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Temporal dissection of beta1-integrin signaling indicates a role for p130Cas-Crk in filopodia formation.
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Fällman)
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Fällman)
Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). (Fällman)
2004 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 22, s. 22893-22901Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Invasin-promoted spreading of beta1-integrin-deficient cells, transfected with the beta1A- or beta1B-integrin splice variants, were used to dissect early beta1-integrin signaling events. The beta1B isoform, which has a different membrane-distal part of the cytoplasmic tail from beta1A, is defective in signaling and function. When plated on surfaces coated with the high affinity ligand invasin, beta1B-integrin-expressing cells spread by forming filopodia with distinct adhesive phosphotyrosine complexes at the tips, without signs of lamellipodia. This suggested that the beta1B-integrin mediated a partial signaling sufficient for formation of filopodia but insufficient for lamellipodia formation. When screening for proteins present in the distal filopodial phosphotyrosine complexes of beta1B cells, p130Cas and the filopodia proteins vasodilator-stimulated phosphoprotein and talin were found, whereas the typical focal complex proteins focal adhesion kinase, paxillin, and vinculin were not. Invasin-promoted adhesion induced complex formation of p130Cas and the adapter Crk. Moreover, Crk together with Dock180 were present at the filopodial tips of beta1B-integrin-expressing cells, and there was a prominent Rac1 activation. Expression of dominant negative variants of p130Cas or CrkII blocked beta1B-integrin-mediated filopodia formation, indicating that this signaling scaffold is central in this process.

sted, utgiver, år, opplag, sider
2004. Vol. 279, nr 22, s. 22893-22901
Emneord [en]
Animals, Antigens; CD29/*physiology, Crk-Associated Substrate Protein, Phosphorylation, Proteins/*physiology, Proto-Oncogene Proteins/physiology, Proto-Oncogene Proteins c-crk, Pseudopodia/*physiology, Rabbits, Rats, Retinoblastoma-Like Protein p130, Signal Transduction
HSV kategori
Identifikatorer
URN: urn:nbn:se:umu:diva-16654DOI: 10.1074/jbc.M309693200PubMedID: 15044442OAI: oai:DiVA.org:umu-16654DiVA, id: diva2:156327
Tilgjengelig fra: 2007-10-08 Laget: 2007-10-08 Sist oppdatert: 2018-06-09bibliografisk kontrollert
Inngår i avhandling
1. Effects of invasin and YopH of Yersinia pseudotuberculosis on host cell signaling
Åpne denne publikasjonen i ny fane eller vindu >>Effects of invasin and YopH of Yersinia pseudotuberculosis on host cell signaling
2004 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Alternativ tittel[sv]
Effekter av proteinerna invasin och YopH från bakterien Yersinia pseudotuberculosis på värdcellen
Abstract [en]

Integrins are a large family of membrane-spanning heterodimeric (αβ) receptors that bind to ligands on other cells or to extracellular matrix (ECM) proteins. These receptors mediate bidirectional signaling over the cell membrane to induce signaling cascades mediating functions as cell adhesion, spreading and migration. This signaling takes place at cell-matrix adhesions, which are sites where clustered and ligand-bound integrins connect to and mediate stabilization of the actin cytoskeleton, and induce signaling cascades. Integrins have a short cytoplasmic tail that is crucial for the bidirectional signaling, and the β1-integrin subunit exists in five splice variants only differing in the membrane-distal part of the cytoplasmic tail. This region of the almost ubiquitously expressed β1-integrin, β1A, contains two protein tyrosine motifs (NPXYs) interspaced with a threonine-rich region, while this region of the β1B splice variant is completely different and lacks known motifs. In contrast to the β1A-integrin, the β1B variant cannot mediate cell-matrix adhesion formation following binding to ECM ligands.

The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1-integrins on the host cell with invasin, and this stimulates uptake of the bacterium. However, upon binding to the host cell, pathogenic Yersinia strains inject virulence effectors that block uptake. One effector responsible for the blocking is a tyrosine phosphatase, YopH. We identified the targets for this effector in the macrophage-like cell line J774A.1, which represent a professional phagocyte and thus is the likely target cell for the antiphagocytic effect of Yersinia. Two YopH target proteins were p130Cas and ADAP, of which the latter interestingly is an adapter protein specifically expressed in hematopoietic cells. ADAP has previously been implicated to participate in Fc-receptor-mediated phagocytosis and in communication between T-cell receptors and integrins.

We also studied the importance of the cytoplasmic tail of β1-integrin for uptake of Yersinia. The GD25 cell line, which is a fibroblast-like cell line that lacks endogenous β1-integrins, was used together with GD25 cells transfected with β1B, β1Α or cytoplasmic tail mutants of β1A. These studies revealed that β1B-integrins could bind to invasin but not mediate uptake of Yersinia, while β1A both bound to invasin and mediated uptake. The first NPXY motif (unphosphorylated) and the double-threonines of the unique part of β1A were important for the ability of integrin to mediate uptake of Yersinia. These studies lead to the interesting finding that, when these cells were allowed to spread on invasin, those that expressed β1A spread as normal fibroblasts while for β1B-integrin-expressing cells, only finger-like protrusions of filopodia were formed. This provided us with a tool to study formation of filopodia without interference of the tightly linked process of lamellipodia formation. Initially, proteins that localized to the tip complex of these filopodia were identified. These were talin, VASP and interestingly the p130Cas-Crk-DOCK180 scaffold, while FAK, paxillin and vinculin were absent. In addition, VASP, p130Cas and Crk were shown to be important for the filopodia formation in GD25β1B. Further, the role of the actin motor myosin X, which previously has been implicated in formation of filopodia, was studied in the GD25Β1B cells and it was shown that myosin X not was important for filopodia formation, but that it recruited FAK and vinculin to the tip complexes of filopodia.

sted, utgiver, år, opplag, sider
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2004. s. 67
Emneord
Molecular biology, β1-integrin, Yersinia pseudotuberculosis, Invasin, YopH, Cell-Matrix Adhesion, Filopodia, Cell Spreading, Molekylärbiologi
HSV kategori
Forskningsprogram
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-183 (URN)91-7305-588-3 (ISBN)
Disputas
2004-03-05, Major Groove, 6L, NUS, Inst f molekylärbiologi, Umeå Universitet, 901 87 Umeå, Umeå, 09:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2004-01-30 Laget: 2004-01-30 Sist oppdatert: 2019-01-22bibliografisk kontrollert

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