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Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane.

Zervas CG, Gregory SL, Brown NH - J. Cell Biol. (2001)

Bottom Line: Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B.ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters.Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK.

View Article: PubMed Central - PubMed

Affiliation: Wellcome/CRC Institute and Department of Anatomy, University of Cambridge, Cambridge CB2 1QR, United Kingdom.

ABSTRACT
Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

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Sequence conservation of Drosophila ILK. (a) A comparison of Drosophila ILK with C. elegans ILK and Homo sapiens ILK. Below each of the three domains is indicated the percent identity between that ILK and Drosophila ILK. (b) Alignment of highly conserved amino acids within kinases. The subdomain numbering is from the alignment of Hanks and Hunter 1995, and amino acids that are generally highly conserved and their counterparts in the ILK sequences are bold. (c) Point mutations in the ilk gene. The mutation in the ilk1 allele changes the codon encoding W211 into a stop codon, truncating the protein as shown. The site-directed mutations we generated in Drosophila ILK replace the invariant lysine at position 219 with methionine, the proline at 358 with serine, and the glutamate at 359 with lysine (b, *). The E359K change has been shown to inactivate the kinase activity of human ILK and v-src (Bryant and Parsons 1984; Delcommenne et al. 1998); the K219M change inactivates the kinase activity of Drosophila Raf (Sprenger et al. 1993), and P358S makes Drosophila Raf temperature sensitive (Hata et al. 1994).
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Figure 1: Sequence conservation of Drosophila ILK. (a) A comparison of Drosophila ILK with C. elegans ILK and Homo sapiens ILK. Below each of the three domains is indicated the percent identity between that ILK and Drosophila ILK. (b) Alignment of highly conserved amino acids within kinases. The subdomain numbering is from the alignment of Hanks and Hunter 1995, and amino acids that are generally highly conserved and their counterparts in the ILK sequences are bold. (c) Point mutations in the ilk gene. The mutation in the ilk1 allele changes the codon encoding W211 into a stop codon, truncating the protein as shown. The site-directed mutations we generated in Drosophila ILK replace the invariant lysine at position 219 with methionine, the proline at 358 with serine, and the glutamate at 359 with lysine (b, *). The E359K change has been shown to inactivate the kinase activity of human ILK and v-src (Bryant and Parsons 1984; Delcommenne et al. 1998); the K219M change inactivates the kinase activity of Drosophila Raf (Sprenger et al. 1993), and P358S makes Drosophila Raf temperature sensitive (Hata et al. 1994).

Mentions: The existence of Drosophila ILK was first revealed by sequence from the Berkeley Drosophila Genome Project. Within the 5′ end sequence of cDNA clone LD02317 is encoded a peptide with 65% identity to residues 1–45 of human ILK. We used this clone to screen an imaginal disc cDNA library and isolated one clone of 1,813 bp 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 (Fig. 1 a; C. elegans Sequencing Consortium 1998). We isolated genomic clones 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, we found that the ilk gene is interrupted by three introns and that the total length of the primary transcript is 2,347 nt.


Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane.

Zervas CG, Gregory SL, Brown NH - J. Cell Biol. (2001)

Sequence conservation of Drosophila ILK. (a) A comparison of Drosophila ILK with C. elegans ILK and Homo sapiens ILK. Below each of the three domains is indicated the percent identity between that ILK and Drosophila ILK. (b) Alignment of highly conserved amino acids within kinases. The subdomain numbering is from the alignment of Hanks and Hunter 1995, and amino acids that are generally highly conserved and their counterparts in the ILK sequences are bold. (c) Point mutations in the ilk gene. The mutation in the ilk1 allele changes the codon encoding W211 into a stop codon, truncating the protein as shown. The site-directed mutations we generated in Drosophila ILK replace the invariant lysine at position 219 with methionine, the proline at 358 with serine, and the glutamate at 359 with lysine (b, *). The E359K change has been shown to inactivate the kinase activity of human ILK and v-src (Bryant and Parsons 1984; Delcommenne et al. 1998); the K219M change inactivates the kinase activity of Drosophila Raf (Sprenger et al. 1993), and P358S makes Drosophila Raf temperature sensitive (Hata et al. 1994).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198807&req=5

Figure 1: Sequence conservation of Drosophila ILK. (a) A comparison of Drosophila ILK with C. elegans ILK and Homo sapiens ILK. Below each of the three domains is indicated the percent identity between that ILK and Drosophila ILK. (b) Alignment of highly conserved amino acids within kinases. The subdomain numbering is from the alignment of Hanks and Hunter 1995, and amino acids that are generally highly conserved and their counterparts in the ILK sequences are bold. (c) Point mutations in the ilk gene. The mutation in the ilk1 allele changes the codon encoding W211 into a stop codon, truncating the protein as shown. The site-directed mutations we generated in Drosophila ILK replace the invariant lysine at position 219 with methionine, the proline at 358 with serine, and the glutamate at 359 with lysine (b, *). The E359K change has been shown to inactivate the kinase activity of human ILK and v-src (Bryant and Parsons 1984; Delcommenne et al. 1998); the K219M change inactivates the kinase activity of Drosophila Raf (Sprenger et al. 1993), and P358S makes Drosophila Raf temperature sensitive (Hata et al. 1994).
Mentions: The existence of Drosophila ILK was first revealed by sequence from the Berkeley Drosophila Genome Project. Within the 5′ end sequence of cDNA clone LD02317 is encoded a peptide with 65% identity to residues 1–45 of human ILK. We used this clone to screen an imaginal disc cDNA library and isolated one clone of 1,813 bp 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 (Fig. 1 a; C. elegans Sequencing Consortium 1998). We isolated genomic clones 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, we found that the ilk gene is interrupted by three introns and that the total length of the primary transcript is 2,347 nt.

Bottom Line: Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B.ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters.Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK.

View Article: PubMed Central - PubMed

Affiliation: Wellcome/CRC Institute and Department of Anatomy, University of Cambridge, Cambridge CB2 1QR, United Kingdom.

ABSTRACT
Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

Show MeSH
Related in: MedlinePlus