JCB Article Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis Isabelle Hoffmann,1 Emmanuel Eugène,2 Xavier Nassif,2 Pierre-Olivier Couraud,1 and Sandrine Bourdoulous1 1Laboratoire d’Immuno-Pharmacologie Moléculaire, Centre National de la Recherche Scientiﬁque, Institut Cochin de Génétique Moléculaire, 75014 Paris, France 2Laboratoire de Microbiologie, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France Efamily of epidermal growth factor (EGF) receptors rbB2 is a receptor tyrosine kinase belonging to the which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by het- erodimerization with the other members of the family. We show here that type IV pilus–mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phos- phorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Speciﬁc inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results pro- vide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efﬁcient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers. Introduction The ErbB family of receptor tyrosine kinases (RTKs)* com- prises four closely related members: EGF receptor (EGFR; also termed ErbB1), ErbB2/Neu, ErbB3, and ErbB4. These receptors are activated by numerous ErbB-specific ligands, each containing an EGF-like domain which confers binding specificity (for review see Olayioye et al., 2000). These ligands, which are produced as transmembrane precursors and are processed and released by proteolysis, bind the extra- cellular domain of ErbB receptors and lead to the formation of both homo- and heterodimers. No soluble ligand for ErbB2 has yet been discovered. However, increasing evi- dence suggests that the primary function of ErbB2 is as a Address correspondence to Sandrine Bourdoulous, Laboratoire d’Im- muno-Pharmacologie Moléculaire, Centre National de la Recherche Sci- entifique UPR 415, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France. Tel.: (33) 1-40-51-64-34. Fax: (33) 1-40- 51-64-30. E-mail: bourdoulous@cochin.inserm.fr *Abbreviations used in this paper: EGFR, EGF receptor; GST, glu- tathione S-transferase; HBMEC, human bone marrow endothelial cell; MAPK, mitogen-activated protein kinase; RTK, receptor tyrosine kinase; SH2, src homology 2. Key words: ErbB2; homodimerization; src; Neisseria meningitidis; invasion  The Rockefeller University Press, 0021-9525/2001/10/133/11 $5.00 The Journal of Cell Biology, Volume 155, Number 1, October 1, 2001 133–143 http://www.jcb.org/cgi/doi/10.1083/jcb.200106148 coreceptor, since ErbB2 is the preferred heterodimerization partner for all other ErbB family members (Tzahar et al., 1996; Graus-Porta et al., 1997) and plays a role in the po- tentiation of ErbB receptor signaling (Beerli et al., 1995; Graus-Porta et al., 1995). Dimerization stimulates the in- trinsic tyrosine kinase activity of the receptors and triggers autophosphorylation of specific tyrosine residues within the cytoplasmic domains. These phosphorylated residues pro- vide docking sites for proteins which contain src homology 2 (SH2) or phosphotyrosine binding domains and which are involved in the regulation of intracellular signaling cascades. All ErbB family members couple via the adaptors Shc and/or Grb2 to the mitogen-activated protein kinase (MAPK) path- way. However, preferential modulation of specific pathways linked to specific homo- or heterodimers, or to specific cell surface sites, have been described (Olayioye et al., 2000; Carraway and Sweeney, 2001). Therefore, the signaling di- versity associated with the ErbB family is dependent upon the repertoire of ErbB ligands, the combinatorial properties of induced receptor dimers, and receptor localization. Ulti- mately, downstream effects on gene expression determine the biological response to receptor activation. ErbB recep- tors are expressed in a variety of tissues of epithelial, mesen- 133