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14-3-3 proteins play an important role in a multitude of signalling pathways. The interactions between 14-3-3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho-specific manner. Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and several proteins, for example 5-phosphatase, p75NTR-associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14-3-3. Furthermore, we show that a peptide derived from ExoS, containing the 14-3-3 interaction site, effectively competes out the interaction between ExoS and 14-3-3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14-3-3 family tested. Moreover, in vivo an ExoS protein lacking the 14-3-3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.