gitidis entry through signaling events distinct from those leading to bacteria-induced actin cytoskeleton reorganiza- tion. Moreover, both pathways cooperate in bacterial entry. Therefore, our results provide evidence for a new mode of activation of ErbB2 by bacteria-induced homodimerization, and for a novel biological function of ErbB2 in supporting bacterial internalization into host cells. Results Adhesion of N. meningitidis to human endothelial cells induces tyrosine phosphorylation of cellular proteins, which can be prevented by AG1478, an ErbB family inhibitor We investigated the tyrosine phosphorylation events occur- ring in a human endothelial cell line (HBMEC) during in- teraction with N. meningitidis. For this purpose, HBMECs were infected with a piliated capsulated strain of N. menin- gitidis (wild-type 2C43), and two derivative mutants of that strain: a highly piliated and adhesive mutant (PilT ) and a nonpiliated (nonadherent) PilE mutant. As shown in Fig. 1 A, infection with the wild-type strain 2C43 for 4 h induced the major tyrosyl phosphorylation of proteins of 80/85 and 180 kD. These phosphorylation events were markedly increased when cells were infected with the PilT mutant of 2C43, whereas they were not observed in cells in con- tact with the nonadherent PilE mutant. Kinetics analysis showed that these signals were detectable as soon as 30 min after infection, maximal at 4 h (during the pilus-mediated localized adhesion). By contrast, they were no longer de- tected at 8 h with the wild-type strain (diffuse adhesion), whereas the signal remained persistent in cells infected with the PilT mutant, which is unable to retract pili and to pro- ceed to the diffuse adhesion step (unpublished data). There- fore, these events occur during the localized pilus-mediated adhesion of N. meningitidis to human endothelial cells. In parallel, experiments were performed with an isolate of a highly virulent strain, designated as ROU, obtained from the cerebrospinal fluid of a patient with fulminant meningo- coccal infection (Fig. 1 B). This strain was highly efficient in inducing tyrosyl phosphorylation of the p80/85 and p180, suggesting a correlation between these cellular events and the infectious process of N. meningitidis. Moreover, similar data were observed with a primary culture of human umbili- cal vein endothelial cells (unpublished data), suggesting that these phosphorylation events might be a general response of endothelial cells to N. meningitidis adhesion. Since ErbB receptors are 180 kD, compatible with that of p180, we hypothesized that tyrosine kinase receptors of the EGFR family might be involved in the cellular response to bacterial adhesion. We tested whether these phosphoryla- tion events would be affected by AG1478, a very potent and selective inhibitor of both EGFR and ErbB2 kinases (Kurokawa et al., 2000). As shown in Fig. 1 C, cell treat- ment with 5 or 10 M AG1478 totally prevented the in- crease of tyrosyl phosphorylation of p180 protein induced by either EGF or infection for 4 h with the PilT mutant, whereas cell treatment with 10 M AG1296, a selective in- hibitor of the PDGF receptor kinase, had no effect. Simi- larly to EGF, N. meningitidis induced the activation of ErbB2 supports N. meningitidis invasion | Hoffmann et al. 135 Figure 1. Localized adhesion of N. meningitidis induces tyrosine phosphorylation events that are prevented by AG1478 treatment. (A) HBMECs (starved for 18 h) were either uninfected ( ) or infected with the wild-type strain (WT), the PilT-defective mutant (PilT ), or the PilE-defective mutant (PilE ) of the 2C43 strain of N. meningitidis for 4 h before lysis. (B) HBMECs starved for 18 h were either uninfected ( ) or infected with the 2C43 wild-type strain (WT) or ROU strain (ROU). (C) HBMECs (starved for 18 h) were pretreated for 2 h with 5 or 10 M AG1478, 10 M AG1296, or left untreated ( ). Cells were then infected for 4 h with the PilT- defective mutant strain in the presence of these inhibitors before lysis. As control, cells pretreated the same way were stimulated with 10 ng/ml EGF for 3 min before lysis. (A–C) Samples of whole cell extracts were analyzed by immunoblotting with an antiphosphoty- rosine antibody (4G10). The electrophoretic mobilities of proteins of 80/85 and 180 kD (arrows) and of molecular weight standards (175, 116, 84, and 58 kD) are indicated. (C) Bottom, blot was reprobed with an antiphosphoErK1/2 antibody, which detects Erk1/2 when activated by phosphorylation. the MAPK Erk1/2 that was also prevented by treatment with AG1478, but not by AG1296. Altogether, these data strongly suggest that the infection by N. meningitidis leads to the activation of member(s) of the ErbB family. Interest- ingly, AG1478 treatment also drastically reduced the phos- phorylation of p80/85 induced by N. meningitidis, suggest- ing these proteins as downstream targets of ErbB signaling cascade. However, phosphorylation of p80/85 was not ob- served in response to EGF stimulation. Adhesion of N. meningitidis to human endothelial cells induces tyrosine phosphorylation and clustering of ErbB2, but not of the other ErbB members Therefore, we analyzed the tyrosyl phosphorylation state of members of the ErbB family in cells infected for 4 h with ei- ther the 2C43 wild-type, the PilT mutant, or the ROU strain in the presence or in the absence of 5 M AG1478. Stimulation of HBMECs with EGF at 10 ng/ml for 3 min