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14-3-3 proteins are required for the inhibition fo Ras by exoenzyme S
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
2000 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 349, p. 697-701Article in journal (Refereed) Published
Abstract [en]

14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine ligands, such as Raf-l kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where 'Xaa' represents 'any residue', and 'pSer' is 'phosphoserine'). However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ib alpha and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS. Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259). In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo. Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.

Place, publisher, year, edition, pages
Portland Press, 2000. Vol. 349, p. 697-701
National Category
Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-2456DOI: 10.1042/bj3490697ISI: 000088712000005PubMedID: 10903129OAI: oai:DiVA.org:umu-2456DiVA, id: diva2:140496
Available from: 2003-01-01 Created: 2003-01-01 Last updated: 2018-12-05Bibliographically approved
In thesis
1. Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S
Open this publication in new window or tab >>Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP-ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity in vitro. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of Yersinia pseudotuberculosis. We found that Ras was ADP-ribosylated in vivo and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily in vivo, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by P. aeruginosa. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 in vivo, and that ExoT shows GAP activity towards RhoA in vitro.

The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids 424DALDL428 on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS in vivo.

Place, publisher, year, edition, pages
Umeå universitet, 2003. p. 51
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 851
Keywords
Cell biology, Pseudomonas aeruginosa, ADP-ribosylation, GAP, Ras superfamily, NAD, ExoS, 14-3-3, Cellbiologi
National Category
Cell and Molecular Biology
Research subject
molecular cell biology
Identifiers
urn:nbn:se:umu:diva-121 (URN)91-7305-505-0 (ISBN)
Public defence
2003-10-31, Betula, 6M, Umeå, 09:00
Opponent
Supervisors
Available from: 2003-01-01 Created: 2003-01-01 Last updated: 2019-01-21Bibliographically approved

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Henriksson, Maria L.Hallberg, Bengt

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