134 The Journal of Cell Biology | Volume 155, Number 1, 2001 chymal, and neuronal origin, where they play fundamental roles in development, proliferation, and differentiation. Re- cently, an expression of ErbB receptors has also been described on endothelial cells, where they play a role in an- giogenesis (Russell et al., 1999). Moreover, deregulated ex- pression of ErbB receptors, in particular ErbB1 and ErbB2, has been implicated in the development and malignancy of numerous types of human cancers (Yarden and Sliwkowski, 2001). ErbB2 overexpression, observed in a significant pro- portion of breast and ovarian cancers, triggers ligand-inde- pendent activation of the kinase domain, apparently as a re- sult of spontaneous dimer formation. A ligand-independent activation of ErbB RTKs can also be triggered by other classes of receptors, such as G protein–coupled receptors, cy- tokine receptors, or adhesion molecules (Carpenter, 1999; Gschwind et al., 2001). In the absence of ErbB kinase activ- ity, phosphorylation of specific tyrosyl residues in the cyto- plasmic tail of the receptor by nonreceptor kinases such as jak or src can provide docking sites for cytoplasmic signaling molecules and promote signaling cascades. In addition, a ligand-dependent transactivation of EGFR by G protein– coupled receptors can also occur by a proteolytic processing of the transmembrane pro-HB-EGF precursor followed by a paracrine activation of EGFR (Gschwind et al., 2001). Therefore, ErbB RTKs are central signal transducers which respond to the binding of specific ligands or via transactiva- tion by distinct receptor systems. Many bacterial pathogens exploit host cell signaling path- ways in order to promote their adherence to or uptake by host cells (Dramsi and Cossart, 1998). For most invasive bacteria, a major consequence of these signaling events is a morphological change of the host cell surface resulting from a reorganization of the actin cytoskeleton. The nature and magnitude of such changes depend highly on the pathogen and its cellular host, although a common feature is the acti- vation of host tyrosine phosphorylation leading to the acti- vation of the small GTPases of the Rho family, key regu- lators of actin cytoskeleton dynamics. The best studied examples are those of Shigella or Salmonella which trigger the induction of large cellular membrane folds or ruffles around bacteria leading to their subsequent uptake by a macropinocytic process (Nhieu and Sansonetti, 1999; Galan and Zhou, 2000). These pathogens have evolved a complex type III secretion system allowing them to inject into host cells bacterial components that can directly initiate the acti- vation of cellular signaling cascades. Alternatively, Listeria monocytogenes, which does not have a type III secretion sys- tem, promotes its entry by engaging cell adhesion receptors (E-cadherins) and the tyrosine kinase receptor Met (Men- gaud et al., 1996; Shen et al., 2000). Neisseria meningitidis (also referred to as meningococcus) is an extracellular, human-specific pathogen responsible for septicemia and meningitis. Despite the lack of type III secre- tion system, virulent capsulated N. meningitidis also initiates host cell signaling cascades leading to their subsequent up- take into host cells that are normally nonphagocytic (Nassif et al., 1999; Merz and So, 2000). This entry is likely to pro- mote their transcytosis through cellular barriers by a mecha- nism that remains to be clearly identified, and therefore plays a major role in bacterial pathogenesis. Capsulated N. meningitidis interact with host cells in a multistep process (Pujol et al., 1997). Bacteria first adhere through their type IV pili, which are long filamentous protein structures that interact with an unidentified cellular receptor. Bacteria then proliferate, locally forming a colony at their site of attach- ment on the cell surface, a step referred to as localized adhe- sion. Subsequently, bacteria loose their pili, disperse from the colony, and spread over the cell surface to form a single monolayer of bacteria covering the cells, a step referred to as diffuse adhesion. The bacterial protein PilT, which is re- sponsible for pilus retraction (Merz et al., 2000), is required for the transition from localized to diffuse adhesion (Pujol et al., 1999). Internalization of these bacteria occurs essentially during the first step of localized adhesion. During this inter- action, N. meningitidis adhering to epithelial cells elicits the formation of specific molecular complexes underneath the bacterial colonies. These structures, called cortical plaques, result from the localized polymerization of cortical actin, re- cruitment of ezrin, a protein that links the cellular mem- brane to the actin cytoskeleton, and clustering of membrane integral proteins, such as the ezrin binding proteins CD44 or ICAM-1 as well as tyrosine phosphorylated proteins (Merz and So, 1997; Merz et al., 1999). These events are ac- companied by the elongation of the epithelial cell microvilli towards the bacteria, leading to their engulfment and inter- nalization into host cells. We recently showed that N. men- ingitidis interaction with endothelial cells also leads to corti- cal actin polymerization underneath the bacterial colonies and the massive recruitment of ezrin and ezrin-binding pro- teins (unpublished data). This recruitment promotes the for- mation of membrane projections, reminiscent of epithelial microvilli structures, which surround single bacteria and provoke their internalization within endothelial intracellular vacuoles. This has been demonstrated both in vitro and in vivo and suggests that this process is essential for the crossing of human endothelium via a transcytosis pathway. We fur- ther showed that N. meningitidis–induced actin polymeriza- tion requires the activation of the small GTPases of the Rho family RhoA and Cdc42, but not Rac1. Inhibition of Rho family GTPases prevented cortical actin polymerization, but only partially inhibited bacterial internalization, suggesting an alternative signaling pathway involved in N. meningitidis internalization. The process by which N. meningitidis inter- acts with the host cell and induces these signaling events has not been elucidated. In this study, we therefore analyzed the tyrosine phos- phorylation events occurring in human endothelial cells dur- ing adhesion of N. meningitidis. We provide evidence that pilus-mediated adhesion of capsulated N. meningitidis in- duces the clustering and tyrosyl phosphorylation of the host cell tyrosine kinase receptor ErbB2, but not of the other ErbBs. ErbB2 activation leads to downstream activation of the protein tyrosine kinase src and the tyrosine phosphoryla- tion of the src-substrate cortactin. Furthermore, we show that ErbB2/src signaling pathway is not involved in the for- mation of cortical plaques per se, but plays a crucial role in facilitating bacterial entry into endothelial cells. By analyz- ing the respective contributions to bacterial entry of both ErbB2/src and Rho GTPases–dependent signaling path- ways, we show that ErbB2/src pathway regulates N. menin-