The interaction of cells with the extracellular matrix regulates cell shape, motility, growth, survival, differentiation and gene expression, through integrin-mediated signal transduction. We used a two-hybrid screen to isolate genes encoding proteins that interact with the beta 1-integrin cytoplasmic domain. The most frequently isolated complementary DNA encoded a new, 59K serine/threonine protein kinase, containing four ankyrin-like repeats. We report here that this integrin-linked kinase (ILK) phosphorylated a beta 1-integrin cytoplasmic domain peptide in vitro and coimmunoprecipitated with beta 1 in lysates of mammalian cells. Endogenous ILK kinase activity was reduced in response to fibronectin. Overexpression of p59ILK disrupted epithelial cell architecture and inhibited adhesion to integrin substrates, while inducing anchorage-independent growth. We propose that ILK is a receptor-proximal protein kinase regulating integrin-mediated signal transduction.

The integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase that can interact directly with the cytoplasmic domains of the ␤1 and ␤3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. Overexpression of constitutively active ILK results in loss of cell-cell adhesion, anchorage-independent growth, and tumorigenicity in nude mice. We now show that modest overexpression of ILK in intestinal epithelial cells as well as in mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of Ecadherin expression, translocation of ␤-catenin to the nucleus, formation of a complex between ␤-catenin and the high mobility group transcription factor, LEF-1, and transcriptional activation by this LEF-1͞␤-catenin complex. We also find that LEF-1 protein expression is rapidly modulated by cell detachment from the extracellular matrix, and that LEF-1 protein levels are constitutively up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by activated H-ras or v-src oncogenes do not result in the activation of LEF-1͞␤-catenin. The results demonstrate that the oncogenic properties of ILK involve activation of the LEF-1͞␤-catenin signaling pathway, and also suggest ILK-mediated cross-talk between cellmatrix interactions and cell-cell adhesion as well as components of the Wnt signaling pathway.The integrin-linked kinase (ILK) was identified from a yeast two-hybrid genetic screen by using as bait the cytoplasmic domain of the ␤ 1 integrin subunit (1). ILK can interact with ␤ 1 and ␤ 3 integrins (1). ILK is a novel ankyrin-repeat containing serinethreonine kinase (1), which also contains sequence motifs found in pleckstrin homology domains capable of interacting with phosphoinositide lipids. The kinase activity of ILK can be modulated by interaction of cells with components of the extracellular matrix (1) or by integrin clustering. The activation or inhibition of ILK activity is cell-type dependent and can be modified by growth factors (M. Delcommenne and S. D., unpublished results). Overexpression of ILK in epithelial cells results in the stimulation of anchorage-independent cell growth (1) and cell cycle progression (2). The latter is caused by the constitutive up-regulation of expression of cyclin D 1 and cyclin A, resulting in the hyperphosphorylation of the retinoblastoma protein (2). Overexpression of ILK in epithelial cells also results in the induction of tumorigenicity in nude mice (3), indicating that ILK is a protooncogene.

Cell adhesion to substratum has been shown to regulate cyclin A expression as well as cyclin D-and E-dependent kinases, the latter via the up-regulation of cyclin D1 and the down-regulation of cyclin-Cdk inhibitors p21 and p27, respectively. This adhesion-dependent regulation of cell cycle is thought to be mediated by integrins. Here we demonstrate that stable transfection and overexpression of the integrin-linked kinase (ILK), which interacts with the ␤1 and ␤3 integrin cytoplasmic domains, induces anchorage-independent cell cycle progression but not serum-independent growth of rat intestinal epithelial cells (IEC18). ILK overexpression results in increased expression of cyclin D1, activation of Cdk4 and cyclin E-associated kinases, and hyperphosphorylation of the retinoblastoma protein. Normal, untransformed epithelial cells require anchorage to a substratum for cell growth and survival. Adhesion to the extracellular matrix (ECM) 1 is required for progression of cells through the G 1 and into the S phase of the cell cycle. When forced to remain in suspension, such cells arrest in the G 1 phase of the cell cycle and undergo apoptosis (1-3). Oncogenic transformation frequently induces anchorage-independent growth, in vitro, and is a specific correlate of tumor growth in vivo (4,5).In fibroblasts, cell adhesion has recently been demonstrated to regulate cell cycle progression by inducing the expression of cyclin D1 (6), the activation of cyclin E-Cdk2 (6, 7), and phosphorylation of the retinoblastoma protein (Rb) (6). Fibroblast adhesion also results in the down-regulation of expression of the Cdk inhibitor proteins, p21 and p27 (6, 7). The combined, adhesion-dependent elevation in cyclin D1 and the decrease in the expression of p21 and p27 result in the stimulation of cyclin D-Cdk4 and cyclin E-Cdk2 activities, both of which can phosphorylate Rb. This latter event relieves the restriction of the entry of cells into S phase, presumably by the release of the transcription factor E2F from phosphorylated Rb (8, 9). In some cell types the expression of cyclin A is also regulated in an anchorage-dependent manner (3, 10, 11), and anchorage-independent growth induced by activated Ras has been shown to depend on cyclin A expression (11). However, in these latter experiments cyclin D1 expression (12) and cyclin E-dependent kinase activity (11) were also dependent on Ras activation. Although mitogens can also activate cyclin D-and cyclin E-dependent kinases, cell adhesion per se can regulate these activities. The regulation of G 1 Cdks, therefore, requires the convergence of signals from both growth factors as well as from the ECM.Anchorage of cells to the ECM is mediated to a large extent by integrins, a large family of heterodimeric cell surface receptors (13, 14). The interaction of integrins with ECM ligands results in the transduction of intracellular signals leading to stimulation of tyrosine phosphorylation (15, 16), turnover of phosphoinositides (17), and activation of the Ras-mitogen-activated protein kinase (MA...

Lipid rafts are plasma-membrane microdomains that are enriched in certain lipids (sphingolipids, glycosphingolipids and cholesterol), as well as in lipid-modified proteins. Rafts appear to exist in the liquid-ordered phase, which contributes to their partitioning from the surrounding liquid-disordered glycerophospholipid environment. DRM (detergent-resistant membrane) fractions isolated from cells are believed to represent coalesced lipid rafts. We have employed extraction using two different non-ionic detergents, Brij-96 and Triton X-100, to isolate detergent-resistant lipid rafts from rat basophilic leukaemia cell line RBL-2H3, and compared their properties with each other and with plasma-membrane vesicles. DRM fractions were isolated as sealed unilamellar vesicles of similar size (135-170 nm diameter), using either sucrose-density-gradient sedimentation or gel-filtration chromatography. Lipid rafts isolated using Brij-96 and Triton X-100 differed in density, protein content and the distribution between high- and low-density fractions of the known raft constituents, Thy-1, and the non-receptor protein tyrosine kinases, Yes and Lyn. Lyn was found in the raft microdomains in predominantly phosphorylated form. The level of enrichment of the protein constituents of the isolated lipid rafts seemed to depend on the ratio of cell lipid/protein to detergent. As indicated by reactivity with anti-Thy-1 antibodies, lipid rafts prepared using Brij-96 appeared to consist of vesicles with primarily right-side-out orientation. Both Brij-96 and Triton X-100 appear to isolate detergent-insoluble raft microdomains from the rat basophilic leukaemia cell line RBL-2H3, but the observed differences suggest that either the detergents themselves play a role in determining the physicochemical characteristics of the resulting DRM fractions, or different subsets of rafts are isolated by the two detergents.

Specific membrane microdomains (including lipid rafts) exist in myelin but have not been fully characterized. Myelin basic protein (MBP) maintains the compactness of the myelin sheath and is highly posttranslationally modified. Thus, it has been suggested that MBP might also have other functions, e.g., in signal transduction. Here, the distribution of MBP and its modified forms was studied, spatially and temporally, by detailed characterization of membrane microdomains from developing and mature bovine myelin. Myelin membranes were extracted with three different detergents (Brij 96V, CHAPS, or Triton X-100) at 4 degrees C. The detergent-resistant membranes (DRMs), representing coalesced lipid rafts, were isolated as low-buoyant-density fractions on a sucrose density gradient. These myelin rafts were disrupted when cholesterol was depleted with methyl-beta-cyclodextrin. The use of CHAPS detergent led to enrichment of several myelin proteins, including phospho-Thr97-MBP, in the DRMs from mature myelin. Citrullinated and methylated MBP remained in "nonraft" microdomains. In contrast, the DRMs from early myelin were enriched in Golli-MBP, Fyn, Lyn, and CNP. The localization of various proteins in DRMs was further supported by the colocalization of these lipid raft components in cultured mouse oligodendrocytes. Thus, there is a developmental regulation of posttranslationally modified forms of MBP into specific membrane microdomains.

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16 O/ 18 O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergentinsoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five non-cysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16 O/ 18 O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content. KeywordsQuantitative proteomics; combined 16 O/ 18 O and ICAT stable isotopic labeling; Triton X-100 and Brij-96 detergent-insoluble membrane proteins

The surface layer (S-layer) protein genes of the uranium mining waste pile isolate Bacillus sphaericus JG-A12 and of its relative B. sphaericus NCTC 9602 were analysed. The almost identical N-termini of the two S-layer proteins possess a unique structure, comprising three N-terminal S-layer homologous (SLH) domains. The central parts of the proteins share a high homology and are related to the S-layer proteins of B. sphaericus CCM 2177 and P-1. In contrast, the C-terminal parts of the S-layer proteins of JG-A12 and NCTC 9602 differ significantly between each other. Surprisingly, the C-terminal part of the S-layer protein of JG-A12 shares a high identity with that of the S-layer protein of B. sphaericus CCM 2177. In both JG-A12 and NCTC 9602 the chromosomal S-layer protein genes are followed by a newly identified putative insertion element comprising three ORFs, which encode a putative transposase, a putative integrase/recombinase and a putative protein containing a DNA binding helix-turn-helix motif, and the S-layer-protein-like gene copies sllA (9602) or sllB (JG-A12). Interestingly, both B. sphaericus strains studied were found to contain an additional, plasmid-located and silent S-layer protein gene with the same sequence as sllA and sllB. The primary structures of the corresponding putative proteins are almost identical in both strains. The N-terminal and central parts of these S-layer proteins share a high identity with those of the chromosomally encoded functional S-layer proteins. Their C-terminal parts, however, differ significantly. These results strongly suggest that the S-layer protein genes have evolved via horizontal transfer of genetic information followed by DNA rearrangements mediated by mobile elements. INTRODUCTIONRegularly structured paracrystalline surface layers (S-layers) are one of the most common surface structures found in bacteria and archaea. Most of them are composed of protein or glycoprotein monomers with the ability to self-assemble in two-dimensional arrays (Sára & Sleytr, 2000;Sidhu & Olsen, 1997). In most cases, they are the major protein species, constituting up to 15 % of the total protein produced by the cells (Kuen et al., 1994). As the outermost cell structure the S-layers serve as an interface between the bacterial cell and the environment. Several functions have been ascribed to S-layers: a molecular sieve (Sára & Sleytr, 1987), virulence factors in several pathogenic bacteria (Ishiguro et al., 1981;Lewis et al., 1987;Mesnage et al., 2001; Mignot et al., 2001), an attachment structure for high-molecular-mass extracellular proteins such as amylases (Matuschek et al., 1994;Egelseer et al., 1995Egelseer et al., , 1996, an adhesin with affinity for human epithelial cells and fibronectin (Hynönen et al., 2002), and sorption of toxic heavy metal ions (Raff, 2002;Merroun et al., 2005).It is essential for micro-organisms to be able to respond rapidly to changes in the environment. One way in which they can do this is to alter their surface properties by varying protein expressio...

In recent years, intense interest has been focussed on the properties and biological functions of specialized membrane domains known as lipid rafts [1,2]. Rafts consist of cholesterol-sphingolipid-rich regions within the plasma membrane, stabilized by interactions between cholesterol and the long saturated acyl chains of sphingolipids. They are thought to exist in the liquid-ordered phase, which has properties intermediate between those of the liquid-crystalline and gel phases [3,4]. Acylated and lipid-modified proteins are P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH R B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM 1 ganglioside-and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-b-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1 ⁄ GM 1 microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM 1 colocalized, while Pgp and caveolin-1, or Pgp and GM 1 , did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.