| 1. |
- Dukuzumuremyi, Jean-Marie, et al.
(författare)
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The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor
- 2000
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 275:45, s. 35281-35290
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Tidskriftsartikel (refereegranskat)abstract
- The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.
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| 2. |
- Henriksson, M L, et al.
(författare)
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Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras
- 2000
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Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 347:1, s. 217-222
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Tidskriftsartikel (refereegranskat)abstract
- We have examined the functional consequences of ADP-ribosyltransferase modification of Ras by the exoenzyme S (ExoS) protein of Pseudomonas aeruginosa. ExoS has been shown previously to ADP-ribosylate a number of proteins, including members of the Ras superfamily, which play an essential role in the processes of cell proliferation, differentiation, motility and cell division. HeLa and NIH3T3 cells were infected with ExoS protein, which was delivered via the type III secretion system of the heterologous host Yersinia pseudotuberculosis. Infection of mammalian cells with ExoS results in a change in the ratio of GTP/GDP bound directly to Ras in vivo. This ADP-ribosylation of Ras in vivo is mediated by the C-terminal domain of ExoS. Further, ExoS ADP-ribosylation of Ras in vivo inhibits activation of Ras and the ability to interact with the Ras binding domain of Raf upon stimulation with epidermal growth factor (EGF). In the present study, we show that ExoS activity does not interfere with EGF receptor phosphorylation itself, nor with the formation of a Grb2-activated Shc complex upon EGF stimulation, consistent with ExoS blockage of this mitogenic signalling pathway at the level of Ras. This is further supported by our observation of a substantial inhibition of extracellular signal-regulated kinase and protein kinase B/Akt kinase activation in response to EGF upon ExoS infection. In conclusion, in the present study, the consequences of ExoS infection on Ras effector pathway in vivo have been defined.
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| 3. |
- Von Pawel-Rammingen, Ulrich, et al.
(författare)
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GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway : a mechanism for disruption of actin microfilament structure.
- 2000
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 36:3, s. 737-748
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Tidskriftsartikel (refereegranskat)abstract
- The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid-encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. Here, we show that YopE exhibits a GTPase-activating protein (GAP) activity and that the presence of YopE stimulates downregulation of Rho, Rac and Cdc42 activity. YopE has an arginine finger motif showing homology with those found in other GAP proteins. Exchange of arginine 144 with alanine, located in this arginine finger motif, results in an inactive form of YopE that can no longer stimulate GTP hydrolysis by the GTPase. Furthermore, a yopE(R144A) mutant is unable to induce cytotoxicity on cultured HeLa cells in contrast to the corresponding wild-type strain. Expression of wild-type YopE in cells of Saccharomyces cerevisiae inhibits growth, while in contrast, expression of the inactive form of YopE, YopE(R144A), does not affect the yeast cells. Co-expression of proteins belonging to the Rho1 pathway of yeast, Rho1, Rom2p, Bck1 and Ste20, suppressed the growth phenotype of YopE in yeast cells. These results provide evidence that YopE exhibits a GAP activity to inactivate RhoGTPases, leading to depolymerization of the actin stress fibres in eukaryotic cells and growth inhibition in yeast.
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