ILK, PINCH and parvin: the tipp of integrin signalling

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1 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 (ECM). A network of secreted proteins and polysaccharides that surrounds all the connective tissues and underlines all the epithelial and the endothelial sheets. It provides a physical support for tissues, as well as a sink for the storage, release and presentation of growth factors. Focal adhesion A highly specialized celladhesion structure that connects actin filaments to the ECM through integrins. Immature focal adhesions are known as focal complexes, and those that are formed through interactions with fibronectin mature into structures known as fibrillar adhesions. Department of Molecular Medicine, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsreid, Germany. s: legate@biochem.mpg.de; montanez@biochem.mpg.de; kudlacek@biochem.mpg.de; faessler@biochem.mpg.de doi: /nrm1789 Published online 21 December 2005 The extracellular matrix (ECM) provides the structural framework for the formation of tissues and organs. The ECM binds to substrate-adhesion molecules on the surface of cells and influences various intracellular signalling pathways that regulate survival, proliferation, polarity and differentiation. An important family of adhesion molecules that bind to the ECM are the integrins. Integrins are heterodimeric transmembrane molecules that consist of α and β subunits, and they are composed of large extracellular domains and relatively small cytoplasmic domains 1. Integrins can switch between active and inactive conformations. In the inactive state, integrins have a low affinity for ligands. Intracellular signalling events such as protein-kinase-c stimulation can prime the integrins, which results in a conformational change that exposes the ligand-binding site (FIG. 1). Ligand binding activates signalling cascades that lead to the assembly of a multiprotein complex at the site of cell adhesion to the ECM. These events have two important impacts on the cell: they forge a connection between the ECM and the actin cytoskeleton, and they alter the fluxes of many intracellular signalling pathways. Among the integrins, β1 integrin contributes to a large number of integrin heterodimers whereas β3 integrin has an important adhesion role in platelets an excellent system in which to study cell ECM adhesion 1. β1 and β3 integrins are widely expressed, and studies on the function of β1 and β3 integrins have provided many general insights into integrin-mediated adhesion. Deletion of the highly conserved β1 integrin gene in different organisms has been associated with defects in adhesion, proliferation, survival and polarity 2 6. However, although these experiments showed that integrins are important for these processes, they provided few insights into how these processes are regulated. Deletions of certain focal-adhesion molecules in different organisms display strikingly similar phenotypes to the β1-integrin-null phenotype, which indicates that certain intracellular proteins might have key roles in the regulation of the function of β1 integrins. Three proteins that have emerged from these studies as important regulators of integrin-mediated signalling are the integrin-linked kinase (ILK), and the adaptor proteins PINCH (particularly interesting Cys-His-rich protein) and parvin. These molecules form a heterotrimeric complex we refer to as the IPP complex, which is named after its components in order of their discovery. Recent reports have provided a wealth of data to expand the known functions of the IPP complex into almost every aspect of cell behaviour and fate. This review will provide an overview of our current knowledge regarding the function of the IPP complex. Interactions between the IPP components and numerous binding partners will be discussed to explain how the IPP complex functions both as an adaptor between integrins and the actin cytoskeleton, and as a hub that regulates several signalling pathways. Furthermore, this review will address the latest results in the ongoing controversy regarding the function of the putative kinase activity of ILK. We will conclude by describing the results of in vivo studies in model organisms, which provide insights into the role of the IPP-mediated integrin-signalling functions during development. Identification, architecture and assembly of IPP ILK was identified in 1996 in a yeast two-hybrid screen for proteins that could bind to the cytoplasmic tail of β1 integrin 7. The protein that was cloned contained three domains (FIG. 2). The N-terminal domain contains three ankyrin repeats, which mediate protein protein interactions, and a putative fourth ankyrin repeat that lacks conserved residues. The C terminus shares significant sequence homology to Ser/Thr protein kinases. A putative pleckstrin homology (PH) domain is situated 20 JANUARY 2006 VOLUME 7

2 Ankyrin repeat A protein protein-interaction module that consists of approximately 30 amino acids. It was first identified in the yeast cell-cycle regulator Swi6/Cdc10 and the D. melanogaster signalling protein Notch, and it was named after the cytoskeletal protein ankyrin. This motif is found in more than 1,700 different proteins. a Inactive state (bent form) Extracellular space Cell membrane Integrins Salt bridge b Primed state (extended form) α β ECM c Active state (ligand-bound form) Vinculin Clustering Src IPP FAK Paxillin Vinculin Pleckstrin homology (PH) domain A phosphoinositide-binding motif that is composed of approximately 100 amino acids and is involved in receiving and transmitting signals at the interface between the membrane and cytosol. PIPKlγ Talin Intracellular signalling PINCH Parvin ILK Paxillin IPP IPP FAK Cytoplasm Gene-expression changes Actin Assembly of the actin cytoskeleton Activation of signalling pathways Senescent Cells that are undergoing a permanent form of cell-cycle arrest that was originally described for post-proliferative primary cells in culture. Senescence can be induced by DNA damage, oxidative stress, chemotherapy and excess mitogenic stimuli, and is controlled by the tumour suppressor proteins, p53 and retioblastoma protein. LIM domain A tandem cysteine-rich Zn 2+ -finger motif that mediates protein protein interactions. It was originally identified in the transcription factors LIN11, ISL1 and MEC3. Calponin homology (CH) domain A relatively small motif that is present in several cytoskeletal proteins and functions as an actin-binding domain, especially when they are presented in tandem. Small inhibitory RNA (sirna). Double-stranded RNA molecules of nucleotides in length that are used as a viral defence mechanism and an endogenous gene-silencing mechanism from plants to humans. Lamellipodium Dynamic actin-mediated cellmembrane protrusions at the front of spreading and migrating cells. They are essential for cell motility, phagocytosis and the development of substrate adhesions. Figure 1 Biogenesis of focal adhesions. a Many integrins that are not bound to the extracellular matrix (ECM) are present on the cell surface in an inactive conformation, which is characterized by bent extracellular domains that mask the ECM-binding pocket. This conformation is stabilized by interactions between integrin transmembrane domains, membrane-proximal extracellular domains and a salt bridge between the cytoplasmic domains. b When talin is recruited to the plasma membrane and activated in association with phosphatidylinositol phosphate kinase type-iγ (PIPKIγ), it binds to the cytoplasmic tail of β integrins 120. This interaction separates the cytoplasmic domains and induces the integrins to adopt the primed conformation. c The integrin extracellular domains extend and unmask the ligand-binding site, allowing the integrin to bind specific ECM molecules. The separated integrin cytoplasmic domains and talin form a platform for the recruitment of other focal-adhesion proteins. Integrin-linked kinase (ILK), and isoforms of particularly interesting Cys-His-rich protein (PINCH) and parvin form the IPP complex (FIG. 2) in the cytoplasm, and this complex is 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 manner 121. The maturation of focal adhesions involves clustering of active, ligand-bound integrins and the assembly of a multiprotein complex that is capable of linking integrins to the actin cytoskeleton and communicating with signalling pathways. between these two domains and partially overlaps them. Cell-culture experiments indicated that the physiological ligand of the ILK PH domain is phosphatidylinositol- 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) 8,9. ILK is the central component of the IPP complex; it binds PINCH proteins through the N-terminal ankyrin-repeat domain and parvins through the kinase domain. It also links the complex to the cytoplasmic tails of β1 and β3 integrins 7,10 14, but it is not known whether it binds to other β integrins. A single ILK isoform has been identified in all species discussed in the following sections. PINCH1 (also known as LIMS1) was originally identified in 1994 as a marker for senescent erythrocytes, and was shown to bind to ILK in 1999 (REFS 15,16). A second isoform, PINCH2 (also known as LIMS2), was predicted by sequence-database mining and was subsequently characterized 17,18. PINCH1 and PINCH2 are adaptor proteins that consist of five LIM domains and tandem nuclear localization signals 17,19, and both isoforms bind to ILK through the N-terminal LIM domain in a mutually exclusive manner 10,18. There is extensive overlap in the expression patterns of PINCH1 and PINCH2 in adult tissues, and both genes are expressed in smooth-muscle layers of the developing embryo 19. Parvins a family of proteins that consists of actopaxin/ch-ilkbp/α-parvin, affixin/β-parvin and γ-parvin bind to ILK through the second of two calponin homology (CH) domains 13,14,20,21. The interaction between α-parvin and ILK is partially dependent on PtdIns(3,4,5)P 3 (REF. 22) and on phosphorylation of α-parvin by CDC2 and mitogen-activated protein kinase (MAPK) It is unclear whether the other parvins are regulated in a similar manner, although β-parvin can also be phosphorylated 14 (see also TABLE 1). α- and β-parvin have overlapping expression patterns in various tissues, but the expression of γ-parvin is restricted to the haematopoietic system 26. It is therefore possible that different IPP complexes can assemble within the same cell. This idea was further supported by a recent study showing that small inhibitory RNA (sirna)-mediated knockdown of α-parvin in HeLa cells stimulated Rac activity and lamellipodium formation. These findings indicate that α-parvin can function as a negative regulator of Rac in cells that express both α- and β-parvin 27. Overexpression of β-parvin in HeLa cells promoted apoptosis, perhaps because of competition with α-parvin for binding to ILK. Monitoring the expression levels of parvins in cells that are exposed to NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 7 JANUARY

3 Extracellular space NCK2 Cytoplasm Growth factors RTKs PtdIns(3,4,5)P 3 ILKAP LIM5 LIM4 LIM3 LIM2 LIM1 RSU1 Tβ4 PINCH RNA interference (RNAi). A method to silence specific gene expression by introducing double-stranded RNA into the cell that matches the nucleotide sequence of the targeted mrna. LD motif Leucine-rich protein-binding sequences with the consensus sequence LDXLLXXL. Kindler syndrome An inheritable epidermal defect that is characterized by blistering, abnormal pigmentation, fragile skin and increased cancer risk. ANK1 ANK2 ANK3 ANK4 Cell membrane ILK PH PDK1 ECM AKT/ PKB Kinase HIC5 TESK1 α-pix CH2 CH1 Paxillin Parvin Actin Integrins Kindlin-2 Migfilin Vinculin α-actinin Filamin Figure 2 Anatomy of the IPP complex and its binding partners. Integrin-linked kinase (ILK) consists of three domains, N-terminal ankyrin (ANK) repeats, a plekstrin 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 ILKassociated phosphatase (ILKAP). The PH domain probably binds to phosphatidylinositol- 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ). The kinase domain of ILK binds to parvins, paxillin, MIG2/kindlin-2, the cytoplasmic tails of β integrins, the kinase substrate AKT/PKB (protein kinase B) and the kinase phosphatidylinositol-3-kinase-dependent kinase-1 (PDK1). PINCH isoforms, which contain five LIM domains, bind to receptor tyrosine kinases (RTKs) through the Src-homology-2 (SH2) SH3 adaptor NCK2, thereby coupling growth-factor signalling to integrin signalling. PINCH1 binds to Ras suppressor-1 (RSU1) and thymosin-β4 (Tβ4) to influence Jun N-terminal kinase (JNK) signalling and cell migration/survival, respectively. α- and β-parvins can bind to F-actin directly, as well as indirectly through binding to paxillin or HIC5 (α-parvin only) or α-actinin (β-parvin only). α-parvin also binds to the Ser/Thr kinase testicular protein kinase-1 (TESK1), whereas β-parvin binds to the guanine nucleotide-exchange factor α-pix, which influences actin remodelling through the GTPases Rac and Cdc42. Interactions with integrins and the cytoskeleton also occur through a MIG2/kindlin-2 migfilin filamin complex. specific conditions, and immunoprecipitation of discrete IPP complexes will determine whether this data is biologically relevant, and will enable the study of the mechanisms that regulate the expression of the different parvin isoforms. IPP-complex assembly and stability. The assembly of the IPP-complex precedes cell adhesion, which indicates that these complexes first form in the cytosol, independently of adhesion signals 28. The stability of the individual IPP components is dependent on complex formation, because RNA interference (RNAi)- mediated depletion of one member of the complex results in degradation of the other components by a proteasome-mediated process 29. This complicates the interpretation of results from genetic-deletion or RNAidepletion studies because the defects that are associated with deletion of one component might be due to diminished levels of other components. Recent work is beginning to address the specific roles of IPP-complex members, taking into account their mutual stabilization and degradation 30. Degradation of ILK or the PINCH proteins can be prevented by expression of the PINCH1 or PINCH2 LIM1 domain in Pinch1-deficient cells 31 or expression of the ILK ankyrin repeats in Ilk-deficient cells, respectively (C. Grashoff and R.F., unpublished data), but only a complex that consists of full-length proteins efficiently assembles into focal adhesions in vertebrate cell culture 20,28,32. Although the full-length IPP complex is required for focal-adhesion assembly in vertebrates, other factors are also required to assemble the IPP complex into focal adhesions, including the adaptor molecule paxillin, and possibly MIG2/kindlin-2 (REFS 28,33). Both of these proteins directly bind to the IPP complex through the kinase domain of ILK 33,34. Many more proteins might also be involved at this stage of IPP-complex assembly and targeting. Interestingly, in Drosophila melanogaster embryos, the PINCH homologue, steamer duck (STCK), is not required to localize ILK to sites of cell adhesion 35. IPP-binding partners There are many molecules that have been shown to interact with the IPP complex 36. The function of the IPP complex as a signalling platform is achieved through its direct interaction with factors that function as upstream regulators of many different signalling pathways (FIG. 3). This section will summarize the known binding partners of the IPP complex and will provide examples where signalling specificity may be achieved through differential binding of molecules to PINCH and parvin isoforms. ILK-interacting partners. As mentioned above, vertebrate ILK can bind directly to the cytoplasmic tails of β1 and β3 integrins 7,11,12, and it is indirectly connected to the actin cytoskeleton through its interaction with parvins (see below). Interactions with the cytoskeleton 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 vinculin 20,37,38. Paxillin binds to ILK through a paxillin-binding site (PBS) within the kinase domain of ILK 33. Furthermore, Caenorhabditis elegans ILK binds to UNC-112, the orthologue of vertebrate MIG2/kindlin-2 (REF. 34). In vertebrates, kindlin-1 binds to the cytoplasmic domains of β1 and β3 integrins 39, and its loss causes Kindler syndrome 40. MIG2/kindlin-2 binds to migfilin, which in turn binds to filamin an adaptor protein that interacts with several molecules, including filamentous (F)-actin and integrins 41,42 and provides another connection between ILK and the actin cytoskeleton. Binding partners of the PINCH isoforms. The signalling specificity of the IPP complexes depends on the presence of the different PINCH or parvin isoforms. When PINCH2 is overexpressed in a basal PINCH1-expressing background, it competes with PINCH1 for binding to ILK but cannot transduce integrin-mediated signals that control cell spreading and migration 18. Also, although the expression of a chimeric PINCH that consists of the PINCH1 LIM domains and the PINCH2 C-terminal tail cannot restore spreading in PINCH1-knockdown HeLa cells 30, expression of full-length PINCH2 in a Pinch1- null background completely restores the adhesion and spreading defects of Pinch1-null fibroblasts 31. Therefore, 22 JANUARY 2006 VOLUME 7

4 Table 1 ILK substrates Substrate Biochemistry Cell culture In vivo References ILK 7 β1 integrin 7 β3 integrin 11 AKT/PKB, *, 71,74 76,111 GSK3β, *, 71,74,75,111,124 Myosin light chain 70 Myelin basic protein 7 β-parvin 14 MYPT1 125 α-nac 126 CPI17 127,128 PHI1 127,128 *Cell culture using Ilk / fibroblasts. Ilk overexpressed in mammary epithelium. Ilk / chondrocytes. CPI17, protein-kinase-c-dependent phosphatase inhibitor of 17 kda; GSK3β, glycogen-synthase kinase-3β; ILK, integrin-linked kinase; MYPT1, myosin-light-chainphosphatase target subunit-1; α-nac, nascent-polypeptide-associated complex and coactivator-α; PHI1, phosphatase-holoenzyme inhibitor-1; PKB, protein kinase B. Thymosins A large family of small peptides that was originally identified in the thymus, but is also found in many tissues. They are divided into three main groups: α-, β-, and γ-thymosins. β-thymosins bind globular actin to maintain a pool of actin monomers in the cell. Calpains A superfamily of multimeric Ca 2+ -dependent cysteine proteases that is implicated in various cellular processes such as proliferation, differentiation and apoptosis. Deregulation of calpain activity has been implicated in various pathological conditions. Sarcolemma The plasma membrane that encloses striated muscle fibres. Adherens junction A highly specialized cell celladhesion complex that contains cadherins and catenins, and which is connected to cytoplasmic actin filaments. accessory proteins that differentially bind to PINCH isoforms are probably responsible for transducing the signals that control cell spreading. Functional differences between PINCH1 and PINCH2 might arise from differential binding of the Ras-suppressor protein RSU1. RSU1 has been shown to function as a negative regulator of growth-factor-induced Jun N-terminal kinase (JNK) activation (FIG. 3). RSU1 is also known to interact with the LIM5 domain of PINCH1 in D. melanogaster 45 and vertebrates 46, but this interaction is specific for PINCH1. PINCH2 does not bind RSU1, because the sequence of the LIM5 domain is different 19. Thymosin-β4 binds to LIM domains -4 and -5 of PINCH1, upregulates ILK activity and positively influences migration and survival of cardiac cells 47. Whether PINCH2 and thymosin-β4 interact is not known, but a conditional knockout of Pinch1 in murine ventricular cardiomyocytes shows no discernable phenotype 48, indicating that PINCH2, which is also expressed in the heart 19, might compensate for the loss of PINCH1. PINCH1 also binds to the receptor-tyrosine-kinase adaptor protein NCK2 in vitro through a LIM4 SH3- domain (Src-homology-3 domain) interaction 49,50. Mutations in vertebrate PINCH1 that disrupt PINCH1 NCK2 binding reduce the amount of PINCH1 that is found in focal adhesions 49, but the relevance of a PINCH1 NCK2 interaction is not clear. There is no evidence for such an interaction in C. elegans or D. melanogaster. Mice that carry a Nck1 or Nck2 genetic deletion, are phenotypically normal, whereas mice that lack both proteins die during embryogenesis. The migration and cytoskeletal defects of Nck1 / Nck2 / fibroblasts 51 are rescued when NCK1 is reintroduced, although the PINCH1 NCK interaction is specific for NCK2. Furthermore, NCK1 has been demonstrated not to bind to PINCH1 (REF. 52). These results indicate that the interaction between PINCH1 and NCK2 might not be essential in vivo. Parvins have overlapping binding partners. α-parvin binds to F-actin directly, as well as indirectly through an interaction with paxillin 20. It is not yet known whether ILK and a parvin isoform bind to the same molecule of paxillin, or if two paxillin molecules bind to a single IPP complex. HIC5, a paxillin-related protein, also binds to α-parvin 20. HIC5 binds to many of the proteins that paxillin binds to, but it also shuttles to the nucleus, where it modulates the expression of several genes 53,54. In addition, α-parvin specifically binds to TESK1, a Ser/Thr kinase that phosphorylates the actin-regulating protein cofilin 55. β-parvin binds to F-actin, but can also bind to the actin-crosslinking protein α-actinin 56 and the guanine nucleotide-exchange factor α-pix 57. It therefore provides a connection between the IPP complex and the actin-regulating GTPases Rac1 and Cdc42 (FIG. 3). α-pix, in turn, binds to PAK1 (REF. 58), a Rac1/Cdc42 effector that regulates cytoskeletal dynamics through the 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 has been shown to cleave talin, and this cleavage is the rate-limiting step in the disassembly of focal adhesions 61. The functions of parvin-binding partners indicate that the parvins have a role in the regulation of actin dynamics and focal-adhesion turnover. β-parvin also binds to the membrane-repair protein dysferlin at the sarcolemma of skeletal muscle; an observation that reveals a potential role for β-parvin in membrane repair 62. Binding partners for the less-well-characterized γ-parvin have yet to be identified. Other roles of the IPP-complex proteins? Deletion of one component of the IPP complex results in degradation of the other components, but this degradation is not complete 29,63, which indicates that the individual components might have extra roles outside the complex. PINCH1 shuttles between the nucleus and the cytoplasm in Schwann cells, and it has been found in the nucleus of the mouse primitive endoderm, and muscle cells of C. elegans. These observations indicate that PINCH1 might have a role in gene regulation or signalling between the cytoplasm and the nucleus 17,63,64. β-parvin localizes to the sarcolemma in skeletal muscle, where it might have a role in Ca 2+ -dependent membrane repair, together with dysferlin 62. ILK redistributes to cell cell contacts in differentiating keratinocytes, where it participates in the early steps of adherens-junction formation 65,66. ILK catalytic activity ILK has been shown to function as an adaptor protein, but recent reports have indicated that ILK also has catalytic activity. The kinase domain of ILK shows significant homology to Ser/Thr protein kinases, with the exception of sequences within the catalytic loop and the conserved DXG motif 67,68. These differences from the canonical kinase sequence are difficult to reconcile with the NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 7 JANUARY

5 Stimulus ECM Growth factors Extracellular space Membrane receptor Integrins RTKs PtdIns(3,4,5)P 3 Cell membrane Rac1 MAP4K α-nac PINCH RSU1 GSK3β NCK2 PI3K PTEN ILK P P P P α-pix Parvin AKT/PKB P ILKAP P Rac1 Cdc42 Cytoplasm MLC JNK TAU1 mtor BAD p70 S6K Caspase-3 Nucleus Caspase-9 Actin Transcription factors AP-1 β-catenin LEF/TCF CREB SNAIL HIF1 NF-κB Target genes expression MMP9 Cyclin D1 E-cadherin VEGF inos COX2 Myc Cell response Tissue Spreading and 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 smallmolecular-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 polypeptideassociated complex and co-activator-α; NF-kB, nuclear factor-kb; p70 S6K, p70 ribosomal S6 kinase; α-pix, activating PAK-interactive exchange factor-α; PtdIns(3,4,5)P 3, phosphatidylinositol-3,4,5-trisphosphate; RSU1, Ras suppressor-1; VEGF, vascular endothelial growth factor. observed kinase activity, because ILK lacks an obvious catalytic base and Mg 2+ -chelating residues. Furthermore, the sequence of the ILK catalytic domain is divergent across different species 69 (FIG. 4). Such a tolerance for mutation indicates that if ILK possesses kinase activity, it is probably unnecessary for its function. Nevertheless, recombinant, purified ILK has been shown to phosphorylate several substrates in vitro 7,70,71 (TABLE 1), and it is possible that ILK retains residual kinase activity that is readily detected in vitro. Because kinetic rates of the kinase reaction have not been reported and the evidence for in vivo kinase activity is weak, it is not known whether ILK possesses sufficient activity to function as a physiologically relevant kinase in vivo. ILK model substrates. The most widely used readouts for ILK activity are the phosphorylation of GSK3β and AKT/PKB (protein kinase B), which regulate many different signalling pathways (FIG. 3). AKT/PKB activation requires phosphorylation of Thr at position 308 by phosphatidylinositol 3-kinase (PI3K)-dependent kinase-1 (PDK1) and Ser at position 473 by PDK2 (which is more appropriately known as hydrophobic-motif kinase (HMK)). The identification of PDK1 is unambiguous, but the identity of PDK2/HMK continues to be debated. ILK, among other candidates (BOX 1), has been proposed to function as a HMK and this idea is further supported by immunoprecipitation assays showing that ILK directly binds to AKT/PKB JANUARY 2006 VOLUME 7

6 Dominant negative Introduction of an inactive mutant gene product, which interferes with the functional endogenous gene product, perhaps by competing for available accessory factors. Consensus TAK1 C. elegans ILK D. melanogaster ILK X. laevis ILK M. musculus ILK I II III IV V L IGXGXXGXL AXKXΦ E Φ ΦΦΦΦΦ V VGRGAFGVV AIKQI E I VCLVM IAESHSGEL VARIL E I LVIIS LSVTPSGET VAKIL E I LVTIS LNDNHSGEL IIKVL E V PVLIT LNENHSGEL VVKVL E V LVIIS Consensus VI VII VIII XI IX HRDLKXXN DFG APE DXWS A XGΦ R TAK1 HRDLKPPN DFG APE DVFSWGI R C. elegans ILK LRFYLLSK --- SPE DMWSFAI R D. melanogaster ILK PTYHLNSH --- SPE DMWSFAI R X. laevis ILK PRHYLNSR --- APE DMASFAV R M. musculus ILK PRHALNSR --- APE DMWSFAV R Figure 4 Divergence of the kinase domain of ILK. Alignment of integrin-linked kinases (ILKs) from several species as well as the related kinase transforming-growthfactor-β-activated kinase-1 (TAK1) with conserved subdomains of protein kinases, numbered according to Hanks et al. 67,68. The subdomain X is poorly conserved and contains no consensus amino acids, so it was not included. Conserved domains that are significantly altered in ILK are shown in red. One of three Gly residues in subdomain I is conserved in all ILK proteins. A Lys in subdomain II that is required for the phosphotransfer reaction 82 is also conserved as a basic residue. However, the catalytic Asp residue in subdomain VI and the DFG sequence in subdomain VII that is required to align the γ-phosphate of ATP, are both missing. The conserved Lys, which neutralizes the charge on the γ-phosphate of ATP, and the conserved Asn, which chelates the secondary Mg 2+ are also missing. All active protein kinases contain a conserved Asp in regions VI and VII except for the haspins a unique family of histone kinases that have a role in mitosis 122, and lack the conserved Asp in subdomain VII but contain the catalytic Asp in subdomain VI 123. ILK is the only protein kinase that is known to be missing both residues. C. elegans, Caenorhabditis elegans; D. melanogaster, Drosophila melanogaster; M. musculus, Mus musculus; X. laevis, Xenopus laevis. Regulation of ILK activity. ILK-dependent phosphorylation is regulated in a PI3K-dependent manner. Inhibitors of PI3K activity reduce ILK activity in cell-lysate immunoprecipitates and impair the phosphorylation of putative ILK substrates in cell culture, whereas overexpression of the PI3K catalytic subunit or the addition of PtdIns(3,4,5)P 3 increase ILK-dependent kinase activity in cell culture and in vitro, respectively 8. The expression of thymosin-β4 in cardiomyocytes also increases the activity of ILK, as measured by phosphorylation of AKT/PKB on Ser Conversely, the catalytic activity of ILK is negatively regulated by the phosphatase ILK-associated protein (ILKAP). ILKAP reduces the kinase activity of ILK in vitro and the phosphorylation of GSK3β in vivo, but the phosphorylation of AKT/PKB remains unaffected 72,73. This demonstrates that the mechanisms of GSK3β and AKT/PKB activation are different, despite the fact that both proteins have been described as ILK substrates. The mechanism by which ILKAP reduces ILK activity towards certain substrates is currently unknown. It has also been reported that ILK can autophosphorylate in vitro 2, but the function of autophosphorylation of ILK in vivo, if present at all, remains to be investigated. Perturbation of ILK function. Despite evidence in favour of ILK having a kinase function, genetic experiments have failed to demonstrate a crucial role for the kinase activity of ILK in several cell types. Alterations in the phosphorylation status of AKT/PKB or GSK3β have not been observed in Ilk / fibroblasts 74. Furthermore, the phosphorylation status of putative ILK substrates is unchanged in chondrocytes or keratinocytes that do not express ILK (K. Lorenz and R.F., unpublished data, and REF. 75). By contrast, deletion of Ilk in immortalized macrophages results in diminished phosphorylation of both AKT/PKB and GSK3β, which is reversed by transfection of wild-type ILK, but not a kinase-deficient ILK mutant 76. This discrepancy might reflect cell-typespecific differences, or it might reveal an increased dependence on the kinase activity of ILK as a result of immortalization. Small-molecule inhibitors that were designed to specifically inhibit the kinase activity of ILK, prevent phosphorylation of AKT/PKB and GSK3β when introduced in ILK-overexpressing cell lines 9,71,77. Further characterization of one of these inhibitors, KP-392, revealed that it disrupts the interaction between ILK and both α-parvin and paxillin, and blocks the accumulation of α-parvin and paxillin into focal adhesions 22. Similarly, several different point mutations in ILK, which were designed to abolish catalytic activity, disrupt protein protein interactions and might function by preventing the assembly of functional IPP complexes. Overexpression of wild-type ILK in cellculture experiments results in increased phosphorylation of AKT/PKB and GSK3β in many cell types. Conversely, overexpression of a dominant-negative mutant of ILK (Glu359Lys) reduces phosphorylation of AKT/PKB and GSK3β. Although the Glu359Lys mutation was reported to impair the kinase activity of ILK 8,78, other studies conclude that this mutation has no effect on the kinase activity of ILK in vitro 14,79. Instead, the Glu359Lys mutation disrupts the interaction between ILK and both α-parvin and paxillin, which results in a failure to assemble ILK into focal adhesions 14,79. Several inactivating point mutations in the kinase domain of ILK have been used to dissect the kinase function, but these mutations (Arg211Ala, Ser343Ala, and Lys220Ala) also disrupt ILK protein interactions, which makes it impossible to derive interpretations that are based solely on the lack of kinase activity. The Arg211Ala mutation results in deficient binding to α-parvin 22, and this interaction is required to target ILK to focal adhesions 28. The Ser343Ala mutation abolishes binding to AKT/PKB, but this mutant protein localizes to focal adhesions 71,80. The Lys220Ala mutant fails to bind to β-parvin 81. Another mutation at this site, Lys220Met binds to β-parvin but shows no catalytic activity 14, but a second activating mutation, Ser343Asp, reverses this defect 69 despite a requirement of Lys220 for protein-kinase activity 68,82. The Phe438Ala mutant, which does not assemble into focal adhesions, has been proposed not to bind to MIG2/kindlin-2 (REF. 28). Furthermore, mutations in ILK that show dominant-negative effects in NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 7 JANUARY

7 Box 1 Kinase candidates responsible for AKT/PKB phosphorylation AKT/PKB (protein kinase B) must be phosphorylated on Thr308 and Ser473 for full activity. The kinases that are involved in this process are called phosphatidylinositol 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. Several candidates have been proposed that do not hold up to scrutiny, including mitogen-activated-protein-kinase-activated kinase-2 (MAPKAPK2), PDK1 bound to a fragment of protein-kinase-c-related kinase-2 (PRK2), and AKT/PKB itself. Integrinlinked kinase (ILK) has also been considered as a candidate for HMK, but there are other candidates that are equally plausible. Two members of the PI3K-related kinase (PIKK) family, the DNA-dependent protein kinase (DNA-PK) 112 and the mammalian target of rapamycin (mtor) rapamycin-insensitive-companion-of-mtor (rictor) complex 113,114 have properties that are consistent with them having AKT/PKB-kinase activity. Each candidate responds positively to insulin treatment and both depend on PI3K activity. 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 mtor rictor can phosphorylate AKT/PKB Ser473 in vitro, although only mtor rictor has been tested on full-length AKT/PKB. A third member of the PIKK family, the gene mutated in ataxia telangiectasia (ATM) 115, also meets most of the requirements for HMK, except that it cannot phosphorylate Ser473 in vitro. However, cell lines that are derived from ataxia telangiectasia patients lack detectable AKT/PKB-Ser473 kinase activity, which can be restored after transfection with a plasmid encoding the ATM gene product. In addition, mice that carry deletions of ATM or AKT/PKB show phenotypic similarities These data indicate that the HMK activity is mediated through ATM. Whether any of these candidates is the bona fide AKT/PKB HMK, or if they can all function as a HMK under specific circumstances, has yet to be determined. The role of the IPP complex in invertebrates To study the contribution of the IPP complex to development, genetic deletion of most components has been achieved in C. elegans, D. melanogaster and mice (see Supplementary information S1 (table)). The phenotypes caused by these deletions confirm a role for the IPP complex as an adaptor complex between the ECM and the actin cytoskeleton, but insights into the signalling function are not as forthcoming, and a role for the ILK catalytic activity has not been established. Differences in phenotypes among IPP components, particularly in mice, reveal that, in addition to a common function as part of the IPP complex, each component has discrete functions. Invertebrates represent relatively simple systems to study integrin-mediated functions. C. elegans has one β integrin (βpat-3) and two α integrin (αpat-2 and αina-1) subunits; D. melanogaster has two β integrin (βps and βv) and five α integrin (αps1 5) subunits. Both organisms have a single orthologue of ILK, PINCH and parvin, which allows straightforward analysis of deletion phenotypes. C. elegans has a second PINCH1- related gene but this shows only 34% identity with PINCH1 and contains a putative endoplasmic reticulum (ER) targeting sequence. Ataxia telangiectasia (ATM). Autosomal recessive hereditary disease associated with DNA-repair defects and caused by mutations in the ATM (ataxia telangiectasia) gene. It is characterized by progressive cerebellar ataxia, dilation of blood vessels in the skin and eyes, chromosomal aberrations, immune dysfunction and an increased risk of cancer malignancy, particularly leukaemia and lymphoma. PAT phenotype A broad phenotypic class of lethal mutations that affect muscle formation in C. elegans. Mutations that cause a PAT (paralyzed and arrested elongation at twofold) phenotype, affect either components of the attachment complex or essential components within the sarcomere. Dense bodies/m-lines Focal-adhesion-like muscleattachment structures in C. elegans. Dense bodies anchor actin filaments to the plasma membrane, and M-lines attach myosin filaments to the plasma membrane. Both structures are essential for the contractility and the maintenance of the muscles. cell culture (Glu359Lys or Lys220Met), or abolish the activity of the protein kinase Raf at non-permissive temperatures (Pro358Ser), completely rescue the phenotype of the ILK loss-of-function mutations in C. elegans (only Glu359Lys was tested) and D. melanogaster (all three ILK mutants). This indicates that kinase activity is dispensable for ILK function in these organisms 34,83. However, residual kinase activity might be sufficient to completely restore a wild-type phenotype. RNAi studies in transformed cells demonstrate that knockdown of ILK, PINCH1 or α-parvin protein expression correlates with reduced phosphorylation of AKT/PKB Ser473 (REFS 29,76,84 86). Furthermore, AKT/PKB Thr308 phosphorylation is also reduced in the absence of PINCH1 (REF.29). AKT/PKB does not localize to the plasma membrane in α-parvindepleted HeLa cells, despite the presence of ILK in focal adhesions, which demonstrates that α-parvin is required for the correct targeting of AKT/PKB before activation 84. However, if AKT/PKB is constitutively targeted to the membrane, phosphorylation of Ser473 does not depend on ILK 29,84. It should be noted that the increased apoptosis that results from knockdown of ILK or PINCH1 is not reversed on restoration of AKT/PKB phosphorylation, which indicates that these molecules regulate numerous survival pathways. Taken together, the data from mutagenesis, smallmolecule inhibition and RNAi all indicate a model whereby ILK activates AKT/PKB indirectly by facilitating the translocation of AKT/PKB to the plasma membrane in an α-parvin-dependent manner. Once AKT/PKB is located at the plasma membrane, it can be phosphorylated by other kinases. Studies in C. elegans. In C. elegans, genetic deletion of βpat-3 (REF. 3), pat-4 (which encodes the ILK orthologue) 34, unc-97 (which encodes the PINCH orthologue) 17 or pat-6 (which encodes the parvin orthologue) 87 all produce mutants with a similar PAT phenotype. Although a direct physical interaction between βpat-3 and PAT-4 has not been reported, deletion of βpat-3 or the ECM component unc-52 (which encodes the orthologue of perlecan) results in mislocalization of PAT-4, which indicates that PAT-4 requires integrins and ECM to localize to dense bodies 34. Recruitment of PAT-4 to dense bodies, and the correct organization of βpat-3 at the cell membrane also depend on UNC-112 (the orthologue of MIG2/kindlin-2) 34,88. However, there is no evidence that PAT-4 regulates the GSK3β signalling pathway in this organism, as the defects that arise from deletion of pat-4 are distinct from the phenotype of the GSK-3β mutant in C. elegans 34. A novel Zn 2+ -finger protein that is unique to C. elegans, UNC-98, binds to UNC-97 at M-lines and dense bodies 89. Both proteins are also detected in the nucleus, which indicates that they might be involved in gene regulation 17,89. Studies in D. melanogaster. In D. melanogaster, deletion of βps, the orthologue of the vertebrate β1 integrin subunit 2, or loss of function of ILK 83 or the PINCH orthologue 35, result in striking embryonic muscle-attachment defects. βps and PINCH-deficient flies also show dorsal-closure defects, which indicates that cell migration is impaired 2,45. Adult chimeric flies display a blister phenotype in wing regions that lack these proteins. These phenotypes are consistent with the loss of cell adhesion to the ECM. However, βps mutants show adhesion defects that result from the detachment of the cell membrane from the ECM, whereas the ILK and PINCH mutants are characterized 26 JANUARY 2006 VOLUME 7

8 Dorsal closure A mid-stage developmental process that involves the movement of lateral dorsal epithelia towards the dorsal midline. This process is required for the sealing of embryonic epidermis in D. melanogaster. Podocyte Highly specialized epithelial cells that cover the outer aspect of the glomerular basement membrane in the kidney. Mature podocytes possess a highly branched array of foot processes that are essential for glomerular filtration. Chondrodysplasia A heterogeneous group of genetic disorders, which are characterized by abnormal skeletal morphogenesis affecting the development and growth of most skeletal elements. by the detachment of the actin cytoskeleton from the cell membrane. Interestingly, ILK localizes normally to muscle-attachment sites in PINCH-mutant embryos, which indicates that the PINCH loss-of-function phenotype is not caused by the mislocalization of ILK 35. The loss-offunction mutations in PINCH affect the LIM domains 3 5, therefore it is possible that the LIM domains 1 and 2 are still expressed, and are sufficient to retain correct ILK localization in this organism. Cytoskeletal reorganization and changes in cellular shape that are required during dorsal closure are coordinated by the JNK signalling pathway (for a review see REF. 90). Therefore, the dorsal-closure defect in PINCHmutant flies might be explained, in part, by disrupted JNK signalling. PINCH might regulate JNK signalling through two distinct pathways. The first involves the interaction between PINCH and RSU1 (FIG. 3). Both PINCH and RSU1 genetically interact with Misshapen, a MAPK-kinase-kinase kinase (MAP4K) in the JNK signalling pathway, which demonstrates that both proteins can negatively regulate this pathway 45. The expression and/or stability of PINCH and RSU1 are mutually dependent on one another, so the relative contributions of PINCH and RSU1 in modulating JNK signalling have not been determined. The second and more-speculative mechanism involves a PINCH Dreadlocks Misshapen pathway. Dreadlocks, the D. melanogaster orthologue of NCK2, interacts with Misshapen 91. Although mammalian PINCH1 binds to NCK2 in vitro 52, an interaction between Dreadlocks and PINCH has not been reported. However, the affinity between mammalian NCK2 and PINCH1 is weak in vitro, and it is possible that it could not be detected. The two main pathways that regulate survival and proliferation involve the phosphorylation of AKT/PKB and the stabilization and nuclear translocation of β-catenin as a consequence of GSK3β phosphorylation (FIG. 3). However, there is no evidence that ILK is involved in these pathways in D. melanogaster as ILK loss-of-function produces a different phenotype to loss of β-catenin or AKT/PKB 34,83,92,93. Furthermore, overexpression of ILK does not affect signalling through β-catenin 83. Functions of IPP in mammalian systems The biological functions of the members of the IPP complex have been examined in several cell types. Many IPP functions can be reconciled with a role for the IPP complex at focal adhesions. For example, the regulation of podocyte adhesion and spreading 23, endothelial cell and cardiomyocyte migration 47,85, platelet aggregation 11,12, neuronal spreading and outgrowth 77,94, and leukocyte recruitment 95 reveal a role for the IPP complex in actin-cytoskeleton dynamics and integrin activation. Studies in mice. Loss of expression of β1 integrin 4,5, ILK 74, PINCH1 (REFS 48,63) or α-parvin (H. Chu and R.F., unpublished data) all result in embryonic lethality. However, these mutants show subtle differences in their phenotypes, which indicates that distinct defects might underlie these phenotypes. β1 Integrin /, Ilk / and Pinch1 / embryos arrest at the peri-implantation stage of embryonic development, but the Pinch1 / embryos die at the embryonic day (E)6.5 E7.5 whereas the β1 integrin / and Ilk / embryos die at E5.5 E6.5. α-parvin / embryos develop the furthest, but die following implantation. The temporal differences in embryonic lethality between β1 integrin / or Ilk / embryos and Pinch1 / embryos are unlikely to be due to PINCH2 compensation, because expression of PINCH2 is not detected until later stages of development 19. On the other hand, Pinch2 / mice are viable, probably as a result of PINCH1 compensation 31. It will be important to determine whether the relatively long survival of α-parvin / embryos is the result of compensation by other members of the parvin family and to investigate whether parvins might have a crucial function either before, or during, peri-implantation development. Embryonic lethality in Pinch1-null mice is associated with an increase in endodermal-cell apoptosis, which demonstrates that PINCH1 regulates cell survival 63. This observation is further supported by PINCH1-knockdown data in HeLa cells 29. However, it remains to be determined whether AKT/PKB is involved in the PINCH1-mediated apoptotic pathway in embryos or if, as in HeLa cells, an AKT/PKB-independent pathway exists. Conditional knockouts of IPP members have been created to overcome the difficulties associated with early embryonic lethality. Mice that carry conditional deletions of β1 integrin or Ilk in chondrocytes show skeletal defects 75,96,97 and develop chondrodysplasia. In culture, β1 integrin- and Ilk-mutant chondrocytes had defective spreading, abnormal F-actin distribution, and impaired adhesion, although the Ilk-mutant chondrocytes had a less-severe adhesion defect. Therefore, deletion of either protein impairs the connection between the ECM and the actin cytoskeleton that is required for cell spreading. In addition, reduced proliferation of β1 integrin- or Ilk-deficient chondrocytes is associated with a defect in the G1 S transition, but impaired cytokinesis in β1 integrin / chondrocytes reveals a defect in the G2 M transition. Conditional deletion of Ilk in endothelial cells results in impaired vascular development and embryonic lethality 98. Integrin activation is reduced in the absence of ILK, which indicates a migration defect, and there is an increase in apoptosis concomitant with decreased AKT/PKB phosphorylation on Ser473. However, apoptosis is reduced when ILK is reintroduced, but not when constitutively active AKT/PKB is introduced. This indicates that, as in HeLa cells, an ILK-dependent, AKT/ PKB-independent pathway might operate in endothelial cells to regulate apoptosis. Immortalized Ilk / macrophages also show decreased AKT/PKB phosphorylation on Ser In addition, inhibition of ILK or PI3K activity, expression of dominant-negative ILK and knockdown of ILK protein levels revealed a role for the IPP complex in AKT/PKB- and GSK3β-mediated signalling in neurons and leukocytes 77,94,95,99,100. However, fibroblasts and chondrocytes that are derived from Ilk / mice do not show altered phosphorylation of AKT/PKB 74,75,97. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 7 JANUARY

9 Box 2 Embryoid bodies as a model for early embryonic development a Wild type Ep Cav b Wild type c Ilk / d Pinch1 / En BM The low number of cells and the inaccessibility of peri-implantation embryos make analysis of the cellular and molecular events that take place during early development difficult. Embryoid bodies that have been derived from embryonic stem cells (ESCs) have emerged as a valuable system for analyzing the processes that occur at the periimplantation stage 119. Embryoid bodies are composed of an outer layer of primitive endodermal (En) cells, which is underlined by a basement membrane (BM), a polarized 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 (see figure, parts a and b), and their differentiation recapitulates the processes of innercell-mass differentiation at the blastocyst stage. Therefore, they provide a useful tool for studying the molecular mechanisms that regulate ESC differentiation, BM assembly, epiblast polarization and cavity formation. Embryoid bodies that have been derived from ESCs that lack integrin-linked kinase (ILK) or particularly interesting Cys-His-rich protein-1 (PINCH1) have been used to address the functions of the ILK, PINCH, parvin (IPP) complex during mammalian development. Ilk / (see figure, part c) and Pinch1 / (see figure, part d) embryoid bodies produce a basement membrane but have defects in epiblast polarization and cavity formation; these defects are less severe in Pinch1 / embryoid bodies, which is consistent with the longer survival time in vivo 63,74. Red staining is for filamentous actin, green staining is for laminin-α1, blue staining (DAPI 4,6- diamidino-2-phenylindole) is for DNA. Part a of the figure is a phase-contrast image under 10 magnification, and parts b d are confocal images under 63 magnification. Inner cell mass Inner cells of the blastocyst that retain pluripotency and give rise to all cell types of the future body. En Ep BM Cav These discrepancies might reflect cell-type-specific differences in the requirement for ILK involvement upstream of AKT/PKB and GSK3β. Insights from studies using embryonic bodies. Because mice that lack β1 integrin, ILK or PINCH1 die early in development, embryoid bodies were used to identify the specific defects that cause peri-implantation lethality (BOX 2). β1 Integrin / embryoid bodies fail to deposit a basement membrane due to a defect in the synthesis of laminin 101,102. By contrast, Ilk / (BOX 2, figure, part c) and Pinch1 / (BOX 2, figure, part d) embryoid bodies produce a basement membrane but have defects in epiblast polarization and cavity formation; these defects are less severe in Pinch1 / embryoid bodies, consistent with the longer survival time in vivo 63,74. The defects in epiblast polarization are accompanied by abnormal localization of F-actin. These studies provide evidence 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 complex could regulate the actin cytoskeleton indirectly through NCK2, which binds to the actin modulators WASP (Wiskott Aldrich syndrome protein), PAK (p21-activated kinase) or DOCK180 (180-kDa protein downstream of CRK) 103. In addition, Pinch1 / embryoid bodies show abnormal cell cell adhesion and impaired endoderm survival 63. A reduction of ILK protein levels in Pinch1 / embryoid bodies, similar to that observed in Pinch1-siRNA-treated HeLa cells 29, provides a possible explanation for the phenotypic similarities between Ilk / and Pinch1 / embryoid bodies 63. However, the defects that are observed in Pinch1 / embryoid bodies represent ILK-independent functions and indicate that the functional interdependence between ILK and PINCH1 is not complete. This is supported by the observation that Ilk / cells still express low levels of PINCH1, and vice versa 31, revealing the existence of different functional pools of these proteins: a main pool in which ILK and PINCH1 are mutually dependent, and a second, smaller pool in which these proteins function independently. Recent data have also shown a role for IPP members in the regulation of cell polarity and cell cell contacts 63,74,104. Cell cell adhesion defects in Pinch1 / embryoid bodies are associated with a diffuse distribution of E-cadherin and the absence of adherens junctions 63. However, how PINCH1 regulates cell cell adhesion is poorly understood. Localization of PINCH1 to cell cell contacts has not been reported so far, but it is possible that PINCH1 regulates cell cell adhesion in an indirect manner. Although a role for ILK in cell cell contacts has been suggested based on studies in keratinocytes 65,66, Ilk / embryoid bodies do not show cell cell adhesion defects. Therefore, PINCH1-mediated regulation of cell cell adhesion must be ILK-independent. Unlike keratinocytes 66 and epiblast cells, trophoectodermal cells or primitive endoderm cells of embryoid bodies can polarize in the absence of ILK 74, which indicates that different epithelial cells might have different requirements for ILK. Conclusions The IPP complex serves as an important transducer of extracellular signals to control many aspects of cell morphology and cell behaviour. Although there are many functions that are likely to be common among all cell types, certain functions seem to be cell-type specific. How does the IPP complex achieve specificity? 28 JANUARY 2006 VOLUME 7