECM adhesion . 1 and 3 tions during development. Medicine, Max-Planck integrins are widely expressed, and studies on the func- Institute of Biochemistry, tion of β1 and β3 integrins have provided many general Identification, architecture and assembly of IPP Am Klopferspitz 18, insights into integrin-mediated adhesion. Deletion of the ILK was identified in 1996 in a yeast two-hybrid screen 82152 Martinsreid, highly conserved β1 integrin gene in different organisms for proteins that could bind to the cytoplasmic tail of Germany. β 7 e-mails: has been associated with defects in adhesion, prolifera- 1 integrin . The protein that was cloned contained three 2–6 [email protected]; tion, survival and polarity . However, although these domains (FIG. 2). The N-terminal domain contains [email protected]; experiments showed that integrins are important for three ankyrin repeats, which mediate protein–protein [email protected]; these processes, they provided few insights into how these interactions, and a putative fourth ankyrin repeat that [email protected] focal-adhe- doi:10.1038/nrm1789 processes are regulated. Deletions of certain lacks conserved residues. The C terminus shares sig- Published online sion molecules in different organisms display strikingly nificant sequence homology to Ser/Thr protein kinases. 21 December 2005 similar phenotypes to the β1-integrin-null phenotype, A putative pleckstrin homology (PH) domain is situated
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a Inactive state b Primed state c Active state Ankyrin repeat (bent form) (extended form) (ligand-bound form) A protein–protein-interaction module that consists of Extracellular space ECM approximately 30 amino acids. α β It was first identified in the Integrins yeast cell-cycle regulator Clustering Swi6/Cdc10 and the Cell D. melanogaster signalling membrane protein Notch, and it was named after the cytoskeletal Salt bridge Src protein ankyrin. This motif is Vinculin IPP FAK found in more than 1,700 Paxillin Vinculin different proteins. Paxillin Pleckstrin homology (PH) IPP PINCH domain Actin A phosphoinositide-binding IPP motif that is composed of PIPKlγ Talin FAK approximately 100 amino ILK Parvin acids and is involved in Cytoplasm receiving and transmitting signals at the interface between the membrane and Intracellular signalling Gene-expression changes • Assembly of the actin cytoskeleton cytosol. •Activation of signalling pathways
Senescent Cells that are undergoing a Figure 1 | Biogenesis of focal adhesions. a | Many integrins that are not bound to the extracellular matrix (ECM) are permanent form of cell-cycle present on the cell surface in an inactive conformation, which is characterized by ‘bent’ extracellular domains that mask arrest that was originally the ECM-binding pocket. This conformation is stabilized by interactions between integrin transmembrane domains, described for post-proliferative membrane-proximal extracellular domains and a salt bridge between the cytoplasmic domains. b | When talin is recruited primary cells in culture. to the plasma membrane and activated in association with phosphatidylinositol phosphate kinase type-Iγ (PIPKIγ), it Senescence can be induced by binds to the cytoplasmic tail of β integrins120. This interaction separates the cytoplasmic domains and induces the DNA damage, oxidative stress, chemotherapy and excess integrins to adopt the ‘primed’ conformation. c | The integrin extracellular domains extend and unmask the ligand-binding mitogenic stimuli, and is site, allowing the integrin to bind specific ECM molecules. The separated integrin cytoplasmic domains and talin form a controlled by the tumour platform for the recruitment of other focal-adhesion proteins. Integrin-linked kinase (ILK), and isoforms of particularly suppressor proteins, p53 and interesting Cys-His-rich protein (PINCH) and parvin form the IPP complex (FIG. 2) in the cytoplasm, and this complex is retioblastoma protein. recruited to focal adhesions through interactions with other factors, such as paxillin. Other proteins such as vinculin and focal adhesion kinase (FAK) are recruited to the nascent focal complex in a sequential manner121. The maturation of focal LIM domain adhesions involves clustering of active, ligand-bound integrins and the assembly of a multiprotein complex that is capable A tandem cysteine-rich of linking integrins to the actin cytoskeleton and communicating with signalling pathways. Zn2+-finger motif that mediates protein–protein interactions. It was originally identified in the transcription factors LIN11, ISL1 and MEC3. between these two domains and partially overlaps them. Parvins — a family of proteins that consists of Cell-culture experiments indicated that the physiological actopaxin/CH-ILKBP/α-parvin, affixin/β-parvin and Calponin homology (CH) ligand of the ILK PH domain is phosphatidylinositol- γ-parvin — bind to ILK through the second of two domain 8,9 calponin homology (CH) domains13,14,20,21 A relatively small motif that is 3,4,5-trisphosphate (PtdIns(3,4,5)P3) . ILK is the central . The interaction present in several cytoskeletal component of the IPP complex; it binds PINCH proteins between α-parvin and ILK is partially dependent on proteins and functions as an (REF. 22) through the N-terminal ankyrin-repeat domain and PtdIns(3,4,5)P3 and on phosphorylation of actin-binding domain, parvins through the kinase domain. It also links the com- α-parvin by CDC2 and mitogen-activated protein kinase especially when they are plex to the cytoplasmic tails of β1 and β3 integrins7,10–14, (MAPK)23–25. It is unclear whether the other parvins are presented in tandem. but it is not known whether it binds to other β integrins. regulated in a similar manner, although β-parvin can Small inhibitory RNA A single ILK isoform has been identified in all species also be phosphorylated14 (see also TABLE 1). α- and (siRNA). Double-stranded RNA discussed in the following sections. β-parvin have overlapping expression patterns in vari- molecules of 21–25 PINCH1 (also known as LIMS1) was originally iden- ous tissues, but the expression of γ-parvin is restricted nucleotides in length that are senescent 26 used as a viral defence tified in 1994 as a marker for erythrocytes, and to the haematopoietic system . It is therefore possible mechanism and an endogenous was shown to bind to ILK in 1999 (REFS 15,16). A second that different IPP complexes can assemble within the gene-silencing mechanism from isoform, PINCH2 (also known as LIMS2), was predicted same cell. This idea was further supported by a recent plants to humans. by sequence-database mining and was subsequently study showing that small inhibitory RNA (siRNA)-medi- characterized17,18. PINCH1 and PINCH2 are adaptor ated knockdown of α-parvin in HeLa cells stimulated Lamellipodium Dynamic actin-mediated cell- proteins that consist of five LIM domains and tandem Rac activity and lamellipodium formation. These find- 17,19 membrane protrusions at the nuclear localization signals , and both isoforms bind ings indicate that α-parvin can function as a nega- front of spreading and to ILK through the N-terminal LIM domain in a mutu- tive regulator of Rac in cells that express both α- and migrating cells. They are ally exclusive manner10,18. There is extensive overlap in β-parvin27. Overexpression of β-parvin in HeLa cells essential for cell motility, phagocytosis and the the expression patterns of PINCH1 and PINCH2 in adult promoted apoptosis, perhaps because of competition development of substrate tissues, and both genes are expressed in smooth-muscle with α-parvin for binding to ILK. Monitoring the adhesions. layers of the developing embryo19. expression levels of parvins in cells that are exposed to
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Extracellular space cells, respectively (C. Grashoff and R.F., unpublished data), but only a complex that consists of full-length Growth factors ECM proteins efficiently assembles into focal adhesions in vertebrate cell culture20,28,32. Although the full-length IPP complex is required for focal-adhesion assembly in RTKs Integrins vertebrates, other factors are also required to assemble Cell membrane the IPP complex into focal adhesions, including the PtdIns(3,4,5)P adaptor molecule paxillin, and possibly MIG2/kind- 3 PDK1 AKT/ ILKAP PKB lin-2 (REFS 28,33). Both of these proteins directly bind Kindlin-2 NCK2 PH Kinase Migfilin to the IPP complex through the kinase domain of 33,34 Paxillin ANK1 ANK2 ANK3 ANK4 ILK . Many more proteins might also be involved Filamin LIM5 LIM4 LIM3 LIM2 LIM1 ILK HIC5 Vinculin at this stage of IPP-complex assembly and targeting. RSU1 Tβ4 CH2 α PINCH TESK1 -actinin Interestingly, in Drosophila melanogaster embryos, α-PIX the PINCH homologue, steamer duck (STCK), is not Cytoplasm CH1 required to localize ILK to sites of cell adhesion35. Parvin Actin IPP-binding partners There are many molecules that have been shown Figure 2 | Anatomy of the IPP complex and its binding partners. Integrin-linked to interact with the IPP complex36. The function of kinase (ILK) consists of three domains, N-terminal ankyrin (ANK) repeats, a plekstrin the IPP complex as a signalling platform is achieved homology (PH) domain and a C-terminal kinase domain. ANK1 binds to the LIM1 domain of particularly interesting Cys-His-rich protein (PINCH) isoforms as well as to the ILK- through its direct interaction with factors that func- associated phosphatase (ILKAP). The PH domain probably binds to phosphatidylinositol- tion as upstream regulators of many different signal- ling pathways (FIG. 3). This section will summarize the 3,4,5-trisphosphate (PtdIns(3,4,5)P3). The kinase domain of ILK binds to parvins, paxillin, MIG2/kindlin-2, the cytoplasmic tails of β integrins, the kinase substrate AKT/PKB known binding partners of the IPP complex and will (protein kinase B) and the kinase phosphatidylinositol-3-kinase-dependent kinase-1 provide examples where signalling specificity may be (PDK1). PINCH isoforms, which contain five LIM domains, bind to receptor tyrosine achieved through differential binding of molecules to kinases (RTKs) through the Src-homology-2 (SH2)–SH3 adaptor NCK2, thereby coupling PINCH and parvin isoforms. growth-factor signalling to integrin signalling. PINCH1 binds to Ras suppressor-1 (RSU1) β β and thymosin- 4 (T 4) to influence Jun N-terminal kinase (JNK) signalling and cell ILK-interacting partners. As mentioned above, verte- α β migration/survival, respectively. - and -parvins can bind to F-actin directly, as well as brate ILK can bind directly to the cytoplasmic tails of indirectly through binding to paxillin or HIC5 (α-parvin only) or α-actinin (β-parvin only). β β 7,11,12 α-Parvin also binds to the Ser/Thr kinase testicular protein kinase-1 (TESK1), whereas 1 and 3 integrins , and it is indirectly connected β-parvin binds to the guanine nucleotide-exchange factor α-PIX, which influences actin to the actin cytoskeleton through its interaction with remodelling through the GTPases Rac and Cdc42. Interactions with integrins and the parvins (see below). Interactions with the cytoskeleton cytoskeleton also occur through a MIG2/kindlin-2–migfilin–filamin complex. can also occur through the LD motif and LIM-domain adaptor protein paxillin, which binds to F-actin through interactions with α-parvin and the actin-binding adaptor molecule vinculin20,37,38. Paxillin binds to ILK through a specific conditions, and immunoprecipitation of discrete IPP paxillin-binding site (PBS) within the kinase domain of complexes will determine whether this data is biologically ILK33. Furthermore, Caenorhabditis elegans ILK binds to relevant, and will enable the study of the mechanisms that UNC-112, the orthologue of vertebrate MIG2/kindlin-2 regulate the expression of the different parvin isoforms. (REF. 34). In vertebrates, kindlin-1 binds to the cytoplas- mic domains of β1 and β3 integrins39, and its loss causes IPP-complex assembly and stability. The assembly of Kindler syndrome40. MIG2/kindlin-2 binds to migfilin, the IPP-complex precedes cell adhesion, which indi- which in turn binds to filamin — an adaptor protein that cates that these complexes first form in the cytosol, interacts with several molecules, including filamentous 28 41,42 RNA interference independently of adhesion signals . The stability (F)-actin and integrins — and provides another con- (RNAi). A method to silence of the individual IPP components is dependent on nection between ILK and the actin cytoskeleton. specific gene expression by complex formation, because RNA interference (RNAi)- introducing double-stranded mediated depletion of one member of the complex Binding partners of the PINCH isoforms. The signalling RNA into the cell that matches the nucleotide sequence of the results in degradation of the other components by a specificity of the IPP complexes depends on the pres- 29 targeted mRNA. proteasome-mediated process . This complicates the ence of the different PINCH or parvin isoforms. When interpretation of results from genetic-deletion or RNAi- PINCH2 is overexpressed in a basal PINCH1-expressing LD motif depletion studies because the defects that are associ- background, it competes with PINCH1 for binding to Leucine-rich protein-binding ated with deletion of one component might be due to ILK but cannot transduce integrin-mediated signals that sequences with the consensus 18 sequence LDXLLXXL. diminished levels of other components. Recent work is control cell spreading and migration . Also, although beginning to address the specific roles of IPP-complex the expression of a chimeric PINCH that consists of the Kindler syndrome members, taking into account their mutual stabilization PINCH1 LIM domains and the PINCH2 C-terminal tail An inheritable epidermal and degradation30. Degradation of ILK or the PINCH cannot restore spreading in PINCH1-knockdown HeLa defect that is characterized by 30 blistering, abnormal proteins can be prevented by expression of the PINCH1 cells , expression of full-length PINCH2 in a Pinch1- 31 pigmentation, fragile skin and or PINCH2 LIM1 domain in Pinch1-deficient cells or null background completely restores the adhesion and increased cancer risk. expression of the ILK ankyrin repeats in Ilk-deficient spreading defects of Pinch1-null fibroblasts31. Therefore,
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Table 1 | ILK substrates bind to PINCH1 (REF. 52). These results indicate that the interaction between PINCH1 and NCK2 might not be Substrate Biochemistry Cell culture In vivo References essential in vivo. ILK 7 β1 integrin 7 Parvins have overlapping binding partners. α-Parvin binds to F-actin directly, as well as indirectly through an β3 integrin 11 interaction with paxillin20. It is not yet known whether AKT/PKB , ×* ‡, ק 71,74–76,111 ILK and a parvin isoform bind to the same molecule GSK3β , ×* ‡, ק 71,74,75,111,124 of paxillin, or if two paxillin molecules bind to a single IPP complex. HIC5, a paxillin-related protein, also binds Myosin light chain 70 to α-parvin20. HIC5 binds to many of the proteins that Myelin basic protein 7 paxillin binds to, but it also shuttles to the nucleus, where β-parvin 14 it modulates the expression of several genes53,54. In addi- α MYPT1 125 tion, -parvin specifically binds to TESK1, a Ser/Thr kinase that phosphorylates the actin-regulating protein α -NAC 126 cofilin55. β-Parvin binds to F-actin, but can also bind to CPI17 127,128 the actin-crosslinking protein α-actinin56 and the gua- α 57 PHI1 127,128 nine nucleotide-exchange factor -PIX . It therefore provides a connection between the IPP complex and –/– ‡ § –/– *Cell culture using Ilk fibroblasts. Ilk overexpressed in mammary epithelium. Ilk the actin-regulating GTPases Rac1 and Cdc42 (FIG. 3). chondrocytes. CPI17, protein-kinase-C-dependent phosphatase inhibitor of 17 kDa; α GSK3β, glycogen-synthase kinase-3β; ILK, integrin-linked kinase; MYPT1, myosin-light-chain- -PIX, in turn, binds to PAK1 (REF. 58), a Rac1/Cdc42 phosphatase target subunit-1; α-NAC, nascent-polypeptide-associated complex and effector that regulates cytoskeletal dynamics through the coactivator-α; PHI1, phosphatase-holoenzyme inhibitor-1; PKB, protein kinase B. LIM-kinase–ADF–cofilin pathway (where ADF stands for actin-depolymerizing factor) 59. Furthermore, α-PIX binds to the protease subunit calpain-4 (REF. 60). Calpain-4 accessory proteins that differentially bind to PINCH has been shown to cleave talin, and this cleavage is the isoforms are probably responsible for transducing the rate-limiting step in the disassembly of focal adhesions61. signals that control cell spreading. The functions of parvin-binding partners indicate that Functional differences between PINCH1 and the parvins have a role in the regulation of actin dynam- PINCH2 might arise from differential binding of the ics and focal-adhesion turnover. β-Parvin also binds to Ras-suppressor protein RSU1. RSU1 has been shown to the membrane-repair protein dysferlin at the sarcolemma function as a negative regulator of growth-factor-induced of skeletal muscle; an observation that reveals a potential Jun N-terminal kinase (JNK) activation43–46 (FIG. 3). RSU1 role for β-parvin in membrane repair62. Binding partners is also known to interact with the LIM5 domain of for the less-well-characterized γ-parvin have yet to be Thymosins PINCH1 in D. melanogaster45 and vertebrates46, but this identified. A large family of small peptides interaction is specific for PINCH1. PINCH2 does not that was originally identified in the thymus, but is also found in bind RSU1, because the sequence of the LIM5 domain Other roles of the IPP-complex proteins? Deletion of one 19 many tissues. They are divided is different . component of the IPP complex results in degradation into three main groups: α-, β-, Thymosin-β4 binds to LIM domains -4 and -5 of of the other components, but this degradation is not and γ-thymosins. β-Thymosins PINCH1, upregulates ILK activity and positively influ- complete29,63, which indicates that the individual com- bind globular actin to maintain 47 a pool of actin monomers in ences migration and survival of cardiac cells . Whether ponents might have extra roles outside the complex. the cell. PINCH2 and thymosin-β4 interact is not known, but PINCH1 shuttles between the nucleus and the cytoplasm a conditional knockout of Pinch1 in murine ventricu- in Schwann cells, and it has been found in the nucleus Calpains lar cardiomyocytes shows no discernable phenotype48, of the mouse primitive endoderm, and muscle cells of A superfamily of multimeric indicating that PINCH2, which is also expressed in the C. elegans. These observations indicate that PINCH1 Ca2+-dependent cysteine 19 proteases that is implicated in heart , might compensate for the loss of PINCH1. might have a role in gene regulation or signalling between 17,63,64 various cellular processes such PINCH1 also binds to the receptor-tyrosine-kinase the cytoplasm and the nucleus . β-Parvin localizes to as proliferation, differentiation adaptor protein NCK2 in vitro through a LIM4–SH3- the sarcolemma in skeletal muscle, where it might have a and apoptosis. Deregulation of domain (Src-homology-3 domain) interaction49,50. role in Ca2+-dependent membrane repair, together with calpain activity has been 62 implicated in various Mutations in vertebrate PINCH1 that disrupt dysferlin . ILK redistributes to cell–cell contacts in dif- pathological conditions. PINCH1–NCK2 binding reduce the amount of PINCH1 ferentiating keratinocytes, where it participates in the that is found in focal adhesions49, but the relevance of early steps of adherens-junction formation65,66. Sarcolemma a PINCH1–NCK2 interaction is not clear. There is The plasma membrane that no evidence for such an interaction in C. elegans or ILK catalytic activity encloses striated muscle fibres. D. melanogaster. Mice that carry a Nck1 or Nck2 genetic ILK has been shown to function as an adaptor protein, Adherens junction deletion, are phenotypically normal, whereas mice that but recent reports have indicated that ILK also has cata- A highly specialized cell–cell- lack both proteins die during embryogenesis. The migra- lytic activity. The kinase domain of ILK shows significant adhesion complex that tion and cytoskeletal defects of Nck1–/– Nck2–/– fibrob- homology to Ser/Thr protein kinases, with the exception contains cadherins and 51 catenins, and which is lasts are rescued when NCK1 is reintroduced, although of sequences within the catalytic loop and the conserved 67,68 connected to cytoplasmic actin the PINCH1–NCK interaction is specific for NCK2. DXG motif . These differences from the canoni- filaments. Furthermore, NCK1 has been demonstrated not to cal kinase sequence are difficult to reconcile with the
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Stimulus ECM Growth factors Extracellular space
Membrane Integrins RTKs receptor PtdIns(3,4,5)P3 Cell membrane
PI3K P ILKAP NCK2 PTEN Cytoplasm PINCH ILK P Rac1 RSU1 P P P P α-PIX MAP4K Parvin α-NAC GSK3β AKT/PKB Rac1 Cdc42 MLC
JNK TAU1 mTOR BAD p70S6K Caspase-3 Nucleus Caspase-9 Actin
Transcription AP-1β-catenin CREB SNAIL HIF1 NF-κB factors LEF/TCF
Target MMP9Cyclin D1 E-cadherin VEGF iNOS COX2 genes Myc expression
Cell Tissue Spreading and response Invasion morphogenesis Proliferation EMT Angiogenesis Survival migration Motility
Figure 3 | Signalling through the IPP complex. The integrin-linked kinase (ILK), particularly interesting Cys-His-rich protein (PINCH), parvin (IPP) complex has been implicated in the control of signalling pathways through both phosphorylation of downstream targets (most notably AKT/protein kinase B (PKB) and glycogen-synthase kinase-3β (GSK3β) and binding to upstream effectors of the Jun N-terminal kinase (JNK) signalling pathway and regulators of small- molecular-weight GTPases. The activity of the complex is upregulated by phosphatidylinositol 3-kinase (PI3K) and downregulated by the phosphatases ILK-associated protein (ILKAP) and phosphatase and tensin homologue deleted in chromosome 10 (PTEN). Growth-factor-mediated signalling through receptor tyrosine kinases (RTKs) might be transduced to the IPP complex through the receptor-tyrosine-kinase adaptor protein NCK2. The signalling pathways that are shown are limited to those that have been experimentally described to be influenced by the IPP complex. AP-1, activator protein-1; BAD, BCL2-antagonist of cell death; COX2, cyclooxygenase-2; CREB, cAMP-response-element-binding protein; ECM, extracellular matrix; HIF1, hypoxia-inducible factor-1; iNOS, inducible nitric-oxide synthase; LEF/TCF, lymphoid enhancer factor/T-cell factor; MAP4K, mitogen-activated-protein-kinase-kinase-kinase kinase; MLC, myosin light chain; MMP9, matrix metalloprotease-9; mTOR, mammalian target of rapamycin; α-NAC, nascent polypeptide- associated complex and co-activator-α; NF-kB, nuclear factor-kB; p70S6K, p70 ribosomal S6 kinase; α-PIX, activating α PAK-interactive exchange factor- ; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; RSU1, Ras suppressor-1; VEGF, vascular endothelial growth factor.
observed kinase activity, because ILK lacks an obvious ILK model substrates. The most widely used readouts catalytic base and Mg2+-chelating residues. Furthermore, for ILK activity are the phosphorylation of GSK3β and the sequence of the ILK catalytic domain is divergent AKT/PKB (protein kinase B), which regulate many dif- across different species69 (FIG. 4). Such a tolerance for ferent signalling pathways (FIG. 3). AKT/PKB activation mutation indicates that if ILK possesses kinase activity, requires phosphorylation of Thr at position 308 by phos- it is probably unnecessary for its function. Nevertheless, phatidylinositol 3-kinase (PI3K)-dependent kinase-1 recombinant, purified ILK has been shown to phospho- (PDK1) and Ser at position 473 by PDK2 (which is rylate several substrates in vitro7,70,71 (TABLE 1), and it is more appropriately known as hydrophobic-motif kinase possible that ILK retains residual kinase activity that (HMK)). The identification of PDK1 is unambiguous, is readily detected in vitro. Because kinetic rates of the but the identity of PDK2/HMK continues to be debated. kinase reaction have not been reported and the evi- ILK, among other candidates (BOX 1), has been proposed dence for in vivo kinase activity is weak, it is not known to function as a HMK and this idea is further supported whether ILK possesses sufficient activity to function as by immunoprecipitation assays showing that ILK directly a physiologically relevant kinase in vivo. binds to AKT/PKB71.
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I II III IV V Perturbation of ILK function. Despite evidence in favour L Consensus IGXGXXGXL AXKXΦ E Φ ΦΦΦΦΦ of ILK having a kinase function, genetic experiments V TAK1 VGRGAFGVV AIKQI E I VCLVM have failed to demonstrate a crucial role for the kinase C. elegans ILK IAESHSGEL VARIL E I LVIIS activity of ILK in several cell types. Alterations in the D. melanogaster ILK LSVTPSGET VAKIL E I LVTIS phosphorylation status of AKT/PKB or GSK3β have X. laevis ILK LNDNHSGEL IIKVL E V PVLIT not been observed in Ilk–/– fibroblasts74. Furthermore, M. musculus ILK LNENHSGEL VVKVL E V LVIIS the phosphorylation status of putative ILK substrates VI VII VIII XI IX S Φ is unchanged in chondrocytes or keratinocytes that do Consensus HRDLKXXN DFG APE DXWAXG R TAK1 HRDLKPPN DFG APE DVFSWGI R not express ILK (K. Lorenz and R.F., unpublished data, C. elegans ILK LRFYLLSK --- SPE DMWSFAI R and REF. 75). By contrast, deletion of Ilk in immortalized D. melanogaster ILK PTYHLNSH --- SPE DMWSFAI R macrophages results in diminished phosphorylation X. laevis ILK PRHYLNSR --- APE DMASFAV R β M. musculus ILK PRHALNSR --- APE DMWSFAV R of both AKT/PKB and GSK3 , which is reversed by transfection of wild-type ILK, but not a kinase-deficient Figure 4 | Divergence of the kinase domain of ILK. ILK mutant76. This discrepancy might reflect cell-type- Alignment of integrin-linked kinases (ILKs) from several species as well as the related kinase transforming-growth- specific differences, or it might reveal an increased factor-β-activated kinase-1 (TAK1) with conserved dependence on the kinase activity of ILK as a result of subdomains of protein kinases, numbered according to immortalization. Hanks et al.67,68. The subdomain X is poorly conserved and Small-molecule inhibitors that were designed to contains no consensus amino acids, so it was not included. specifically inhibit the kinase activity of ILK, prevent Conserved domains that are significantly altered in ILK are phosphorylation of AKT/PKB and GSK3β when intro- shown in red. One of three Gly residues in subdomain I is duced in ILK-overexpressing cell lines9,71,77. Further conserved in all ILK proteins. A Lys in subdomain II that is characterization of one of these inhibitors, KP-392, 82 required for the phosphotransfer reaction is also revealed that it disrupts the interaction between ILK and conserved as a basic residue. However, the catalytic Asp both α-parvin and paxillin, and blocks the accumulation residue in subdomain VI and the DFG sequence in α 22 subdomain VII that is required to align the γ-phosphate of of -parvin and paxillin into focal adhesions . ATP, are both missing. The conserved Lys, which neutralizes Similarly, several different point mutations in the charge on the γ-phosphate of ATP, and the conserved ILK, which were designed to abolish catalytic activ- Asn, which chelates the secondary Mg2+ are also missing. All ity, disrupt protein–protein interactions and might active protein kinases contain a conserved Asp in regions VI function by preventing the assembly of functional IPP and VII except for the haspins — a unique family of histone complexes. Overexpression of wild-type ILK in cell- kinases that have a role in mitosis122, and lack the conserved culture experiments results in increased phosphor- Asp in subdomain VII but contain the catalytic Asp in ylation of AKT/PKB and GSK3β in many cell types. 123 subdomain VI . ILK is the only protein kinase that is known Conversely, overexpression of a dominant-negative to be missing both residues. C. elegans, Caenorhabditis mutant of ILK (Glu359Lys) reduces phosphorylation elegans; D. melanogaster, Drosophila melanogaster; β M. musculus, Mus musculus; X. laevis, Xenopus laevis. of AKT/PKB and GSK3 . Although the Glu359Lys mutation was reported to impair the kinase activity of ILK8,78, other studies conclude that this mutation Regulation of ILK activity. ILK-dependent phosphoryla- has no effect on the kinase activity of ILK in vitro14,79. tion is regulated in a PI3K-dependent manner. Inhibitors Instead, the Glu359Lys mutation disrupts the inter- of PI3K activity reduce ILK activity in cell-lysate action between ILK and both α-parvin and paxillin, immunoprecipitates and impair the phosphorylation which results in a failure to assemble ILK into focal of putative ILK substrates in cell culture, whereas over- adhesions14,79. Several inactivating point mutations in expression of the PI3K catalytic subunit or the addition the kinase domain of ILK have been used to dissect the
of PtdIns(3,4,5)P3 increase ILK-dependent kinase activ- kinase function, but these mutations (Arg211Ala, ity in cell culture and in vitro, respectively 8. The expres- Ser343Ala, and Lys220Ala) also disrupt ILK–protein sion of thymosin-β4 in cardiomyocytes also increases interactions, which makes it impossible to derive inter- the activity of ILK, as measured by phosphorylation of pretations that are based solely on the lack of kinase AKT/PKB on Ser47347. Conversely, the catalytic activ- activity. The Arg211Ala mutation results in deficient ity of ILK is negatively regulated by the phosphatase binding to α-parvin22, and this interaction is required ILK-associated protein (ILKAP). ILKAP reduces the to target ILK to focal adhesions28. The Ser343Ala kinase activity of ILK in vitro and the phosphorylation mutation abolishes binding to AKT/PKB, but this of GSK3β in vivo, but the phosphorylation of AKT/PKB mutant protein localizes to focal adhesions71,80. The remains unaffected72,73. This demonstrates that the Lys220Ala mutant fails to bind to β-parvin81. Another mechanisms of GSK3β and AKT/PKB activation are mutation at this site, Lys220Met binds to β-parvin but different, despite the fact that both proteins have been shows no catalytic activity14, but a second activating Dominant negative described as ILK substrates. The mechanism by which mutation, Ser343Asp, reverses this defect69 despite a Introduction of an inactive ILKAP reduces ILK activity towards certain substrates requirement of Lys220 for protein-kinase activity68,82. mutant gene product, which is currently unknown. It has also been reported that The Phe438Ala mutant, which does not assemble interferes with the functional 2 endogenous gene product, ILK can autophosphorylate in vitro , but the function into focal adhesions, has been proposed not to bind perhaps by competing for of autophosphorylation of ILK in vivo, if present at all, to MIG2/kindlin-2 (REF. 28). Furthermore, muta- available accessory factors. remains to be investigated. tions in ILK that show dominant-negative effects in
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Box 1 | Kinase candidates responsible for AKT/PKB phosphorylation The role of the IPP complex in invertebrates To study the contribution of the IPP complex to devel- AKT/PKB (protein kinase B) must be phosphorylated on Thr308 and Ser473 for full opment, genetic deletion of most components has been activity. The kinases that are involved in this process are called phosphatidylinositol achieved in C. elegans, D. melanogaster and mice (see 3-kinase (PI3K)-dependent kinase-1 (PDK1) and hydrophobic-motif kinase (HMK), respectively. While PDK1 has been identified, the identity of the HMK is not clear. Supplementary information S1 (table)). The phenotypes Several candidates have been proposed that do not hold up to scrutiny, including caused by these deletions confirm a role for the IPP mitogen-activated-protein-kinase-activated kinase-2 (MAPKAPK2), PDK1 bound to a complex as an adaptor complex between the ECM and fragment of protein-kinase-C-related kinase-2 (PRK2), and AKT/PKB itself. Integrin- the actin cytoskeleton, but insights into the signalling linked kinase (ILK) has also been considered as a candidate for HMK, but there are other function are not as forthcoming, and a role for the ILK candidates that are equally plausible. Two members of the PI3K-related kinase (PIKK) catalytic activity has not been established. Differences family, the DNA-dependent protein kinase (DNA-PK)112 and the mammalian target of in phenotypes among IPP components, particularly in rapamycin (mTOR)–rapamycin-insensitive-companion-of-mTOR (rictor) complex113,114 mice, reveal that, in addition to a common function as have properties that are consistent with them having AKT/PKB-kinase activity. Each part of the IPP complex, each component has discrete candidate responds positively to insulin treatment and both depend on PI3K activity. functions. Small interfering RNA (siRNA) knockdown of DNA-PK, mTOR or rictor results in a decrease in AKT/PKB phosphorylation on Ser473. Importantly, both DNA-PK and Invertebrates represent relatively simple systems to mTOR–rictor can phosphorylate AKT/PKB Ser473 in vitro, although only mTOR–rictor study integrin-mediated functions. C. elegans has one β β α α has been tested on full-length AKT/PKB. A third member of the PIKK family, the gene integrin ( PAT-3) and two integrin ( PAT-2 and mutated in ataxia telangiectasia (ATM)115, also meets most of the requirements for HMK, αINA-1) subunits; D. melanogaster has two β integrin except that it cannot phosphorylate Ser473 in vitro. However, cell lines that are derived (βPS and βv) and five α integrin (αPS1–5) subunits. from ataxia telangiectasia patients lack detectable AKT/PKB-Ser473 kinase activity, Both organisms have a single orthologue of ILK, PINCH which can be restored after transfection with a plasmid encoding the ATM gene and parvin, which allows straightforward analysis of product. In addition, mice that carry deletions of ATM or AKT/PKB show phenotypic deletion phenotypes. C. elegans has a second PINCH1- 116–118 similarities . These data indicate that the HMK activity is mediated through ATM. related gene but this shows only 34% identity with Whether any of these candidates is the bona fide AKT/PKB HMK, or if they can all PINCH1 and contains a putative endoplasmic reticulum function as a HMK under specific circumstances, has yet to be determined. (ER) targeting sequence.
Studies in C. elegans. In C. elegans, genetic deletion of β 34 Ataxia telangiectasia cell culture (Glu359Lys or Lys220Met), or abolish the pat-3 (REF. 3), pat-4 (which encodes the ILK orthologue) , 17 (ATM). Autosomal recessive activity of the protein kinase Raf at non-permissive unc-97 (which encodes the PINCH orthologue) or hereditary disease associated temperatures (Pro358Ser), completely rescue the pat-6 (which encodes the parvin orthologue)87 all pro- with DNA-repair defects and phenotype of the ILK loss-of-function mutations in duce mutants with a similar PAT phenotype. Although a caused by mutations in the β ATM (ataxia telangiectasia) C. elegans (only Glu359Lys was tested) and D. mela- direct physical interaction between PAT-3 and PAT-4 gene. It is characterized by nogaster (all three ILK mutants). This indicates that has not been reported, deletion of βpat-3 or the ECM progressive cerebellar ataxia, kinase activity is dispensable for ILK function in component unc-52 (which encodes the orthologue of dilation of blood vessels in the these organisms34,83. However, residual kinase activity perlecan) results in mislocalization of PAT-4, which skin and eyes, chromosomal might be sufficient to completely restore a wild-type indicates that PAT-4 requires integrins and ECM to aberrations, immune 34 dysfunction and an increased phenotype. localize to dense bodies . Recruitment of PAT-4 to dense risk of cancer malignancy, RNAi studies in transformed cells demonstrate bodies, and the correct organization of βPAT-3 at the cell particularly leukaemia and that knockdown of ILK, PINCH1 or α-parvin protein membrane also depend on UNC-112 (the orthologue of lymphoma. expression correlates with reduced phosphorylation MIG2/kindlin-2)34,88. However, there is no evidence that (REFS 29,76,84–86) β PAT phenotype of AKT/PKB Ser473 . Furthermore, PAT-4 regulates the GSK3 signalling pathway in this A broad phenotypic class of AKT/PKB Thr308 phosphorylation is also reduced organism, as the defects that arise from deletion of pat-4 lethal mutations that affect in the absence of PINCH1 (REF.29). AKT/PKB does are distinct from the phenotype of the GSK-3β mutant in muscle formation in C. elegans. not localize to the plasma membrane in α-parvin- C. elegans34. A novel Zn2+-finger protein that is unique Mutations that cause a PAT depleted HeLa cells, despite the presence of ILK in to C. elegans, UNC-98, binds to UNC-97 at M-lines and (paralyzed and arrested α 89 elongation at twofold) focal adhesions, which demonstrates that -parvin is dense bodies . Both proteins are also detected in the phenotype, affect either required for the correct targeting of AKT/PKB before nucleus, which indicates that they might be involved in components of the attachment activation84. However, if AKT/PKB is constitutively gene regulation17,89. complex or essential targeted to the membrane, phosphorylation of Ser473 components within the 29,84 sarcomere. does not depend on ILK . It should be noted that Studies in D. melanogaster. In D. melanogaster, deletion of the increased apoptosis that results from knockdown βPS, the orthologue of the vertebrate β1 integrin subunit2, Dense bodies/M-lines of ILK or PINCH1 is not reversed on restoration of or loss of function of ILK83 or the PINCH orthologue35, Focal-adhesion-like muscle- AKT/PKB phosphorylation, which indicates that these result in striking embryonic muscle-attachment defects. attachment structures in molecules regulate numerous survival pathways. βPS and PINCH-deficient flies also show dorsal-closure C. elegans. Dense bodies 2,45 anchor actin filaments to the Taken together, the data from mutagenesis, small- defects, which indicates that cell migration is impaired . plasma membrane, and molecule inhibition and RNAi all indicate a model Adult chimeric flies display a blister phenotype in wing M-lines attach myosin whereby ILK activates AKT/PKB indirectly by facili- regions that lack these proteins. These phenotypes are filaments to the plasma tating the translocation of AKT/PKB to the plasma consistent with the loss of cell adhesion to the ECM. membrane. Both structures are α β essential for the contractility membrane in an -parvin-dependent manner. Once However, PS mutants show adhesion defects that result and the maintenance of the AKT/PKB is located at the plasma membrane, it can be from the detachment of the cell membrane from the ECM, muscles. phosphorylated by other kinases. whereas the ILK and PINCH mutants are characterized
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by the detachment of the actin cytoskeleton from the cell Pinch1–/– embryos arrest at the peri-implantation stage of membrane. Interestingly, ILK localizes normally to mus- embryonic development, but the Pinch1 –/– embryos die at cle-attachment sites in PINCH-mutant embryos, which the embryonic day (E)6.5–E7.5 whereas the β1 integrin–/– indicates that the PINCH loss-of-function phenotype is and Ilk–/– embryos die at E5.5–E6.5. α-Parvin–/– embryos not caused by the mislocalization of ILK35. The loss-of- develop the furthest, but die following implantation. The function mutations in PINCH affect the LIM domains temporal differences in embryonic lethality between 3–5, therefore it is possible that the LIM domains 1 and 2 β1 integrin–/– or Ilk–/– embryos and Pinch1–/– embryos are are still expressed, and are sufficient to retain correct ILK unlikely to be due to PINCH2 compensation, because localization in this organism. expression of PINCH2 is not detected until later stages Cytoskeletal reorganization and changes in cellular of development19. On the other hand, Pinch2 –/– mice are shape that are required during dorsal closure are coor- viable, probably as a result of PINCH1 compensation31. dinated by the JNK signalling pathway (for a review see It will be important to determine whether the relatively REF. 90). Therefore, the dorsal-closure defect in PINCH- long survival of α-parvin–/– embryos is the result of mutant flies might be explained, in part, by disrupted compensation by other members of the parvin family JNK signalling. PINCH might regulate JNK signalling and to investigate whether parvins might have a crucial through two distinct pathways. The first involves the function either before, or during, peri-implantation interaction between PINCH and RSU1 (FIG. 3). Both development. PINCH and RSU1 genetically interact with Misshapen, Embryonic lethality in Pinch1-null mice is associated a MAPK-kinase-kinase kinase (MAP4K) in the JNK with an increase in endodermal-cell apoptosis, which signalling pathway, which demonstrates that both pro- demonstrates that PINCH1 regulates cell survival63. This teins can negatively regulate this pathway45. The expres- observation is further supported by PINCH1-knockdown sion and/or stability of PINCH and RSU1 are mutually data in HeLa cells29. However, it remains to be determined dependent on one another, so the relative contributions whether AKT/PKB is involved in the PINCH1-mediated of PINCH and RSU1 in modulating JNK signalling have apoptotic pathway in embryos or if, as in HeLa cells, an not been determined. The second and more-speculative AKT/PKB-independent pathway exists. mechanism involves a PINCH–Dreadlocks–Misshapen Conditional knockouts of IPP members have been pathway. Dreadlocks, the D. melanogaster orthologue created to overcome the difficulties associated with of NCK2, interacts with Misshapen91. Although mam- early embryonic lethality. Mice that carry conditional malian PINCH1 binds to NCK2 in vitro52, an interaction deletions of β1 integrin or Ilk in chondrocytes show between Dreadlocks and PINCH has not been reported. skeletal defects75,96,97 and develop chondrodysplasia. In However, the affinity between mammalian NCK2 and culture, β1 integrin- and Ilk-mutant chondrocytes had PINCH1 is weak in vitro, and it is possible that it could defective spreading, abnormal F-actin distribution, and not be detected. impaired adhesion, although the Ilk-mutant chondro- The two main pathways that regulate survival and cytes had a less-severe adhesion defect. Therefore, dele- proliferation involve the phosphorylation of AKT/PKB tion of either protein impairs the connection between and the stabilization and nuclear translocation of the ECM and the actin cytoskeleton that is required β-catenin as a consequence of GSK3β phosphorylation for cell spreading. In addition, reduced proliferation of Dorsal closure (FIG. 3). However, there is no evidence that ILK is involved β1 integrin- or Ilk-deficient chondrocytes is associated A mid-stage developmental in these pathways in D. melanogaster as ILK loss-of-func- with a defect in the G1–S transition, but impaired cyto- process that involves the tion produces a different phenotype to loss of β-catenin kinesis in β1 integrin–/– chondrocytes reveals a defect in movement of lateral dorsal 34,83,92,93 epithelia towards the dorsal or AKT/PKB . Furthermore, overexpression of the G2–M transition. 83 midline. This process is ILK does not affect signalling through β-catenin . Conditional deletion of Ilk in endothelial cells results required for the sealing of in impaired vascular development and embryonic embryonic epidermis in Functions of IPP in mammalian systems lethality98. Integrin activation is reduced in the absence D. melanogaster. The biological functions of the members of the IPP of ILK, which indicates a migration defect, and there is Podocyte complex have been examined in several cell types. an increase in apoptosis concomitant with decreased Highly specialized epithelial Many IPP functions can be reconciled with a role AKT/PKB phosphorylation on Ser473. However, apop- cells that cover the outer for the IPP complex at focal adhesions. For example, tosis is reduced when ILK is reintroduced, but not when aspect of the glomerular the regulation of podocyte adhesion and spreading23, constitutively active AKT/PKB is introduced. This indi- basement membrane in the 47,85 kidney. Mature podocytes endothelial cell and cardiomyocyte migration , cates that, as in HeLa cells, an ILK-dependent, AKT/ 11,12 possess a highly branched platelet aggregation , neuronal spreading and out- PKB-independent pathway might operate in endothelial array of foot processes that are growth77,94, and leukocyte recruitment95 reveal a role for cells to regulate apoptosis. essential for glomerular the IPP complex in actin-cytoskeleton dynamics and Immortalized Ilk–/– macrophages also show decreased filtration. integrin activation. AKT/PKB phosphorylation on Ser47376. In addi- Chondrodysplasia tion, inhibition of ILK or PI3K activity, expression 4,5 A heterogeneous group of Studies in mice. Loss of expression of β1 integrin , of dominant-negative ILK and knockdown of ILK genetic disorders, which are ILK74, PINCH1 (REFS 48,63) or α-parvin (H. Chu and protein levels revealed a role for the IPP complex in characterized by abnormal R.F., unpublished data) all result in embryonic lethality. AKT/PKB- and GSK3β-mediated signalling in neu- skeletal morphogenesis 77,94,95,99,100 affecting the development and However, these mutants show subtle differences in their rons and leukocytes . However, fibroblasts –/– growth of most skeletal phenotypes, which indicates that distinct defects might and chondrocytes that are derived from Ilk mice do elements. underlie these phenotypes. β1 Integrin–/–, Ilk–/– and not show altered phosphorylation of AKT/PKB74,75,97.
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basement membrane due to a defect in the synthesis of Box 2 | Embryoid bodies as a model for early embryonic development laminin101,102. By contrast, Ilk–/– (BOX 2, figure, part c) a Wild type b Wild type and Pinch1–/– (BOX 2, figure, part d) embryoid bodies produce a basement membrane but have defects in epiblast polarization and cavity formation; these defects En are less severe in Pinch1–/– embryoid bodies, consistent Ep BM with the longer survival time in vivo63,74. The defects in epiblast polarization are accompanied by abnormal En localization of F-actin. These studies provide evidence Cav Ep that β1 integrins have separate functions from ILK and PINCH1 at the peri-implantation stage and highlight an important role for the IPP complex in organizing the actin cytoskeleton. Evidence also indicates that the IPP BM complex could regulate the actin cytoskeleton indirectly Cav through NCK2, which binds to the actin modulators WASP (Wiskott–Aldrich syndrome protein), PAK (p21-activated kinase) or DOCK180 (180-kDa protein –/– –/– c Ilk d Pinch1 downstream of CRK)103. In addition, Pinch1–/– embryoid bodies show abnormal cell–cell adhesion and impaired endoderm survival63. A reduction of ILK protein levels in Pinch1–/– embryoid bodies, similar to that observed in Pinch1-siRNA-treated HeLa cells29, provides a possible explanation for the phe- notypic similarities between Ilk–/– and Pinch1–/– embry- oid bodies63. However, the defects that are observed in Pinch1–/– embryoid bodies represent ILK-independent functions and indicate that the functional interdepend- ence between ILK and PINCH1 is not complete. This is supported by the observation that Ilk–/– cells still express low levels of PINCH1, and vice versa31, revealing the existence of different functional pools of these proteins: The low number of cells and the inaccessibility of peri-implantation embryos make a main pool in which ILK and PINCH1 are mutually analysis of the cellular and molecular events that take place during early development dependent, and a second, smaller pool in which these difficult. Embryoid bodies that have been derived from embryonic stem cells (ESCs) proteins function independently. have emerged as a valuable system for analyzing the processes that occur at the peri- Recent data have also shown a role for IPP members 119 implantation stage . Embryoid bodies are composed of an outer layer of primitive in the regulation of cell polarity and cell–cell con- endodermal (En) cells, which is underlined by a basement membrane (BM), a polarized tacts63,74,104. Cell–cell adhesion defects in Pinch1–/– embry- epiblast (Ep) layer (a primitive ectoderm that will differentiate into the three germinal layers) and a central cavity (Cav). They resemble early embryos at the two-layered stage oid bodies are associated with a diffuse distribution of 63 (see figure, parts a and b), and their differentiation recapitulates the processes of inner- E-cadherin and the absence of adherens junctions . cell-mass differentiation at the blastocyst stage. Therefore, they provide a useful tool for However, how PINCH1 regulates cell–cell adhesion is studying the molecular mechanisms that regulate ESC differentiation, BM assembly, poorly understood. Localization of PINCH1 to cell–cell epiblast polarization and cavity formation. Embryoid bodies that have been derived from contacts has not been reported so far, but it is possible ESCs that lack integrin-linked kinase (ILK) or particularly interesting Cys-His-rich that PINCH1 regulates cell–cell adhesion in an indirect protein-1 (PINCH1) have been used to address the functions of the ILK, PINCH, parvin manner. Although a role for ILK in cell–cell contacts has –/– –/– (IPP) complex during mammalian development. Ilk (see figure, part c) and Pinch1 been suggested based on studies in keratinocytes65,66, (see figure, part d) embryoid bodies produce a basement membrane but have defects in Ilk–/– embryoid bodies do not show cell–cell adhesion epiblast polarization and cavity formation; these defects are less severe in Pinch1 –/– defects. Therefore, PINCH1-mediated regulation of embryoid bodies, which is consistent with the longer survival time in vivo63,74. Red staining is for filamentous actin, green staining is for laminin-α1, blue staining (DAPI — 4′,6- cell–cell adhesion must be ILK-independent. Unlike 66 diamidino-2-phenylindole) is for DNA. Part a of the figure is a phase-contrast image keratinocytes and epiblast cells, trophoectodermal under 10× magnification, and parts b–d are confocal images under 63× magnification. cells or primitive endoderm cells of embryoid bodies can polarize in the absence of ILK74, which indicates that dif- ferent epithelial cells might have different requirements These discrepancies might reflect cell-type-specific for ILK. differences in the requirement for ILK involvement upstream of AKT/PKB and GSK3β. Conclusions The IPP complex serves as an important transducer Insights from studies using embryonic bodies. Because of extracellular signals to control many aspects of cell Inner cell mass mice that lack β1 integrin, ILK or PINCH1 die early in morphology and cell behaviour. Although there are Inner cells of the blastocyst embryoid bodies that retain pluripotency and development, were used to identify the many functions that are likely to be common among give rise to all cell types of the specific defects that cause peri-implantation lethality all cell types, certain functions seem to be cell-type future body. (BOX 2). β1 Integrin–/– embryoid bodies fail to deposit a specific. How does the IPP complex achieve specificity?
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β β Blastocyst The answer is not clear, but differential binding of ILK proliferator-activated receptor- (PPAR )-dependent An early stage of embryonic to PINCH and parvin isoforms is likely to be involved. upregulation of ILK expression in keratinocytes correlates development, during which Titration of binding sites by one isoform might lead to with increased phosphorylation of AKT/PKB on Ser473 cells begin to commit to trans-dominant effects on other isoforms. Therefore, (REFS 106,107). Interestingly, ILK expression is upregulated developmental lineages. understanding how cells that express numerous isoforms in many human tumours108–110, and in vivo models have Embryoid bodies of PINCH and parvin control the composition of the IPP revealed that ILK overexpression leads to the develop- Three-dimensional spherical complex to achieve spatial and temporal control over ment of mammary tumours in mice111. Differentiating aggregates of differentiated IPP function will be an important direction for future the functions of ILK under normal and pathological con- cells that are derived from investigations. ditions will provide insights into how ILK contributes to embryonic stem cells. The differentiation of embryoid In addition, the role of ILK as a kinase that is involved disease, and under which conditions ILK can be exploited bodies recapitulates many in the phosphorylation of AKT/PKB and GSK3β is therapeutically. aspects of the early course of still unclear. Immortalized cell lines or overexpressed More fundamentally, careful enzymatic analysis of embryonic development ILK have been used in most of the studies, and few the kinase activity of ILK and determining the substrate in vivo. reports have examined the role of ILK in primary cells. specificity will address whether ILK is a biologically Basement membrane Many of the effects of ILK-specific inhibitors or PI3K relevant AKT/PKB HMK. Furthermore, the elucida- Specialized extracellular matrix inhibitors that are observed in ILK-overexpressing cell tion of the three-dimensional structure of ILK is critical that first appears during the lines do not manifest in cells that express basal levels to adequately address the functions of ILK as a kinase peri-implantation stage in of ILK. For example, cyclin-D1-promoter activity in and as an adaptor molecule. A structure will provide vertebrates and during gastrulation in invertebrates. epithelial cells is reduced in response to PI3K inhibitors insights into how a kinase activity can be achieved in only when ILK is overexpressed, which indicates that the absence of conserved subdomains, and will identify PI3K-mediated pathways are not involved in the basal potential mechanisms whereby ILK activity is regulated 105 control of cyclin-D1 expression . On the other hand, by PtdIns(3,4,5)P3 and ILKAP. Extending a structural cyclin-D1 expression in endothelial cells is upregulated analysis to PINCH and parvin isoforms will allow us in the presence of vascular endothelial growth factor to identify the differences in their structures that lead to (VEGF) in an ILK-dependent manner85, and peroxisome- their distinct biological functions.
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Full activation of PKB/Akt in Framework Programme to K.R.L, an Erwin Schroedinger fellow- of basement membrane component deposition in β1 response to insulin or ionizing radiation is mediated ship from the Austrian Science Foundation (FWF) to O.K, and integrin-deficient embryoid bodies. J. Cell Sci. 113, through ATM. J. Biol. Chem. 280, 4029–4036 the Deutsche Forschungsgemeinschaft, Fonds der Chemischen 259–268 (2000). (2005). Industrie and the Max-Planck Society. The authors would like 102. Li, S. et al. Matrix assembly, regulation, and survival 116. Barlow, C. et al. Atm-deficient mice: a paradigm of to thank C. Zervas and members of the Fässler laboratory for functions of laminin and its receptors in embryonic ataxia telangiectasia. Cell 86, 159–171 (1996). critical reading of the manuscript. stem cell differentiation. J. Cell Biol. 157, 117. Cho, H. et al. 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