136 The Journal of Cell Biology | Volume 155, Number 1, 2001 Figure 2. Localized adhesion of N. meningitidis induces the tyrosyl phosphorylation of ErbB2, but not of the other ErbB members. HBMECs (starved for 24 h) were pretreated for 2 h with 5 M AG1478 ( ) or left untreated ( ). Cells were either noninfected (NT) or infected with the wild-type (WT), the PilT-defective mutant (PilT ) of the 2C43 strain, or the ROU strain of N. meningitidis, as indicated, for 4 h before lysis. AG1478 treatment was maintained during infection. As control, cells pretreated the same way were stimulated with 10 ng/ml EGF for 3 min before lysis. (A) EGF recep- tor was immunoprecipitated and immunoblotted with an antiphos- photyrosine antibody (4G10) (top). Blot was reprobed with an anti- EGFR antibody (bottom) to show that similar levels of receptors were immunoprecipitated. (B and C) ErbB2 receptor was immuno- precipitated and immunoblotted with an antiphosphotyrosine antibody (4G10) (top). Blots were reprobed with an anti-ErbB2 antibody (bottom). Three independent experiments were carried out with similar results and representative results from one experi- ment are shown. highly stimulated EGFR tyrosine phosphorylation as ex- pected, whereas N. meningitidis adhesion had no effect on tyrosine phosphorylation of the EGFR (Fig. 2 A). Analysis of not only EGFR, but also ErbB3 or ErbB4 phosphor- ylation over several hours of infection by N. meningitidis failed to reveal any modification (unpublished data). By contrast, bacterial infection induced tyrosyl phosphorylation of ErbB2, which could be prevented by AG1478 treatment (Fig. 2), indicating that N. meningitidis leads to the selective activation of ErbB2, whereas EGF stimulated both EGFR and ErbB2 phosphorylation, consistent with EGFR/ErbB2 heterodimerization (Fig. 2 C). Therefore, cellular localization of the ErbB receptors was analyzed in cells infected for 4 h with the 2C43 wild-type strain. As shown in Fig. 3, although ErbB2 displayed a Figure 3. Localized adhesion of N. meningitidis induces the clustering of ErbB2, but not of the other ErbB members. (A) Top, uninfected cells were stained for ErbB2; bottom, cells infected for 3 h with the 2C43 wild-type strain of N. meningitidis were double- stained for ErbB2 (left) and bacterial colonies (middle). Merged images (overlay) of the same fields are presented on the right. (B) Cells infected for 3 h with the 2C43 wild-type strain of N. meningi- tidis were double-stained for either EGFR, ErbB3, or ErbB4 (top, as indicated) and bacterial colonies (bottom). Panels show receptor and bacterial staining in the same fields. punctuated staining in uninfected cells, it was highly re- cruited underneath the bacterial colonies in infected cells, whereas EGFR, ErbB3, and ErbB4 staining was not af- fected. Altogether, these data show that adhesion of N. men- ingitidis to human endothelial cells induces tyrosine phos- phorylation and clustering of ErbB2, but not of the other ErbB members, suggesting that most likely N. meningitidis induces ErbB2 activation by promoting the formation of ErbB2 homodimers. ErbB2 activation is not required for cortical plaque formation induced by N. meningitidis We have reported previously that N. meningitidis induces ac- tin cytoskeleton rearrangements and cortical plaque forma- tion in human endothelial cells (unpublished data). Since stimulation of ErbBs may lead to Rho GTPase activation