Integrin linked kinase
Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. The molecular architecture of PINCH (Particularly Interesting New Cysteine-Histidine rich protein), which consists exclusively of multiple LIM domains suggests that it may function as a platform for the docking and/or productive juxtaposition of proteins involved in integrin signaling. In order to evaluate the role of PINCH in integrin-mediated cellular events, function of PINCH in Drosophila melanogaster was directly tested in vivo. The steamer duck (stck) alleles, that were first identified in a screen for potential integrin effectors, represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with ßPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, it is shown that integrins are required for proper localization of PINCH at the myotendinous junction. Integrin-linked kinase (Ilk), is also essential for integrin function. Drosophila PINCH and Ilk are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, Ilk localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of Ilk. These results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion (Clark, 2003).
The genetic analysis of PINCH function has led to four main conclusions: (1) Drosophila PINCH is encoded by the stck locus and is essential for embryonic development and maintenance of tissue architecture; (2) PINCH is necessary for stable actin-membrane anchorage in muscle and contributes to integrin-dependent adhesion in muscle cells and epithelial cells; (3) integrins are required for the stable association of PINCH with muscle-attachment sites; and (4) the lethal stck mutant phenotype cannot be attributed to mislocalization of the PINCH-binding partner, Ilk, whose recruitment to muscle-attachment sites appears normal in stck mutant embryos (Clark, 2003).
Genetic analyses of the roles of integrins in Drosophila have clearly highlighted the importance of integrins for adhesion and signaling in vivo. Drosophila PINCH is colocalized with integrins in both muscle and epithelial cells. Integrins retain the capacity to accumulate at muscle-attachment sites in stck mutants, illustrating that PINCH does not have an obligatory role in the proper processing and membrane targeting of integrins in vivo. The integrin staining in stck mutants does lack the high degree of order and lateral registration observed in wild-type embryos. In the Drosophila system, it is difficult to distinguish whether this modest disorganization simply reflects the underlying disturbance of the musculature or if it is revealing some contribution of PINCH to maintenance of spatially restricted integrin localization. In C. elegans embryos in which PINCH function is compromised by unc-97 mutation, both integrin and vinculin spread laterally beyond their normal zones of accumulation in dense plaques, suggesting a role for PINCH in clustering of adhesive junction components in this system (Clark, 2003).
Interestingly, PINCH depends on the presence of integrins for its stable accumulation at muscle-attachment sites. Several other proteins, including Talin, Ilk, Myosin II and Short stop colocalize with ßPS integrin at Drosophila muscle-attachment sites. These proteins display variable levels of dependence on integrins for their localization. Like Talin, a well-established integrin effector, PINCH depends on the presence of integrins for its concentration at muscle-attachment sites. The reliance of PINCH and Talin on integrins for their spatially restricted accumulation in muscle emphasizes their connection to the integrin receptors (Clark, 2003).
Integrins must establish links to both extracellular determinants and to intracellular cytoskeletal elements in order to support strong adhesion. Examination of the cellular defects in stck mutant muscle suggests that PINCH contributes to the stabilization of actin-membrane linkages at integrin-rich adhesion sites. In a stck mutant muscle cell, the actin filaments lose their linear organization and eventually accumulate in clumps at one end of the cell. These defects are interpreted to mean that a primary consequence of disturbed PINCH function is a destabilization of the linkage between the actin cytoskeleton and the muscle membrane; it appears that the actin-membrane attachments in stck mutants lack the mechanical strength to remain intact during cyclic muscle contraction. Because integrin functionality relies on the ability of the receptors to establish a transmembrane link between the cytoskeletal elements and the extracellular matrix, reduced substratum attachment strength and/or stability might also be expected to occur if membrane cytoskeletal linkages were compromised. Consistent with this prediction, loss of adhesion is evident in the stck17-/- wing cell clones and, to some extent, in muscles of stck mutant embryos (Clark, 2003).
The molecular architecture of PINCH suggests that it may function as a platform for the docking and/or productive juxtaposition of protein partners. Ilk, a binding partner of PINCH, is thus a candidate to collaborate with PINCH in the stabilization of integrin-cytoskeletal linkages. Consistent with the view that PINCH and Ilk cooperate to promote stable actin anchorage at sites of integrin-mediated adhesion, the phenotypes that result from compromised function of either protein in Drosophila are very similar (Zervas, 2001; Clark, 2003). Moreover, PINCH and Ilk are colocalized in Drosophila embryos and are recovered in a protein complex isolated from embryos by immunoprecipitation. Drosophila PINCH also interacts directly with Ilk using two-hybrid methods. These results are consistent with findings for vertebrate PINCH and Ilk. PINCH and Ilk also colocalize at actin-membrane anchorage sites in C. elegans muscle, and elimination of either gene product was shown to produce a paralyzed at twofold stage (PAT) phenotype similar to that seen for ß-integrin mutants. Collectively, results in both invertebrate and vertebrate systems illustrate that the capacity to form a PINCH/Ilk complex has been conserved through evolution (Clark, 2003 and references therein).
Given the fact that Ilk and PINCH colocalize, co-precipitate and have similar loss of function phenotypes, it is possible that disturbed PINCH function could adversely affect Ilk localization and that such mislocalization might account for the stck mutant phenotype. To explore this possibility the localization of Ilk was examined in stck mutant embryos; Ilk was found to be unperturbed in its ability to accumulate at muscle-attachment sites, even when a dramatic lethal phenotype is evident in stck mutant embryos. As noted above, ßPS integrin also accumulates at muscle-attachment sites in stck mutant embryos. These findings illustrate that the proper localization of integrin and Ilk is not sufficient to stabilize actin membrane linkages at sites of integrin-dependent adhesion, and define PINCH as a critical component of the molecular machinery necessary for the tethering of actin to the integrin-rich membranes (Clark, 2003).
The demonstration that single ilk and stck mutants both display deficiencies in integrin-dependent processes illustrates that neither PINCH nor Ilk is sufficient on its own to support full integrin function. It is possible that PINCH acts as a positive regulator of Ilk function, either by modulating Ilk function by direct binding or by recruitment of an Ilk-modifying factor. Alternatively, Ilk may activate some PINCH function that is crucial for stabilization of actin-membrane linkages. Finally, a PINCH-Ilk protein complex may be a key component of the platform necessary for the recruitment of other proteins required to achieve stable actin-membrane associations. In this regard, it is of interest that PINCH and Ilk can be recovered in a complex with the Ilk-binding partner, CH-IlkBP, a calponin domain-containing protein related to Affixin and Actopaxin that could provide the link to actin filaments. Because the localization of Drosophila PINCH is dependent on integrins, the establishment of PINCH-Ilk complexes at muscle-attachment sites is not be supported in the absence of integrin function. This dependence of PINCH localization on integrins could provide a means to couple integrin adhesive function to its role in cytoskeletal anchorage (Clark, 2003).
In vertebrate cells, PINCH and Ilk appear to be mutually dependent on each other for their localization to integrin-rich focal adhesions (Zhang, 2002b). However, as noted above, despite their ability to interact with each other, PINCH and Ilk show distinct requirements for their recruitment to specific subcellular domains in Drosophila. In particular, it is shown that PINCH requires functional integrins for its localization to muscle-attachment sites, whereas it has previously been demonstrated that Drosophila Ilk fails to bind integrins directly and localizes normally in an integrin mutant. Rather than employing an association with integrins, Ilk may rely on a protein such as Paxillin for its targeting to integrin-rich sites. Although Drosophila PINCH requires integrins for its stable accumulation at muscle-attachment sites, there is no evidence that PINCH can associate directly with integrin cytoplasmic domains, therefore additional proteins probably act as a bridge (Clark, 2003 and references therein).
Many kinases that act in specific processes during development have been found to be ubiquitously expressed. By contrast, the mRNA distribution of Ilk is both temporally and spatially regulated. At the cellular blastoderm stage, Ilk expression is low, and it becomes stronger during gastrulation, mainly in the presumptive mesoderm. Its expression continues to increase through stage 13 within the somatic mesoderm, the midgut endoderm, and the surrounding visceral mesoderm. High levels of Ilk expression are maintained in the somatic and visceral muscles through the end of stage 16, when the embryos are almost fully developed (Zervas, 2001).
The finding that Ilk mRNA is expressed mainly in mesodermal tissues was confirmed by the localization of Ilk-GFP protein. However, low levels of Ilk are found to be distributed throughout the embryo. Some of these correspond to sites of integrin expression, such as at the leading edge of the epidermis and amnioserosa during dorsal closure. Strong expression of Ilk-GFP in the visceral mesoderm is first detected at stage 12, and it accumulates steadily during embryogenesis, following the level of mRNA expression. In mid–stage 16 embryos, Ilk-GFP is particularly strong in the midgut constrictions and the pharyngeal muscles. Low levels of Ilk-GFP were found in the ventral nerve cord and throughout the epidermis. The most striking feature of Ilk localization during embryogenesis is its tight localization at muscle attachment sites, where PS integrins are strongly expressed. These data show that high levels of Ilk-GFP are found at the places where integrins are found and the two proteins are tightly colocalized. There is not strong expression of Ilk at epidermal sites where wingless signaling through ß-catenin is particularly active, but low levels of Ilk are detectable throughout the embryo, so this expression pattern does not exclude the possibility of the suggested interaction between Ilk and ß-catenin/T cell factor signaling occurring in Drosophila (Zervas, 2001).
The colocalization of Ilk-GFP with integrins raises the question as to whether this is due to the binding of Ilk to the cytoplasmic tail of the ßPS subunit. The distribution of Ilk-GFP was examined in embryos mutant for the ßPS subunit and it was found that Ilk-GFP is still concentrated at the muscle attachments. In addition, no interaction between Drosophila Ilk and the ßPS subunit cytoplasmic domain could be detected by two-hybrid analysis, although the interaction between human ILK and human ß1 integrin could be reproduced. Weak interaction is seen between Drosophila Ilk and the ß1 integrin cytoplasmic tail, but not between human ILK and ßPS, indicating that the differences between the cytoplasmic tails (11 of 47 amino acids) have caused the loss of this interaction. Thus, no evidence could be provided for a direct interaction between Drosophila Ilk and the PS integrins, either in yeast or in the Drosophila embryonic muscles (Zervas, 2001).
The striking colocalization observed between Ilk and the PS integrins was not particularly surprising given that ILK was identified by its ability to bind integrins and their colocalization at focal contacts. It was more surprising to find that Ilk is localized normally to the muscle ends in the absence of integrins. This refutes one obvious possible mechanism for Ilk localization: apparently it is not recruited to the muscle ends by binding the cytoplasmic domain of ßPS. In cell culture, there is evidence to suggest that the NH2 terminus of Ilk may also play a part in localization (Li, 1999), and this is currently being testing in flies. Consequently, although no data supporting a direct interaction between Ilk and ßPS in vivo could be presented, the fact that loss of Ilk funtion gives a phenotype similar to that engendered from the loss of integrins strongly suggests that they interact. The only place where Ilk and integrins colocalize where Ilk does not cause an integrin-like phenotype is in the leading edge of the epidermis during dorsal closure. There are several possible explanations for this, the simplest being that the function of Ilk in these cells is redundant. It is worth noting that even the function of integrins in this process is not clear but is probably different from their assembly of strong adhesive junctions, as seen in muscles and wings (Zervas, 2001).
To assess the function of Ilk during development, mutations were sought in the Ilk gene. The region containing the Ilk gene was characterized genetically as part of the studies of the nearby gene Ecdysone-induced protein 78C (Eip78C). A genetic screen for lethal mutations uncovered by the deficiency Df(3L)Pc-14d, which deletes 78C2;D1, has identified two new lethal complementation groups: l(3)78Ca and l(3)78Cb. By mapping the genes within a genomic clone, it was found that Eip78C is proximal to Ilk, and deficiency mapping has shown that Eip78C is distal to l(3)78Cb, and proximal to l(3)78Ca, making the latter the best candidate for the Ilk locus. The Ilk coding region was sequenced from the DNA of flies containing the l(3)78Ca mutation and it was compared to the sequence of the gene in the strain that was mutagenized in the genetic screen. Sequences from three independent PCR amplifications of the l(3)78Ca mutant DNA have a single change, the nucleotide transition, which changes W211 to a stop codon. Therefore, this mutant gene will produce a truncated form of Ilk that lacks the kinase domain, which is also the region in human ILK (but not Drosophila Ilk) that binds to the integrin cytoplasmic tail by yeast two-hybrid interaction (Zervas, 2001).
A second allele, ilk2, was isolated in a screen for genes required for integrin-mediated adhesion in the adult wing. A single allele was isolated, which is homozygous lethal and causes a dominant wing blister phenotype. This mutation is lethal over Ilk1, although a few adult escapers (<5%) are seen. Genetic and cytological analysis reveals that ilk2 is associated with a reciprocal translocation between the second and third chromosomes. The dominant phenotype of ilk2 is not shared by other Ilk mutations (Ilk1 and Ilk deficiencies), so it is not certain whether the phenotype is caused by the aberration in the Ilk gene or a second site mutation (Zervas, 2001).
Embryos homozygous for Ilk1 die at the end of embryogenesis. To check whether the Ilk mutant embryos have defects similar to the pattern defects caused by the loss of the Wnt signal through ß-catenin (armadillo), or the reduced cuticle caused by loss of PKB, the cuticle secreted by the epidermis was examined. The cuticle of Ilk mutant embryos is completely normal with, for example, no indication of the dramatic pattern changes observed when ß-catenin is defective. The development of the midgut was also found to be normal, ruling out a requirement for Ilk in the ß-catenin signaling that occurs in the visceral mesoderm (Zervas, 2001).
Ilk is required for muscle adhesion in embryos. Having observed a relatively mild phenotype for Ilk, which is detectable later in development than the PS integrin phenotype, it was of interested to establish with certainty that this mutation completely removes Ilk function. A test was performed to see whether Ilk1 is an amorphic (null) allele by comparing the homozygous Ilk1 phenotype to that of Ilk1/Df(3L)Pc-14d embryos; the phenotypes were found to be identical, demonstrating that Ilk1 is an amorphic allele. Embryos transheterozygous for the two overlapping deficiencies Df(3L)Pc-14d and Df(3L)ME-107, and therefore completely deficient for Ilk, also have an equivalent muscle phenotype. Another possibility is that some Ilk mRNA or protein that is deposited in the egg during oogenesis persists until late stages of embryogenesis, masking a complete loss of function phenotype. The maternal product was removed by making germ-line clones of the Ilk1 mutation. It was found that embryos lacking both maternal and zygotic Ilk have a modestly more severe muscle phenotype, with clumping of the actin first visible a little earlier, at the end of stage 16. These embryos also have normal cuticles and do not display any additional defects compared with embryos lacking zygotic Ilk function. Therefore, maternal contribution of Ilk does not significantly compensate for the loss of Ilk synthesized during embryogenesis (Zervas, 2001).
Because Ilk has been proposed to act as an effector for integrin signaling, it was of interest to test whether it is required for this role in Drosophila. Two genes expressed in the Drosophila midgut that are targets of integrin signaling have been identified, providing the first transcriptional assay for integrin signaling in this organism. One of these targets, 258, was tested in embryos lacking Ilk function (also in the absence of maternal product) and 258 was found to be expressed normally, demonstrating that Ilk is not required for this integrin-signaling pathway. Complete loss of integrin function causes additional defects in midgut morphogenesis and dorsal closure, which are not seen in the Ilk mutant embryos (Zervas, 2001).
Overexpression of Ilk in cell culture has been proposed to affect several signaling molecules, including GSK3ß (Delcommenne, 1998); therefore, it was of interest to test whether this also occurs in a whole organism. The Gal4 system was used to drive additional expression of Ilk in the wing, on top of the endogenous protein levels, since this tissue shows clear phenotypes for the different signaling pathways. A UAS::Ilk construct was prepared and a variety of GAL4 drivers was used. No phenotype was detected: neither wing blisters indicative of an effect on integrins nor differentiation defects that might have revealed an effect on ß-catenin signaling. It was demonstrated that this construct does express functional Ilk by using it to rescue the embryonic lethality of Ilk1/Df(3L)Pc-14d flies. Expression of UAS::Ilk primarily in the mesoderm with the driver 24B::Gal4 (which is also expressed in the epidermal tendon cells in the embryo) is sufficient to rescue the embryonic lethality of the Ilk mutation, and viable adults were obtained. The surviving adults have blistered wings, perhaps due to insufficient expression of UAS::Ilk by 24B in the wings. Several points can be concluded from these experiments: the UAS::Ilk construct is functional, the lethality of the Ilk1 mutation is rescued by expression of the Ilk protein alone; Ilk is only required in the embryo in those cells where the 24B driver is expressed, and additional expression of Ilk does not perturb signaling through ß-catenin in the wing (Zervas, 2001).
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date revised: 20 January 2007
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