ErbB2 supports N. meningitidis invasion | Hoffmann et al. 137 affect its own massive recruitment underneath the bacte- rial colonies, thus suggesting that N. meningitidis–induced ErbB2 clustering may precede receptor activation. Cellular localization of ErbB2 was then analyzed in the presence of 30 M Y27632, a selective inhibitor of the Rho effector Rho-kinase (Ishizaki et al., 2000), which prevents N. meningitidis–induced actin cytoskeleton rearrangement, but does not affect ezrin recruitment underneath the bacte- rial colonies (Fig. 4 B). Interestingly, Y27632 did not affect either ErbB2 clustering (Fig. 4 B) or phosphorylation (un- published data) induced by bacterial interaction. Therefore, the Rho-GTPase–dependent cortical actin polymerization induced by N. meningitidis is not required for ErbB2 recruit- ment underneath the bacterial colonies or ErbB2 activation. ErbB2 activation induced by N. meningitidis leads to the activation of the tyrosine kinase src and the subsequent phosphorylation of the src substrate cortactin To define the role of ErbB2 in the interaction process of N. meningitidis with human endothelial cells, we attempted to identify the tyrosyl-phosphorylated proteins p80/85 affected by AG1478 treatment described in Fig. 1. The migration pattern of p80/85 was suggestive of a few candidate proteins, including the actin-binding protein cortactin, ezrin, and the p85 subunit of the PI-3 kinase. Indeed, as shown in Fig. 5 A, infection for 4 h with the 2C43 wild-type strain of N. Figure 4. ErbB2 activation is not required for cortical plaque formation induced by N. meningitidis. HBMECs were pretreated for 2 h with 5 M AG1478 (A) or with 30 M Y27632 (B) before infection for 3 h with the 2C43 wild-type strain of N. meningitidis in the presence of the inhibitors. Cells were double-stained for ErbB2 and bacterial colonies (top) or ezrin and actin (bottom). Merged images (overlay) of the same fields are presented on the right. and actin cytoskeleton rearrangements, we investigated the contribution of ErbB2 activation in the HBMEC response to N. meningitidis. Immunofluorescence analysis of ezrin, actin, and ErbB2 was performed in cells infected for 4 h with the 2C43 wild-type strain of N. meningitidis in the presence of 5 M AG1478. As shown in Fig. 4 A, AG1478 did not prevent the massive recruitment of ezrin and cortical actin polymerization underneath the bacterial colonies that we described previously in the absence of inhibitor. This ob- servation indicates that ErbB2 activation is not required for cortical plaque formation induced by N. meningitidis. More- over, we observed that inhibition of ErbB2 activity did not Figure 5. ErbB2 activation induced by N. meningitidis leads to src activation and the phosphorylation of cortactin. (A and B) HBMECs (starved for 18 h) were either uninfected ( ) or infected with the wild- type (WT), the PilT-defective mutant (PilT ), or the PilE-defective mutant (PilE ) of the 2C43 strain of N. meningitidis for 4 h before lysis. (C–E) HBMECs starved for 18 h were pretreated for 2 h with either 5 M PP2 or 5 M AG1478 ( ) or left untreated ( ). Cells were either noninfected (NT) or infected with the wild-type 2C43 or the ROU strains as indicated for 4 h before lysis. PP2 and AG1478 treatments were maintained during infection. As control, cells pretreated the same way were stimulated with 10 ng/ml EGF for 3 min before lysis. (A, D, and E) Cortactin was immunoprecipitated and immunoblotted with an antiphosphotyrosine antibody (4G10) (top). Blots were reprobed with an anticortactin antibody (bottom) to show that similar protein levels were immunoprecipitated. (B and C) Src was immunoprecipitated and subjected to either an autophosphorylation assay (B) or an in vitro kinase assay using acid-denaturated enolase as a substrate (C). Three independent experiments were carried out with similar results and representative results from one experiment are shown.