BIOLOGICAL OVERVIEW

Drosophila Integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane. Two-hybrid screening in yeast has been used to identify intracellular proteins that bind to the cytoplasmic tails of integrins. Mammalian integrin-linked kinase (ILK; Hannigan, 1996) was identified using the yeast two-hybrid technique. ILK contains three clear ankyrin repeats at the NH2 terminus, followed by a kinase domain most similar in sequence to the kinase domain of Raf serine/threonine protein kinase. The ankyrin repeats provide modules for protein-protein interaction and have been shown to bind to the first LIM domain in the protein PINCH (Tu, 1999). The kinase domain overlaps at its NH2 terminus with a short sequence proposed to bind phosphoinositides as it shares some similarity to a pleckstrin homology domain, and phosphoinositides have been shown to activate ILK activity (Delcommenne, 1998). Overexpression of wild-type and mutant forms of ILK in cell culture has suggested diverse roles for ILK. It has been implicated in both positive and negative regulation of integrin function and has been shown to colocalize with integrins at focal adhesions (Hannigan, 1996; Delcommenne, 1998; Li, 1999). Expression of high levels of ILK in cells has been shown to cause increased accumulation of ß-catenin with T cell factor (Drosophila homolog: Pangolin) in the nucleus (Novak, 1998). The translocation of these molecules to the nucleus is usually associated with a cellular response to signals from the Wnt family of secreted proteins. Wnt signaling downregulates a negative regulator of the Wnt pathway, GSK3ß, and ILK has been proposed to act similarly, by increasing the phosphorylation of GSK3ß (Delcommenne, 1998). Overexpression of ILK also results in the increased phosphorylation of protein kinase B (PKB), and has been proposed to represent the elusive serine473 kinase activity, which is required for full activation of PKB (Delcommenne, 1998; Zervas, 2001 and references therein).

Although the COOH terminus of ILK is clearly related to kinase domains, there are a few results suggesting that kinase activity may not be the main function of this domain. The ILK sequence diverges from other kinases at some extremely well-conserved positions, such as the aspartic acid in subdomain VIb, which is involved in the transfer of the phosphate. In contrast, several lines of biochemical evidence support the view that ILK is an active kinase: ILK has been shown to phosphorylate peptides on serine and threonine residues, as well as other standard kinase substrates such as myelin basic protein (Hannigan, 1996; Morimoto, 2000); replacement of a conserved residue in the substrate binding loop of the kinase domain (E359K) results in a mutant ILK protein that has lost its in vitro kinase activity and causes dominant-negative effects (Delcommenne, 1998; Wu, 1998) or loss of wild-type overexpression effects (Novak, 1998) when overexpressed in cell culture. A mutation in the ATP binding site (K219M) has been shown to eliminate the ability of ILK to stimulate phosphorylation of PKB on Ser473 (Lynch, 1999). However, introducing a second mutation, predicted to mimic autophosphorylation at a potential site in ILK (S342E), partially restores PKB phosphorylation despite the mutation in the ILK ATP binding site (Lynch, 1999). This has led to a proposal that the primary substrate of ILK may be itself, and that the main function of ILK is that of an adaptor rather than a kinase (Zervas, 2001 and references therein).

With the identification of a putative ILK ortholog in the expressed sequence tag sequences of the Drosophila genome project, a genetic analysis of Ilk function was initiated. The main goal of this work was to determine whether Ilk is essential for the pathways it has been proposed to interact with: integrins, ß-catenin, and PKB. Phenotypes corresponding to each of these pathways have been described in Drosophila, and they are each distinct, allowing an unambiguously determination of whether Ilk is required for any of these pathways. The results show that Ilk is required for integrin-mediated adhesion, but not signaling involving integrins, ß-catenin (Armadillo), or PKB. In support of this, an Ilk-green fluorescent protein (GFP) fusion protein has been found to be concentrated at sites of integrin adhesion. No defects have been found in the development or viability of flies in which the wild-type Ilk gene has been replaced with one containing a mutation that inactivates the kinase activity of human ILK. These results suggest that the main function of ILK may be as a structural adaptor between the plasma membrane and the cytoskeleton at sites of integrin-mediated cell adhesion (Zervas, 2001).

Drosophila Ilk is required for two cell-ECM adhesion events that also require integrin function: muscle attachment and adhesion between the two surfaces of the wing. Since Ilk is particularly highly expressed in the mesoderm, the defects in mesodermal derivatives were examined. A particularly prominent phenotype associated with mutations in the ßPS subunit (see Myospheroid) or the alphaPS2 subunit evinces detachment of the somatic muscles. The muscle detachment begins in stage 15 and is well advanced by stage 16 in embryos lacking the ßPS subunit, but starts later in the absence of alphaPS2, with only some of the muscles detaching during stage 16. Ilk mutant embryos were stained with an antibody against muscle myosin, but no detachment during stage 16 could be detected. The deposition of the cuticle at the end of stage 16 prevents staining with antibodies in stage 17 and later, but the embryos are still permeable to phalloidin, which stains filamentous actin. Phalloidin staining of stage 17 Ilk mutant embryos shows defects in most of the muscles by this point in development, with the actin clumped together rather than extended along the length of the muscles as seen in wild-type embryos. Embryos lacking the alphaPS2 subunit have a more severe phenotype at this stage, with all the muscles detached and rounded up or spindle shaped (Zervas, 2001).

The phenotype of Ilk¹ in the embryo is consistent with a role for Ilk in integrin-mediated adhesion; whether this is also true during adult development was assessed. Although Ilk activity is required for viability, it is possible to assay the role of Ilk after embryogenesis by making mosaic mutant animals by mitotic recombination. Clones of cells within the wing that are homozygous for the Ilk¹ allele cause a wing blister. This phenotype appears identical to that caused by clones mutant for the integrin subunits ßPS, alphaPS1, and alphaPS2, as well as a number of other loci. The marked Ilk¹ clones allowed the determination that wing blisters are associated with clones on either side of the wing blade, indicating either that Ilk is essential for both PS1 and PS2 integrin-mediated adhesion, whose functions are primarily restricted to the dorsal and ventral sides, respectively, or that lkK is required for a parallel pathway required for wing adhesion. The localization of Ilk-GFP within the pupal wing was examined and it was found that Ilk is concentrated in a series of discs corresponding to the sites of adhesion between the dorsal and ventral surfaces. Clones within the wing mutant for ß-catenin, or GSK3, cause a clear phenotype (loss/gain of wing margin), which is not seen in any of the Ilk mutant wings, providing additional evidence that Ilk is not required for the function of this pathway in Drosophila (Zervas, 2001).

In summary, the phenotype of the Ilk mutations indicates that Ilk is essential for two cell-ECM adhesion events that also require integrin function: muscle attachment and adhesion between the two surfaces of the wing. These data do not allow a determination of whether Ilk is required for integrin function directly or for some other hypothetical adhesive mechanism that is also essential for adhesion at these sites. If Ilk functions directly with integrins, the difference between the Ilk mutant phenotype and that of the integrins suggests that it mediates some integrin intracellular interactions, but not all. Ilk is not required for pathways involving ß-catenin or PKB, although this does not rule out a redundant role for Ilk in these pathways. Many of the effects of Ilk on these pathways have been seen when Ilk is overexpressed in cells in culture, so tests were made to determine whether overexpression is required to reveal the role of Ilk in these processes (Zervas, 2001).

There are a number of possible roles that Ilk could have in the generation of the normal actin configuration within the muscles. It could be required for the extracellular adhesion of the muscle to the ECM, for example by modifying integrin adhesion to the ECM, or it could be required for the link between the actin filaments and the intracellular face of the muscle membrane. To distinguish between these two possibilities, the plasma membrane of muscle cells was marked so the membrane attachment to the ECM as well as the actin filaments in the muscle cells could be examined. GFP fused to the NH₂-terminal myristylation signal of Src kinase was used to mark the membrane. In wild-type embryos, the actin filaments extend to the very ends of the muscles so that the signal from rhodamine-phalloidin and Src-GFP overlap. By contrast, in the Ilk¹/Df(3L)Pc-14d mutant embryos, muscles were seen where the actin had detached from the plasma membrane and the membrane remained attached at its normal position adjacent to the ECM. This is visible because the filamentous actin retracts to one end of the muscle, presumably due to the contraction of the actin/myosin fibers. Muscles were also seen where both the plasma membrane and actin had retracted, although they were still separate. This detachment of the membrane from the ECM-containing attachment site is seen in muscles lacking PS integrin function, but in that case the actin filaments are still anchored to the membrane. Thus, both Ilk and integrins are required for the ECM-cytoskeletal link, but the point at which breakage occurs differs: in the absence of integrins, the membrane pulls away from the ECM as a severe, early defect; whereas, in the absence of Ilk, the cytoskeleton also pulls away from the membrane as a later defect after the integrins have been localized and bind to the ECM. This phenotype allows a clear distinction between the two possible roles for Ilk in muscle attachment: Ilk is not required for the stage 16 adhesion of integrins to the ECM, but instead is required later to maintain the link between the contractile actin filaments and the plasma membrane at the ends of the muscles (Zervas, 2001).

Although overexpression of wild-type Ilk does not have an effect in Drosophila, it is possible that Ilk functions, normally masked by redundancy, would be seen if a dominant-negative form were expressed. Expression of Ilk-containing mutations in conserved residues within the kinase domain, that inhibit kinase activity in human ILK, has been shown to cause effects that are consistent with these ILK mutants acting in dominant-negative fashion. One of these mutations was made in Drosophila Ilk, replacing glutamic acid 359 with a lysine. This amino acid is located in subdomain VIII, is invariant in all known protein kinases, and this change has been shown to reduce drastically the kinase activity of v-Src and human ILK (Delcommenne, 1998). However, when the Gal4 system was used to express the E359K mutant Ilk with a variety of drivers, no effects were seen. This raises the possibility that inactivation of the Ilk kinase domain does not disturb its function in Drosophila; therefore, whether this mutant construct is able to rescue the embryonic lethality of the Ilk¹ mutation, when driven by 24B::Gal4, was tested. Surprisingly, this construct is as good at rescuing the Ilk mutation as the wild-type Ilk. To avoid any effects of overexpression, this mutation was introduced into the genomic rescue construct, which expresses Ilk from its own promoter. The kinase-defective Ilk protein expressed from ilk^E359K is also able to fully rescue the embryonic lethality of Ilk¹ mutant flies to adult viability, indistinguishable from the wild-type version of Ilk gene. To confirm that these results are not due to some peculiarity in the E359K mutation, the same rescue construct was generated with different mutations. The invariable lysine 219, which is located in subdomain II and mediates interaction with ATP, was changed to methionine and the highly conserved proline 358 was changed to serine. The K219M change causes the closely related Drosophila Raf kinase to completely lose kinase activity and P358S causes it to become temperature sensitive, with normal activity at 16°C and no activity above 20°C. However, both Ilk^K219M and Ilk^P358S mutant genes are able to rescue the lethality of Ilk¹ mutant flies to adult viability. The Ilk^P358S mutant gene rescues Ilk¹ equally well at 18°, 25°, and 29°C, demonstrating that, unlike Raf, this mutation does not cause Ilk to become temperature sensitive for the functions required for viability (Zervas, 2001).

The ability of the kinase-defective Ilk to function normally is surprising, but not unprecedented, since Abl is a bona fide kinase for which a kinase-dead form can rescue loss of function mutations (Henkemeyer, 1990). In the case of Abl, there are functions that require kinase activity, but they are redundant, and only apparent when additional genes are mutated. This may certainly also be true for Ilk, but a screen for mutations that enhance the Ilk phenotype may be required before any such kinase-dependant function can be assayed. However, the kinase-defective rescue is much more dramatic in Ilk than in Abl, and Ilk is significantly diverged from the consensus kinase sequence, so the straightforward interpretation that Ilk is not required to act as a kinase in vivo must be considered a possibility. If no kinase activity is needed, some explanation is required for the conservation between ILK and the kinase domains of functional kinases. One explanation could be that the sequence conservation reflects a structural requirement rather than an enzymatic one. Perhaps having lost enzymatic activity it retains the ability to bind 'target' sites. This has been seen in 'anti-phosphatases,' which retain the phosphatase domain structure and bind phosphorylated residues but lack catalytic activity. This is consistent with the observation that the integrin-binding activity of ILK was found in the kinase domain (Hannigan, 1996). A detailed biochemical investigation into what molecules bind Ilk, and how, will hopefully be able to shed light on this question (Zervas, 2001).

GENE STRUCTURE

cDNA clone length - 1813

Bases in 5' UTR - 170

Exons - 4

Bases in 3' UTR - 296

PROTEIN STRUCTURE

Amino Acids - 448

Structural Domains

The existence of Drosophila Ilk was first revealed by sequence from the Berkeley Drosophila Genome Project. Encoded within the 5' end sequence of cDNA clone LD02317 is a peptide with 65% identity to residues 1-45 of human ILK. This clone was used to screen an imaginal disc cDNA library and one clone of 1,813 bp was isolated that encodes a 448 amino acid protein that is similar throughout its length to human ILK and the ILK encoded in the genome sequence of Caenorhabditis elegans. Genomic clones were isolated containing the ilk gene by screening a filter of gridded P1 clones, which also served to map the gene to cytological interval 78C1--4. By sequencing the gene, it was found that the ilk gene is interrupted by three introns and that the total length of the primary transcript is 2,347 nt (Zervas, 2001).

The predicted Drosophila Ilk protein (Lynch, 1999) is 60% identical and 75% similar overall to human ILK, and these two are more similar to each other than to any other protein kinase, indicating that they are orthologs. All three domains in ILK, the ankyrin repeats, pleckstrin homology-like domain, and kinase domain, are conserved in the three species. The high conservation between Drosophila, C. elegans, and human ILK strongly suggests that its function has been conserved during evolution (Zervas, 2001).

Human ILK differs in sequence from other kinases at several residues that are otherwise invariant, so the conservation at these positions is of particular interest. These residues were found to differ from the consensus in all three ILK sequences, but are not conserved among ILKs in the different species. In kinase subdomain I, the kinase consensus is GxGxxG, with the middle glycine invariant, and the first two glycines different in each ILK sequence. The invariant aspartic acid in subdomain VIb, which is involved in the transfer of the phosphate, is not conserved, and again is different in the three sequences. By contrast, the invariant lysine in subdomain II, which is a key residue in ATP binding, and the motif A/SPE in subdomain VIII, which is involved in substrate recognition, are conserved in all three ILK sequences. Thus, the divergence of human ILK from other kinases is not a recent change, but occurred before the separation of invertebrates and vertebrates (Zervas, 2001).

date revised: 23 June 2023

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