In vivo modifications of small GTPase Rac and Cdc42 by Bordetella dermonecrotic toxin

Abstract

Bordetella dermonecrotic toxin (DNT) is known to activate the small GTPase Rho through deamidation or polyamination. In this study, we examined whether Rac and Cdc42, the two other members of the Rho family, serve as intracellular targets for the toxin. Immunoprecipitation and immunoblot assays revealed that DNT deamidated or polyaminated intracellular Rac and Cdc42. After the modifications, both Rac and Cdc42 lost their GTP-hydrolyzing, but not GTP-binding, activities. The interactions of the modified Rac and Cdc42 with their respective effectors were strictly dependent on GTP. MC3T3-E1 cells treated with DNT at high concentrations demonstrated extensive formations of lamellipodia and filopodia, which indicate the intracellular activation of Rac and Cdc42, respectively.

FIG. 1.

In vivo deamidation (A) and polyamination (B) of Rac and Cdc42 by DNT. (A) C3H10T1/2 cells expressing FLAG-tagged Rac1 or Cdc42 were treated with DNT at 5 ng/ml for 20 h and subjected to immunoprecipitation with anti-FLAG antibody. The precipitates were subjected to SDS-PAGE followed by immunoblot analysis with anti-63E antibody. The arrows and asterisk indicate the deamidated GTPases and nonspecifically reactive bands, respectively. (B) C3H10T1/2 cells expressing the FLAG-GTPases were preloaded with [¹⁴C]putrescine and treated with DNT at 10 ng/ml for 24 h. The cell lysates or the immunoprecipitated (IP) FLAG-GTPases were subjected to SDS-PAGE followed by autoradiography. Lane 2 shows that several kinds of endogenous proteins were ¹⁴C-polyaminated in the DNT-treated cells. The arrow indicates ¹⁴C-polyaminated FLAG-Rac1 and FLAG-Cdc42.

FIG. 2.

The GTP-binding (upper panels) and GTPase (lower panels) activities of the Rho GTPases treated with DNT. The recombinant GTPases (5 μM) were treated with DNT (50 nM) in the presence or absence of 250 μM spermidine and examined for γ-³⁵S-labeled GTP binding and GTPase activity. Ordinates express the amounts of γ-³⁵S-labeled GTP bound to the GTPases for 2 h (upper panels) and the percentage of [γ-³²P]GTP remaining bound to the GTPases after the indicated periods of incubation (lower panels). Open squares, control; closed diamonds, polyaminated GTPase; open circles, deamidated GTPase. Three independent experiments were carried out, and representative data are shown.

FIG. 3.

Binding of Rac to PAK (left panels) and Cdc42 to N-WASP (right panels) monitored by a BIAcore system.

FIG. 4.

Formation of actin cytoskeletons in MC3T3-E1 cells treated with DNT. MC3T3-E1 cells in a subconfluent state were incubated in the presence (B) or absence (A) of DNT at 5 μg/ml for 24 h, and the actin fibers with rhodamine-phalloidin were observed under a fluorescence microscope. Arrowheads: S, stress fibers; L, lamellipodia; F, filopodia.