Focal Adhesion Complex Proteins Research Articles

Modeling cellular adhesion.

Cellular adhesion strength, described by the fraction of cells that detach from a surface for a range of applied shear stresses, is predictive of cancer cell migration in 2D and 3D environments and the overall metastatic potential of these cells. Simply stated, cell adhesion strength depends on the balance between cell-surface bond formation and breakage rates. This has been previously modeled as such using the concentration of adhesion proteins such as integrins, the cell-surface interaction area, and a shear force dependent bond breakage rate between the surface ligands and adhesion proteins. However, we have shown that changes in intracellular myosin motor activity, actin stress fibers and force responsive focal adhesion complex proteins can also influence cell adhesion strength and alter cell migration behavior. Using a computational model, we explore how various intracellular factors beyond the concentration of adhesion proteins, govern cell adhesion strengths as measured by shear flow analysis. Overall, our model focuses on 5 key cytoskeletal aspects and their impact on cellular adhesion because of their apparent relevance from previous studies. These include: average number of stress fibers per cell (dictated by f-actin and myosin concentrations), available integrins per stress fiber (dictated by integrin concentration and actin branching), likelihood of bound paxillin, active myosin motors (dictated by myosin light chain phosphorylation), and finally actin polymerization and depolymerization dynamics (governed by time between depolymerization and repolymerization events). The model is further used as a predictive tool in order to determine the particular intracellular factors differentiating cancer strains. The goal of this research is to unravel biomechanical and consequently biochemical differences between cytoskeletal elements of highly metastatic and non-metastatic cancer cells.

FRMD3 inhibits the growth and metastasis of breast cancer through the ubiquitination-mediated degradation of vimentin and subsequent impairment of focal adhesion

Recurrence and metastasis are the main causes of breast cancer (BRCA)-related death and remain a challenge for treatment. In-depth research on the molecular mechanisms underlying BRCA progression has been an important basis for developing precise biomarkers and therapy targets for early prediction and treatment of progressed BRCA. Herein, we identified FERM domain-containing protein 3 (FRMD3) as a novel potent BRCA tumor suppressor which is significantly downregulated in BRCA clinical tissue and cell lines, and low FRMD3 expression has been closely associated with progressive BRCA and shortened survival time in BRCA patients. Overexpression and knockdown experiments have revealed that FRMD3 significantly inhibits BRCA cell proliferation, migration, and invasion in vitro and suppresses BRCA xenograft growth and metastasis in vivo as well. Mechanistically, FRMD3 can interact with vimentin and ubiquitin protein ligase E3A(UBE3A) to induce the polyubiquitin-mediated proteasomal degradation of vimentin, which subsequently downregulates focal adhesion complex proteins and pro-cancerous signaling activation, thereby resulting in cytoskeletal rearrangement and defects in cell morphology and focal adhesion. Further evidence has confirmed that FRMD3-mediated vimentin degradation accounts for the anti-proliferation and anti-metastasis effects of FRMD3 on BRCA. Moreover, the N-terminal ubiquitin-like domain of FRMD3 has been identified as responsible for FRMD3-vimentin interaction through binding the head domain of vimentin and the truncated FRMD3 with the deletion of ubiquitin-like domain almost completely loses the anti-BRCA effects. Taken together, our study indicates significant potential for the use of FRMD3 as a novel prognosis biomarker and a therapeutic target of BRCA and provides an additional mechanism underlying the degradation of vimentin and BRCA progression.

Microenvironmental stiffness mediates cytoskeleton re-organization in chondrocytes through laminin-FAK mechanotransduction

Microenvironmental biophysical factors play a fundamental role in controlling cell behaviors including cell morphology, proliferation, adhesion and differentiation, and even determining the cell fate. Cells are able to actively sense the surrounding mechanical microenvironment and change their cellular morphology to adapt to it. Although cell morphological changes have been considered to be the first and most important step in the interaction between cells and their mechanical microenvironment, their regulatory network is not completely clear. In the current study, we generated silicon-based elastomer polydimethylsiloxane (PDMS) substrates with stiff (15:1, PDMS elastomer vs. curing agent) and soft (45:1) stiffnesses, which showed the Young’s moduli of ~450 kPa and 46 kPa, respectively, and elucidated a new path in cytoskeleton re-organization in chondrocytes in response to changed substrate stiffnesses by characterizing the axis shift from the secreted extracellular protein laminin β1, focal adhesion complex protein FAK to microfilament bundling. We first showed the cellular cytoskeleton changes in chondrocytes by characterizing the cell spreading area and cellular synapses. We then found the changes of secreted extracellular linkage protein, laminin β1, and focal adhesion complex protein, FAK, in chondrocytes in response to different substrate stiffnesses. These two proteins were shown to be directly interacted by Co-IP and colocalization. We next showed that impact of FAK on the cytoskeleton organization by showing the changes of microfilament bundles and found the potential intermediate regulators. Taking together, this modulation axis of laminin β1-FAK-microfilament could enlarge our understanding about the interdependence among mechanosensing, mechanotransduction, and cytoskeleton re-organization.

Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates \u03b15\u03b21 integrin endocytosis

Endosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility.

Altered Actin Dynamics in Cell Migration of GNE Mutant Cells

Cell migration is an essential cellular process that requires coordination of cytoskeletal dynamics, reorganization, and signal transduction. The actin cytoskeleton is central in maintaining the cellular structure as well as regulating the mechanisms of cell motility. Glycosylation, particularly sialylation of cell surface proteins like integrins, regulates signal transduction from the extracellular matrix to the cytoskeletal network. The activation of integrin by extracellular cues leads to recruitment of different focal adhesion complex proteins (Src, FAK, paxillin, etc.) and activates the signal including Rho GTPases for the regulation of actin assembly and disassembly. During cell migration, the assembly and disassembly of actin filament provides the essential force for the cell to move. Abnormal sialylation can lead to actin signaling dysfunction leading to aberrant cell migration, one of the main characteristics of cancer and myopathies. In the present study, we have reported altered F-actin to G-actin ratios in GNE mutated cells. These cells exhibit pathologically relevant mutations of GNE (UDP N-acetylneuraminic 2-epimerase/N-acetylmannosamine kinase), a key sialic acid biosynthetic enzyme. It was found that GNE neither affects the actin polymerization nor binds directly to actin. However, mutation in GNE resulted in increased binding of α-actinin to actin filaments. Further, through confocal imaging, GNE was found to be localized in focal adhesion complex along with paxillin. We further elucidated that mutation in GNE resulted in upregulation of RhoA protein and Cofilin activity is downregulated, which could be rescued with Rhosin and chlorogenic acid, respectively. Lastly, mutant in GNE reduced cell migration as implicated from wound healing assay. Our study indicates that molecules altering Cofilin function could significantly revert the cell migration defect due to GNE mutation in sialic acid-deficient cells. We propose cytoskeletal proteins to be alternate drug targets for disorders associated with GNE such as GNE myopathy.

Augmenting cancer cell proteomics with cellular images - A semantic approach to understand focal adhesion.

If monolayers of cancer cells are exposed to microgravity, some of the cells cease adhering to the bottom of a culture flask and join three-dimensional aggregates floating in the culture medium. Searching reasons for this change in phenotype, we performed proteome analyses and learnt that accumulation and posttranslational modification of proteins involved in cell-matrix and cell-cell adhesion are affected. To further investigate these proteins, we developed a methodology to find histological images about focal adhesion complex (FA) proteins. Selecting proteins expressed by human FTC-133 and MCF-7 cancer cells and known to be incorporated in FA, we transformed the experimental data to RDF to establish a core semantic knowledgebase. Applying iterative SPARQL queries to Linked Open Databases, we augmented these data with additional functional, transformation- and aggregation-related relationships. Using reasoning, we retrieved publications with images about the spatial arrangement of proteins incorporated in FA. Contextualizing those images enabled us to gain insights about FA of cells changing their site of growth, and to independently validate our experimental results. This new way to link experimental proteome data to biomedical knowledge from various sources via searching images may generally be applied in science when images are a tool of knowledge dissemination.

Prostate transglutaminase (TGase-4, TGaseP) enhances the adhesion of prostate cancer cells to extracellular matrix, the potential role of TGase-core domain

BackgroundTransglutaminase-4 (TGase-4), also known as the Prostate Transglutaminase, is an enzyme found to be expressed predominately in the prostate gland. The protein has been recently reported to influence the migration and invasiveness of prostate cancer cells. The present study aimed to investigate the influence of TGase-4 on cell-matrix adhesion and search for the candidate active domain[s] within the protein.MethodsHuman prostate cancer cell lines and prostate tissues were used. Plasmids that encoded different domains and full length of TGase-4 were constructed and used to generate sublines that expressed different domains. The impact of TGase-4 on in vitro cell-matrix adhesion, cell migration, growth and in vivo growth were investigated. Interactions between TGase-4 and focal adhesion complex proteins were investigated using immunoprecipitation, immunofluorescence and phosphospecific antibodies.ResultsTGase-4 markedly increased cell-matrix adhesion and cellular migration, and resulted in a rapid growth of prostate tumours in vivo. This effect resided in the Core-domain of the TGase-4 protein. TGase-4 was found to co-precipitate and co-localise with focal adhesion kinase (FAK) and paxillin, in cells, human prostate tissues and tumour xenografts. FAK small inhibitor was able to block the action mediated by TGase-4 and TGase-4 core domain.ConclusionTGase-4 is an important regulator of cell-matrix adhesion of prostate cancer cells. This effect is predominately mediated by its core domain and requires the participation of focal adhesion complex proteins.

Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.

KIAA1797/FOCAD encodes a novel focal adhesion protein with tumour suppressor function in gliomas

In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.

Gambogic acid inhibits tumor cell adhesion by suppressing integrin β1 and membrane lipid rafts-associated integrin signaling pathway

Cell adhesion plays an important role in the steps of cancer metastasis. Regulation of cell–cell (intercellular) and cell–matrix adhesion is a promising strategy for cancer progression. Gambogic acid is a xanthone derived from the resin of the Chinese plant Garciania hanburyi, with potent anti-metastasis activity on highly metastatic cells. The aim of this study was to investigate the function and mechanism of gambogic acid on tumor adhesion. We found that gambogic acid strongly inhibited the adhesion of human cancer cells to fibronectin. This inhibition was associated with the deformation of focal adhesion complex, which was mediated by suppressing the expression of integrin β1 and integrin signaling pathway. In vitro, cell lipid rafts clustering was inhibited following treatment of gambogic acid, which induced the suppression of integrin β1 and focal adhesion complex proteins colocalization within rafts. Moreover, gambogic acid significantly decreased cellular cholesterol content, whereas cholesterol replenishment lessened the inhibitory effect of gambogic acid on cell adhesion. Real-time PCR analysis showed that gambogic acid reduced mRNA levels of hydroxymethylglutaryl-CoA reductase and sterol regulatory element binding protein-2, while increased acetyl-CoA acetyltransferase-1/2. Taken together, these results demonstrate that gambogic acid inhibits cell adhesion via suppressing integrin β1 abundance and cholesterol content as well as the membrane lipid raft-associated integrin function, which provide new evidence for the anti-cancer activity of gambogic acid.

A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis

Cleft formation is the initial step of branching morphogenesis in many organs. We previously demonstrated that ROCK 1 regulates a nonmuscle myosin II-dependent mechanochemical checkpoint to transition initiated clefts to progressing clefts in developing submandibular salivary glands. Here, we report that ROCK-mediated integrin activation and subsequent formation of focal adhesion complexes comprise this mechanochemical checkpoint. Inhibition of ROCK1 and nonmuscle myosin II activity decreased integrin β1 activation in the cleft region and interfered with localization and activation of focal adhesion complex proteins, such as focal adhesion kinase (FAK). Inhibition of FAK activity also prevented cleft progression, by disrupting recruitment of the focal adhesion proteins talin and vinculin and subsequent fibronectin assembly in the cleft region while decreasing ERK1/2 activation. These results demonstrate that inside-out integrin signaling leading to a localized recruitment of active FAK-containing focal adhesion protein complexes generates a mechanochemical checkpoint that facilitates progression of branching morphogenesis.

Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach

BackgroundWe and others have previously reported that resveratrol (RSV) suppresses colon cancer cell proliferation and elevates apoptosis in vitro and/or in vivo, however molecular mechanisms are not fully elucidated. Particularly, little information is available on RSV's effects on metabolic pathways and the cell-extra cellular matrix (ECM) communication that are critical for cancer cell growth. To identify important targets of RSV, we analyzed whole protein fractions from HT-29 advanced human colon cancer cell line treated with solvent control, IGF-1 (10 nM) and RSV (150 μM) using LC/MS/MS-Mud PIT (Multidimensional Protein Identification Technology).ResultsPentose phosphate pathway (PPP), a vital metabolic pathway for cell cycle progression, was elevated and suppressed by IGF-1 and RSV, respectively in the HT-29 cell line. Enzymatic assays confirmed RSV suppression of glucose-6 phosphate dehydrogenase (rate limiting) and transketolase, key enzymes of the PPP. RSV (150 μM) suppressed, whereas IGF-1 (10 nM) elevated focal adhesion complex (FAC) proteins, talin and pFAK, critical for the cell-ECM communication. Western blotting analyses confirmed the suppression or elevation of these proteins in HT-29 cancer cells treated with RSV or IGF-1, respectively.ConclusionsProteomic analysis enabled us to establish PPP and the talin-pFAK as targets of RSV which suppress cancer cell proliferation and induce apoptosis in the colon cancer cell line HT-29. RSV (150 μM) suppressed these pathways in the presence and absence of IGF-1, suggesting its role as a chemo-preventive agent even in obese condition.

Abstract 3269: Calpain knockdown in human breast carcinoma cells reduced xenograft tumor growth rates and inhibited in vitro cell migration and proliferation

Abstract Ubiquitously expressed µ- and m-calpain are calcium dependent intracellular cysteine proteases that have been implicated in the regulation of cell proliferation, survival, apoptosis, migration, and invasion. Stable expression and proteolytic activity of the µ- and m-calpain catalytic subunits (encoded by capn1 and capn2 genes, respectively) requires a common regulatory subunit encoded by the capn4/capns1 gene; thus both µ- and m-calpain activities can be blocked by genetic knockout or knockdown of capn4 expression. Calpain is also regulated by an endogenously expressed inhibitor peptide, calpastain. We screened a panel of human breast cancer and normal epithelial cell lines for expression of calpain and calpastatin. µ- and m-calpain and calpastatin were abundantly expressed in MDA-MB-231, SKBR3, T47D, and MCF-7 breast cancer lines, as well as the normal human breast epithelial cell line MCF-10A. A stably expressed lentivirally encoded shRNA directed against the capn4 transcript was used to effectively knockdown µ- and m-calpain expression and activity in MDA-MB-231 breast cancer cells. This correlated with attenuated proliferation, migration and invasion, but resistance to anoikis. Biochemical analysis also suggests a role for calpain in the regulation of focal adhesion complex proteins such FAK, PTP-1B, talin and F-actin. Finally, knockdown of calpain in MDA-MB-231 cells significantly reduced their ability to form tumors when orthotopically injected into the mammary fat pads of nude mice. These observations provide direct genetic evidence that calpain plays a pro-tumorigenic role in the MDA-MB-231 breast cancer cell model system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3269.

Correlation between the interaction of the vinculin tail domain with lipid membranes, its phosphorylation and cell mechanical behaviour

Cell adhesion and cell-cell contacts are a pre-requisite for proper metabolism, protein synthesis and cell survival. Integrins are the transmembrane receptors that link the extracellular matrix via the FAC (focal adhesion complex) with the cytoskeleton. Vinculin is a pivotal FAC protein that has not only been implicated in regulating FAC formation and transmitting mechanical forces, but also in associating with membranous lipids in biological systems.

Talin1 Promotes Tumor Invasion and Metastasis via Focal Adhesion Signaling and Anoikis Resistance

Talin1 is a focal adhesion complex protein that regulates integrin interactions with ECM. This study investigated the significance of talin1 in prostate cancer progression to metastasis in vitro and in vivo. Talin1 overexpression enhanced prostate cancer cell adhesion, migration, and invasion by activating survival signals and conferring resistance to anoikis. ShRNA-mediated talin1 loss led to a significant suppression of prostate cancer cell migration and transendothelial invasion in vitro and a significant inhibition of prostate cancer metastasis in vivo. Talin1-regulated cell survival signals via phosphorylation of focal adhesion complex proteins, such as focal adhesion kinase and Src, and downstream activation of AKT. Targeting AKT activation led to a significant reduction of talin1-mediated prostate cancer cell invasion. Furthermore, talin1 immunoreactivity directly correlated with prostate tumor progression to metastasis in the transgenic adenocarcinoma mouse prostate mouse model. Talin1 profiling in human prostate specimens revealed a significantly higher expression of cytoplasmic talin1 in metastatic tissue compared with primary prostate tumors (P < 0.0001). These findings suggest (a) a therapeutic significance of disrupting talin1 signaling/focal adhesion interactions in targeting metastatic prostate cancer and (b) a potential value for talin1 as a marker of tumor progression to metastasis.

Correlation of dystrophin–glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscle

The dystrophin-glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures in muscle fibres that link the intracellular cytoskeleton to the extracellular matrix. DGC and FAC proteins are abundant in slow-type muscles, indicating the structural reinforcement which play a pivotal role in continuous force output to maintain posture for long periods. The aim of the present study was to examine the expression of these structures across fast-type muscles containing different myosin heavy chain (MHC) isoform patterns which reflect the fatigue-resistant characteristics of skeletal muscle. We measured the expression of dystrophin and beta1 integrin (representative proteins of DGC and FAC respectively) in plantaris, extensor digitorum longus, tibialis anterior, red and white portions of gastrocnemius, superficial portion of vastus lateralis and diaphragm, in comparison with soleus (SOL) and cardiac muscle from rats. The expression of dystrophin and beta1 integrin correlated positively with the percentage of type I, IIa and IIx MHC isoforms and negatively with that of type IIb MHC isoform in fast-type skeletal muscles, and their expression was abundant in SOL and cardiac muscle. Our results support the idea that DGC and FAC are among the factors that explain the fatigue-resistant property not only of slow-type but also of fast-type skeletal muscles.

High-Throughput Phosphotyrosine Profiling Using SH2 Domains

Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. One of the major consequences of tyrosine phosphorylation is the creation of binding sites for proteins containing Src homology 2 (SH2) domains. To profile the global tyrosine phosphorylation state of the cell, we have developed proteomic binding assays encompassing nearly the full complement of human SH2 domains. Here we provide a global view of SH2 domain binding to cellular proteins based on large-scale far-western analyses. We also use reverse-phase protein arrays to generate comprehensive, quantitative SH2 binding profiles for phosphopeptides, recombinant proteins, and entire proteomes. As an example, we profiled the adhesion-dependent SH2 binding interactions in fibroblasts and identified specific focal adhesion complex proteins whose tyrosine phosphorylation and binding to SH2 domains are modulated by adhesion. These results demonstrate that high-throughput comprehensive SH2 profiling provides valuable mechanistic insights into tyrosine kinase signaling pathways.

Emodin Inhibits Tumor Cell Adhesion through Disruption of the Membrane Lipid Raft-Associated Integrin Signaling Pathway

Cell adhesion and spreading is a crucial step in the metastatic cascade of cancer cells, and interruption of this step is considered to be a logical strategy for prevention and treatment of tumor metastasis. Emodin is the major active component of the rhizome of Rheum palmatum L., with known anticancer activities. Here, we first found that emodin significantly inhibited cell adhesion of various human cancer cells. This inhibition was achieved through suppressing the recruitment of focal adhesion kinase (FAK) to integrin beta(1) as well as the phosphorylation of FAK followed by the decreased formation of focal adhesion complex (FAC). In understanding the underlying mechanisms, we found that emodin inhibited the lipid raft clustering and subsequent colocalization of integrin beta(1) and FAC proteins within lipid rafts. Lipid profile analysis revealed significant decrease of cholesterol and sphingolipids in raft fraction after emodin treatment. Cholesterol replenishment abolished the adverse effect of emodin on the translocation of integrin beta(1) and FAC proteins into the lipid raft fraction and cell adhesion. Therefore, data from this study provide novel evidence that emodin inhibits cell adhesion and spreading through disruption of the membrane lipid raft-associated integrin signaling pathway.

Hsp72 Interacts with Paxillin and Facilitates the Reassembly of Focal Adhesions during Recovery from ATP Depletion

The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in both the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p < 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion.

Phosphorylation-Dependent Paxillin-ERK Association Mediates Hepatocyte Growth Factor-Stimulated Epithelial Morphogenesis

Activation of the hepatocyte growth factor (HGF) receptor c-met results in the regulation of cell-matrix interactions, including the MAPK-dependent stimulation of epithelial cell morphogenesis. In the present study we demonstrate that HGF stimulates the localization of ERK to sites of cell-matrix interactions and that this is mediated by the tyrosine phosphorylation-dependent association of inactive ERK and the focal adhesion complex protein paxillin. In addition, paxillin was found to associate with the upstream MAP kinases Raf and MEK, resulting in a complex that can mediate localized ERK activation. Mutation of the ERK binding site in paxillin prevented HGF-stimulated ERK-paxillin association and eliminated HGF-induced cell spreading and branching process formation. These experiments reveal that paxillin-dependent ERK activation at sites of cell-matrix interaction is critical for HGF-stimulated epithelial morphogenesis.