Abstract
Pseudomonas aeruginosa exoenzyme S double ADP-ribosylates Ras at Arg(41) and Arg(128). Since Arg(41) is adjacent to the switch 1 region of Ras, ADP-ribosylation could interfere with Ras-mediated signal transduction via several mechanisms, including interaction with Raf, or guanine nucleotide exchange factor-stimulated or intrinsic nucleotide exchange. Initial experiments showed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interacted with Raf with equal efficiencies, indicating that ADP-ribosylation did not interfere with Ras-Raf interactions. While ADP-r-Ras and Ras possessed equivalent intrinsic nucleotide exchange rates, guanine nucleotide exchange factor (Cdc25) stimulated the nucleotide exchange of ADP-r-Ras at a 3-fold slower rate than Ras. ADP-r-Ras did not affect the nucleotide exchange of Ras, indicating that the ADP-ribosylation of Ras was not a dominant negative phenotype. Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates as Ras, indicating that ADP-ribosylation at Arg(128) did not inhibit Cdc25-stimulated nucleotide exchange. Consistent with the slower nucleotide exchange rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucleotide exchange factor (Cdc25) less efficiently than Ras in direct binding experiments. Together, these data indicate that ADP-ribosylation of Ras at Arg(41) disrupts Ras-Cdc25 interactions, which inhibits the rate-limiting step in Ras signal transduction, the activation of Ras by its guanine nucleotide exchange factor.
Highlights
Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, can cause severe infections in immunocompromised individuals, cystic fibrosis patients, and burn wound victims (1)
Growth factors, such as epidermal growth factor, stimulate Ras activation through a signal transduction cascade, involving the recruitment of the Ras guanine nucleotide exchange factor to the membrane where the guanine nucleotide exchange factor catalyzes the exchange of GDP with GTP in Ras (12)
The exchange rates of Ras-GDP and Ras R41K-GDP differed by about 20% (Table I), indicating that the Arg to Lys mutation at residue 41 had some affect on Cdc25-catalyzed nucleotide exchange. These results indicate that Exoenzyme S (ExoS) inhibits Cdc25-catalyzed nucleotide exchange by ADP-ribosylating Ras at Arg41
Summary
Materials—Mammalian cell culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD). For analysis of Ras-Cdc interactions and Cdc25-catalyzed nucleotide exchange, nucleotide loading reactions were prepared containing 30 M Ras, 40 mM Tris-HCl (pH 7.6), 2 mM DTT, and 200 M of the respective nucleotide for 30 min at 30 °C and reactions were stopped by adding MgCl2 to a final concentration of 20 mM. Ras Nucleotide Exchange—For the measurement of GTP association rates, reaction mixtures contained 2.5 M of either Ras-GDP or ADPr-Ras-GDP, 500 M GTP␥35S, 40 mM Tris-HCl (pH 7.6), 2 mM DTT, and 10 mM MgCl2 in the presence and absence of GST-Cdc at the ratios indicated in a total volume of 225 l. For the measurement of GDP dissociation rates, reaction mixtures contained: 0.5 M Ras-GDP35S, 5 M Ras-GDP or 5 M ADP-r-Ras-GDP, 500 M GTP␥S, 250 nM Cdc mM Tris-HCl (pH 7.6), 2 mM DTT, and 10 mM MgCl2 in a total volume of 225 l. Precipitates were washed four times in Co-IP wash buffer and resolved by 12.5% SDSPAGE, transferred to polyvinylidene difluoride membranes and blotted with LA069 anti-H-Ras antibody (Quality Biotech) and detected by ECL
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