140 The Journal of Cell Biology | Volume 155, Number 1, 2001 ously described only in a few cases: a ligand-independent ac- tivation of the kinase domain of ErbB2 is observed in a significant proportion of breast and ovarian cancers overex- pressing ErbB2, apparently as a result of spontaneous dimer formation (Yarden and Sliwkowski, 2001). Homodimeriza- tion of this receptor can also be achieved by mutating a single amino acid residue in the transmembrane domain, leading to constitutive ErbB2 dimerization and activation (Bargmann et al., 1986; Olayioye et al., 1998). Alterna- tively, antibody binding to the extracellular domain can also promote ErbB2 homodimerization and its subsequent acti- vation (Sarup et al., 1991; Olayioye et al., 1998). Thus, our results constitute the first example of ErbB2 activation by homodimerization induced in nontumor cells (expressing physiological levels of ErbB2) through a ligand-independent pathway. ErbB2 activation by N. meningitidis infection apparently results from bacteria-induced clustering of this receptor, since ErbB2 tyrosine phosphorylation is dependent on its in- trinsic kinase activity. In contrast, ErbB2 recruitment under- neath the bacterial colonies appears to be activation-inde- pendent, inasmuch as it was not affected by the ErbB2 kinase inhibitor AG1478. Clustering of ErbB2 may result in the spontaneous homodimer formation due to an increased local concentration of ErbB2 molecules, in a similar way to what has been described in tumor cells overexpressing ErbB2. Indeed, previous studies performed with antibody- induced clustering of ErbB2 showed that the activation state of ErbB2 correlates with the size of ErbB2 clusters (Nagy et al., 1999). However, how N. meningitidis interaction leads to ErbB2 clustering is still unclear. No activation of this sig- naling pathway is observed in cells in contact with the non- piliated strain of N. meningitidis (PilE ), thus indicating that these events are not due to the presence of any bacterial- secreted molecule in the infection medium. Moreover, this activation is maximal during the localized adhesion of N. meningitidis and decreases when bacteria loose their pili and disperse at the cell surface, whereas it remains persistent in cells infected with the PilT mutant. This mutant, defective in pili retraction, is highly piliated and cannot disperse at the cell surface (Pujol et al., 1999; Merz and So, 2000). Thus, these data provide evidence that ErbB2 activation requires a pilus-dependent interaction of the bacteria with the host cells. Type IV pili initiate the interaction of virulent, capsu- lated N. meningitidis with human cells by interacting with a cellular receptor that remains to be clearly identified. The membrane protein MCP/CD46 was suggested as a receptor for N. meningitidis pili (Kallstrom et al., 1997). However the weak affinity of the pili–CD46 interaction suggests the exist- ence of an alternative human cell receptor for N. meningiti- dis. Thus, ErbB2 clustering may result from a direct inter- action with bacterial pili. Alternatively, ErbB2 might be recruited underneath the bacterial colonies by interacting in a multimer complex with other cellular components re- cruited after interaction of bacteria with an unknown cellu- lar receptor. Indeed, the evidence provided suggests that an interaction between ErbB2 and other transmembrane glyco- proteins may potentiate ErbB2 activation. In normal and transformed mammary epithelial cells, the transmembrane subunit of sialomucin complex ASGP-2 was shown to form a stable complex through its EGF-like domain with ErbB2, but not EGFR, ErbB3, or ErbB4, and to potentiate neureg- ulin-induced ErbB2–ErbB3 heterodimer activation (Carr- away et al., 1999). Moreover, a direct interaction between CD44 and ErbB2 has been described, leading to a potentia- tion of the constitutive tyrosine phosphorylation of ErbB2 in an ovarian carcinoma cell line overexpressing ErbB2 (Bourguignon et al., 1997), or to a significant increase in neuregulin-induced ErbB2–ErbB3 heterodimerization and ErbB2 phosphorylation in Schwann cells (Sherman et al., 2000). Interestingly, we show here that ErbB2 was recruited together with ezrin and CD44. Further studies are required to assess whether such an interaction of ErbB2 with other transmembrane glycoproteins may occur in endothelial cells in response to N. meningitidis adhesion and may be critical for bacteria-induced ErbB2 clustering and/or activation. How does the activation of ErbB2 lead to the entry of N. meningitidis into host cells that are normally nonphagocytic? We showed that ErbB2 phosphorylation results in the re- cruitment and activation of the tyrosine kinase src, which are dependent on the intrinsic kinase activity of ErbB2. A se- lective inhibition of both ErbB2 and src revealed a major role of the ErbB2/src signaling in N. meningitidis internal- ization. Interestingly, the ErbB2/src pathway is not required for the activation of GTPase-dependent cytoskeletal modifi- cations that we have observed recently in response to the ad- hesion of N. meningitidis (unpublished data). Inhibition of ErbB2 or src activity did not affect the cortical actin reorga- nization induced underneath the bacterial colonies. In- versely, inhibition of bacteria-induced cortical actin po- lymerization did not prevent the activation of ErbB2/src pathway. Therefore, N. meningitidis promotes the activation of two distinct pathways, both of which contribute to an ef- ficient internalization of bacteria. Interestingly, the implica- tion in bacterial internalization of host cell src family tyro- sine kinases and Rho family GTPases has been reported in several cases of invasive pathogens (Dramsi and Cossart, 1998). For example, Opa52-mediated phagocytosis of N. gonorrhoeae by human neutrophils requires, for an efficient uptake, the activity of the related Rho family member Rac, which appears to be controlled by an src-like tyrosine kinase (Fgr or Hck) (Hauck et al., 1998). In the same way, src, to- gether with Rho family GTPases, is involved in the internal- ization of Shigella (Dehio et al., 1995; Adam et al., 1996) and is required for triggering the formation of actin poly- merization foci induced by Shigella (Dumenil et al., 1998). By contrast, invasion by Salmonella, which requires the acti- vation of Cdc42, probably does not involve tyrosine phos- phorylation (Galan and Zhou, 2000). When required, tyro- sine kinase activity is then directly involved in the activation of small GTPases and controls bacterial entry by eliciting cy- toskeleton changes. N. meningitidis seems to have developed an alternative pathway leading to its subsequent uptake into endothelial cells, since we observed that src tyrosine kinase activity controls N. meningitidis entry at a different level than Rho family GTPases. We showed that src activation by N. meningitidis leads to the tyrosyl phosphorylation of a ma- jor susbstrate (p80/85), which we identified as cortactin. Cortactin is an actin-binding protein, which can promote in vitro F-actin crosslinking. We consistently observed that