Identification of a Protein Interacting with ras-p21- by Chemical Cross-Linking

Gunzburg, Jean de; Riehl, R; Weinberg, Robert A.

doi:10.1007/978-1-4757-1235-3_37

Cited by 8 publications

(11 citation statements)

References 19 publications

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“…Surface Hsp60 may activate and modulate the binding activity of mature ␣ 3 ␤ 1 integrin on breast cancer cells during their metastasis to lymph nodes and osteoblasts (2). In addition, Hsp60 enhanced the in vivo GTPase activity of surface p21 ras , a signal transduction protein, in rat fibroblasts (9,21). Similarly, interaction of the L. monocytogenes protein InlB with its receptor, the complement protein gC1q-R, is associated with phosphoinositide 3-kinase (6), whose activation may affect host cell actin polymerization, a key factor in L. monocytogenes uptake and virulence (32).…”

Section: Discussionmentioning

confidence: 99%

Heat Shock Protein 60 Acts as a Receptor for theListeriaAdhesion Protein in Caco-2 Cells

et al. 2004

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The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

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Section: Discussionmentioning

confidence: 99%

Heat Shock Protein 60 Acts as a Receptor for theListeriaAdhesion Protein in Caco-2 Cells

et al. 2004

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“…Hsp60 is a 57-kDa protein that was originally well characterized because of its function as a molecular chaperonin and its association with the mitochondrial matrix. However, a number of recent reports have documented the presence of Hsp60 not only in the mitochondria but also in several extramitochondrial sites including the cytoplasmic membrane (3,5,13,19,21,31,32,42). Although many studies have associated the surface expression of Hsp60 with stressed, apoptotic, or transformed cells, membrane Hsp60 is now considered to be typical of eukaryotic cells in general (33,34).…”

Section: Discussionmentioning

confidence: 99%

“…Phosphorylation of E, A, and Y random polymers by c-Src kinase was stimulated 20-fold in the presence of Hsp60 (1). Also, Hsp60 interacts with p21 ras , a signaling molecule in the cytoplasmic membrane (5,13). Whether any of these activities is involved in internalization of S. aureus into epithelial cells is currently under investigation in our laboratories.…”

Section: Discussionmentioning

confidence: 99%

Staphylococcal Fibronectin Binding Protein Interacts with Heat Shock Protein 60 and Integrins: Role in Internalization by Epithelial Cells

et al. 2000

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Bacteria have developed various mechanisms for inducing internalization into nonprofessional phagocytes (8,9). A shared requirement is that a molecular interaction must occur between a bacterial surface adhesin and a host ligand in the cytoplasmic membrane. This interaction must be of sufficiently high affinity to induce signal transduction through the membrane, resulting in cytoskeletal rearrangements and uptake of the organism (14, 37). For some organisms, a simple model involving the binding of a bacterial adhesin to its host receptor is sufficient to induce uptake. Yersinia species and Listeria monocytogenes are examples of organisms with this pattern of uptake. The adhesins on these facultative intracellular pathogens bind directly to integrins or E-cadherin, respectively, with an affinity that is sufficient to induce internalization (15,24).A number of reports have described bacterial surface adhesins, which adhere to host extracellular matrix (ECM) proteins. The ECM binding proteins are termed MSCRAMMS, and Staphylococcus aureus expresses several of these proteins with different ligand specificities (17,18,27). We demonstrated previously that the S. aureus surface adhesin responsible for stimulating signal transduction upon uptake by nonprofessional phagocytes is one of its MSCRAMMs, fibronectin (Fn) binding protein (FnBP) (6). Our data were confirmed in two additional publications (22,28). This finding raised several questions regarding the nature of the molecular interactions at the host cell surface.Fn is the ECM protein commonly associated with integrins. It is known that Fn is bivalent and can serve as a bridging molecule between FnBP and the host cell integrins (17,26, 39). Although other bacteria use Fn as a link to adhere to host tissues, the mechanisms by which this linkage could induce internalization are less clear. For example, Tran Van Nhieu and Isberg showed that coating of S. aureus with Fn did not lead to efficient internalization (37). It was proposed that the binding affinity between Fn and integrins was not sufficient to induce uptake. Interestingly, at least one organism, Neisseria gonorrhoeae has overcome this limitation by employing a heparin-containing accessory coreceptor, which is necessary to induce maximal internalization by HEp-2 cells (39).The present study was initiated to identify potential cellular ligands and other molecular requirements for uptake of S. aureus by nonprofessional phagocytes. Using a variety of methods, we found that FnBP binds directly to heat shock protein 60 (Hsp60) on the membranes of human and bovine epithelial cells. Fn and ␤ 1 integrins are also required for maximal uptake. Based on these combined results, a potential model to explain the molecular interactions leading to uptake of S. aureus is proposed.

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“…The spheroplasts were incubated in 5 ml of YPD-0.7 M sorbitol-25 mM Tris-HCl (pH 7.5) at 30°C for 20 min, followed by washing the spheroplasts twice with 2 ml of cross-linking buffer (0.4 M sorbitol, 150 mM Na acetate, 2 mM Mg 2ϩ acetate, 20 mM HEPES-KOH [pH 7.5]). The spheroplasts were suspended in 2 ml of cross-linking buffer containing the cross-linking agent dithiobis(succinimidylpropionate) (DSP; Pierce) to a final concentration of 2 mM (20,36). After 30-min incubation at room temperature, the reaction was stopped by incubation for 5 min with 50 mM Tris-HCl (pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

Dpb11 Controls the Association between DNA Polymerases α and ɛ and the Autonomously Replicating Sequence Region of Budding Yeast

Masumoto

Sugino

Araki

2000

Molecular and Cellular Biology

164

176

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Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase (Dpb11-Pol complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Pol associated with these fragments. Association of Dpb11 and Pol with these fragments was mutually dependent, suggesting that the Dpb11-Pol complex associates with the ARS. Moreover, Dpb11 was required for the association of Pol␣-primase with the fragments. Thus, it seems likely that association of the Dpb11-Pol complex with the ARS fragments is required for the association of the Pol␣-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Pol in dpb11-1 cells associated with early-and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases ␣ and and the ARS.

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Identification of a Protein Interacting with ras-p21- by Chemical Cross-Linking

Cited by 8 publications

References 19 publications

Heat Shock Protein 60 Acts as a Receptor for theListeriaAdhesion Protein in Caco-2 Cells

Heat Shock Protein 60 Acts as a Receptor for theListeriaAdhesion Protein in Caco-2 Cells

Staphylococcal Fibronectin Binding Protein Interacts with Heat Shock Protein 60 and Integrins: Role in Internalization by Epithelial Cells

Dpb11 Controls the Association between DNA Polymerases α and ɛ and the Autonomously Replicating Sequence Region of Budding Yeast

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