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2 British Columbia Cancer Agency and The Prostate Center at Vancouver Hospital, Jack Bell Research Center, Vancouver, BC, Canada, V6H3Z6
3 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada, V6T1Z3
Address correspondence to Shoukat Dedhar, BC Cancer Agency and Jack Bell Research Center, 2660 Oak St. Vancouver, BC, Canada, V6H3Z6. Tel.: (604) 875-5655. Fax: (604) 875-5452. E-mail: sdedhar{at}interchange.ubc.ca
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Abstract |
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Key Words: integrins; integrin-linked kinase; cell adhesion; cytoskeleton; signal transduction
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Integrin-linked kinasebinding proteins |
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The COOH-terminal domain of ILK interacts with the ß1 integrin cytoplasmic domain (Hannigan et al., 1996) and at least three different cytoplasmic adaptor proteins. CH-ILKBP, which contains two calponin homology (CH) domains, was identified and cloned in a yeast two-hybrid screen of a human cDNA library using the ILK COOH-terminal domain as bait (Tu et al., 2001). The CH2 domain of CH-ILKBP mediates the interaction with ILK (Tu et al., 2001). Two proteins (actopaxin [Nikolopoulos and Turner, 2000] and -parvin [Olski et al., 2001]) that are closely related structurally to human CH-ILKBP have been identified independently and cloned from rat and mouse cDNA libraries, respectively. Interestingly, actopaxin was identified in a search for proteins that bind to the LD1 motif of paxillin (Nikolopoulos and Turner, 2000), and
-parvin was identified based on its sequence homology with the actin-binding domain of
-actinin (Olski et al., 2001). Although murine actopaxin and
-parvin were identified based on binding activities toward proteins other than ILK, the high degree of sequence similarity of CH-ILKBP, actopaxin, and
-parvin at both the protein level (98% identical) and the cDNA level (90% identical) suggest that the human, rat, and mouse proteins are orthologues and therefore are likely to share the ILK-binding activity. In an independent study, Yamaji et al. (2001) identified and cloned another human protein, affixin, that also binds to the ILK COOH-terminal domain. CH-ILKBP and affixin are encoded by two different genes, but they share significant sequence similarity, particularly in the CH2 domains that mediate the ILK binding, suggesting that they likely recognize a common site on ILK. Affixin is the human orthologue of mouse ß-parvin, another recently described actin-binding protein (Olski et al., 2001). These recent studies define a new family of ILK-binding proteins that include CH-ILKBPactopaxin
-parvin and affixinß-parvin. In addition to interacting with the CH2 domains of CH-ILKBPactopaxin
-parvin and affixinß-parvin, the ILK COOH-terminal domain can also be recognized by the paxillin LD1 motif (Nikolopoulos and Turner, 2001).
Some, although probably not all, of the interactions described above occur simultaneously in cells. It has been demonstrated recently that ILK binds to PINCH and CH-ILKBP simultaneously through two separate domains (the NH2-terminal ANK domain and the COOH-terminal kinase domain), resulting in the formation of a multicomponent PINCHILKCH-ILKBP complex in cells (Tu et al., 2001). On the other hand, paxillin was not detected as part of the multicomponent ILK complex, despite its ability to interact with both ILK and CH-ILKBP through the LD1 motif.
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Localization of ILK to cell matrix contact sites |
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A crucial role of ILK in anchoring the actin filaments to cell matrix contact sites |
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Are there other linkages between ILK and the actin cytoskeleton? The answer is almost certainly yes. One potentially important connection could be provided by PINCH. In C. elegans, PINCH like ILK is required for the assembly of muscle-dense bodies (Hobert et al., 1999). Paxillin can interact with ILK and actin-binding proteins, including viculin and CH-ILKBPactopaxin-parvin, and therefore could provide another linkage to the actin filaments. An additional connection could be provided by affixinß-parvin. Overexpression of an affixin fragment containing the ILK-binding CH2 domain inhibits initial spreading of the cells (Yamaji et al., 2001). However, despite extensive efforts a direct interaction between affixinß-parvin and actin has not been detected in vitro (Yamaji et al., 2001). Finally, ILK can interact with the ß1 integrin cytoplasmic domain and thereby could be potentially linked to the actin filaments through other actin- and ß1 integrinbinding proteins such as talin. It is worth noting that an interaction between Drosophila ILK and the cytoplasmic domain of integrin ßPS was not detected in yeast two-hybrid analyses (Zervas et al., 2001). Consistent with this, Drosophila ILK is localized normally to muscle attachment sites in the absence of the ßPS integrin (Zervas et al., 2001). However, in C. elegans integrin is required for the proper localization of ILK (Mackinnon, A.C., and B.D. Williams. 2000. 40th American Society for Cell Biology Annual Meeting. 2664 [Abstr.]), suggesting a connection between integrin and ILK in this organism.
The multiple interactions between ILK and the actin cytoskeleton have at least two important implications. First, it could allow cells to modulate the physical strength of the connection at the cell matrix contact sites. Second, it could facilitate signal transduction and regulation through the cell matrix contact sites. In addition to binding to ILK through the LIM1 domain, PINCH can interact with Nck-2 (also known as Nckß or Grb4), an adaptor protein containing three Src homology (SH)3 domains and one SH2 domain, through the LIM4 domain (Tu et al., 1998) (Fig. 1). In turn, Nck-2 could potentially help to bring other components of the growth factor and small GTPase signaling pathways into proximity of the adhesion sites through interactions mediated by the SH2 and SH3 domains.
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Signaling role of ILK |
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Despite having a somewhat unusual kinase catalytic domain (Dedhar et al., 1999; Lynch et al., 1999; Dedhar, 2000), ILK has been shown recently to directly phosphorylate proteins such as PKB (PKB/Akt) on ser 473 (Persad et al., 2001a), glycogen synthase kinase 3 (GSK-3) on ser 9 (Persad et al., 2001b), myosin light chain (MLC) on ser 18/thr 19 (Deng et al., 2001), and ILK-binding protein affixin (Yamaji et al., 2001). In addition, ILK can phosphorylate the cytoplasmic domain of ß1 integrin subunit in vitro (Hannigan et al., 1996), although whether this occurs in intact cells is not clear. On the other hand, Lynch et al. (1999) have suggested that ILK regulates the phosphorylation of PKB/Akt on ser 473 indirectly. This suggestion was based on their inability to detect kinase activity in ILK immunoprecipitates and on the reversal of a dominant negative form of ILK to wild type by mutation of ser 343 to aspartic acid. However, more recent work by Persad et al. (2001a) has shown that a similar mutation in another dominant negative form of ILK does not cause this reversal and that ser 343 is critical for ILK kinase activity. Thus, the issue of whether ILK has kinase activity remains controversial, although the recent demonstration of ILK as a kinase (Deng et al., 2001; Persad et al., 2001a) seems to tip the balance in favor of ILK being a bona fide serine/threonine protein kinase. However, it is also likely that certain functions of ILK may not require its kinase activity. This is apparent from the suggestion that the regulation of integrinactin cytoskeleton interaction by ILK in Drosophila may not require kinase activity (Zervas et al., 2001), although it should be pointed out that the mutation in the ILK kinase domain on which this conclusion was based results in the retention of substantial amount of residual ILK activity (Persad et al., 2001a; Yamaji et al., 2001).
The role of ILK in the stimulation of various signaling pathways (Fig. 1 B) has been demonstrated by analyzing ILK activity in cells stimulated by ECM or growth factors and in cells overexpressing ILK. Overexpression of ILK in epithelial cells resulted in an increased phosphorylation of PKB/Akt and GSK-3 (Lynch et al., 1999; Dedhar, 2000), activating the former and inhibiting the latter. The hallmarks of overexpressing ILK in normal epithelial cells is the loss of cellcell adhesion, due to the downregulation of E-cadherin expression, and nuclear translocation of ß-catenin (Dedhar, 2000; Somasiri et al., 2001). The downregulation of E-cadherin expression may involve ILK-mediated activation of the E-cadherin repressor Snail (Tan et al., 2001), and nuclear ß-catenin activation may involve ILK-mediated inhibition of GSK-3 activity, resulting in ß-catenin stabilization (Persad et al., 2001b). The inhibition of GSK-3 by ILK also results in the activation of the transcription factor AP-1 (Dedhar, 2000), which along with ß-catenin/Tcf and the transcription factor CREB may also be responsible for ILK-mediated upregulation of cyclin D1 expression (D'Amico et al., 2000). ILK-induced AP-1 activity also results in the stimulation of expression of matrix metalloproteinase-9 (Troussard et al., 2000). It should be pointed out that these readily demonstrated signaling functions of ILK in mammalian cells appear not to be conserved in Drosophila, since inactivating mutation of Drosophila ILK does not appear to affect PKB/Akt, GSK-3, or ß-catenin pathways (Zervas et al., 2001).
One of the consequences of constitutive ILK activation, or overexpression, in mammalian cells is suppression of apoptosis and anoikis (Attwell et al., 2000; Persad et al., 2000). Both of these effects involve ILK-mediated activation of PKB/Akt and suppression of activation of caspase 3. The central role of ILK in these pathways can be inferred from experiments in which inhibiting ILK with a dominant negative form of ILK or by a pharmacological inhibitor of ILK induced apoptosis in cells in which PKB/Akt is activated constitutively (Persad et al., 2000). It is interesting that mammary gland tumors arise in ILK transgenic mice, and the tumor tissues exhibit many of the hallmarks of ILK overexpression in tissue culture cells, namely phosphorylation of PKB/Akt and GSK-3 but also downregulation of expression of E-cadherin and phosphorylation and activation of extracellular signalregulated kinase (Erk) (West et al., 2001). ILK also appears to regulate muscle differentiation by activating Erk, which suppresses transcription factors required for myogenic differentiation (Huang et al., 2000). The signaling pathway involved in the ILK-mediated Erk activation remains to be determined.
Therefore, it is apparent that ILK can control the activities of key signaling pathways, leading to the stimulation of downstream effector kinases and transcription factors, which either activate or repress the expression of genes encoding proteins involved in the regulation of cell survival, the cell cycle, cell adhesion, and ECM modification (Fig. 1 B). In addition, ILK may also be involved in the regulation of cell migration, cell motility, and contractility by directly phosphorylating proteins such as MLC (Deng et al., 2001) and affixin (Yamaji et al., 2001). The phosphorylation of the latter may also affect early stages of cell spreading by modulating the interaction of affixin with actin and ILK (Yamaji et al., 2001). Although not demonstrated yet, it is likely that ILK can also phosphorylate CH-ILKBP, since affixin and CH-LIKBP share significant sequence similarity and appear to bind to ILK via similar domains. ILK may also modulate adhesion and spreading by directly or indirectly regulating the phosphorylation of the ß1 integrin cytoplasmic domain, since serine phosphorylation of this subunit has been implicated in integrin-mediated regulation of cell adhesion, spreading, and formation of focal adhesion plaques (Mulrooney et al., 2000).
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Conclusion |
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Finally, dysregulation of ILK activity and/or expression appears to play important roles in certain disease states. Overexpression, or constitutive activation of ILK, due to inactivating mutations in tumor suppressors such as PTEN or APC lead to oncogenic transformation of cell lines and tumor formation in transgenic mouse models (Dedhar, 2000; West et al., 2001). In addition, ILK expression and activity are upregulated in Ewings sarcomas, advanced prostate cancers (Graff et al., 2001), and colonic poyposis and colorectal cancers (Marotta et al., 2001). ILK has also been demonstrated recently to be upregulated in the kidney glomeruli in patients with diabetic nephropathy (Guo et al., 2001), from children with congenital nephrotic syndrome, and in kidney podocytes from two murine models of proteinuria (Kretzler et al., 2001).
Given the central role of ILK in connecting integrins to the actin cytoskeleton and regulation of cell growth, dysregulation of ILK is likely to have much wider implications in many developmental and somatic diseases, underscoring the need to understand fully the mechanism of its regulation and activation.
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Footnotes |
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Acknowledgments |
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Submitted: 16 August 2001
Revised: 1 October 2001
Accepted: 2 October 2001
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References |
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