138 The Journal of Cell Biology | Volume 155, Number 1, 2001 meningitidis induced the tyrosyl phosphorylation of cortac- tin, whereas ezrin or the p85 subunit of the PI-3 kinase were not tyrosine phosphorylated under the same conditions (unpublished data). In agreement with the fact that cortactin is a known substrate of the tyrosine kinase src, an autophos- phorylation assay demonstrated that src was indeed activated in cells infected for 4 h with the 2C43 wild-type (Fig. 5 B). As expected, no src activity nor tyrosyl phosphorylation of cortactin was detected in cells in contact with the 2C43 PilE mutant, whereas the PilT mutant was more efficient in inducing both src activity and cortactin phosphorylation. Src activation induced by either 2C43 wild-type or ROU strains of N. meningitidis was largely reduced by treatment with 5 M AG1478 (Fig. 5 C). Moreover, cortactin phos- phorylation was fully inhibited by a treatment with 5 M PP2, a selective inhibitor of src family kinases (Fig. 5 D), and was also largely inhibited by treatment with 5 M AG1478 (Fig. 5 E), whereas EGF stimulation did not stimu- late cortactin phosphorylation. Together, these results dem- onstrate that N. meningitidis induces src activation and sub- sequent cortactin phosphorylation and point to src as a downstream signaling effector of ErbB2 in this pathway. In addition, since AG1478 does not fully inhibit src activity and cortactin phosphorylation, whereas it completely pre- vents ErbB2 phosphorylation, an alternative pathway, inde- pendent of ErbB2, may also contribute to the activation of src induced by N. meningitidis. Therefore, we examined whether src was associated with phosphorylated ErbB2 in infected cells. As shown in Fig. 6 A, an increased amount of src was detected in ErbB2 immu- noprecipitates in cells infected for 4 h with either the 2C43 wild-type or PilT mutant. These interactions were largely prevented by treatment with 5 M AG1478, thus confirm- ing that ErbB2 phosphorylation induced by N. meningitidis results in the recruitment and activation of src. To further confirm that bacteria- or EGF-induced ErbB2 phosphoryla- tion can provide a docking site for the SH2 domain of src, we performed a pull-down assay with a recombi- nant gluthatione S-transferase (GST)–src SH2 fusion pro- tein (Fig. 6, B and C). Analysis of the pattern of tyrosine- phosphorylated proteins precipitated with the fusion protein showed that both N. meningitidis and EGF induced the in- teraction of src–SH2 with a 185-kD phosphoprotein identi- fied by Western blotting as ErbB2, whereas only EGF stim- ulation induced the interaction of src-SH2 with a prominent tyrosine-phosphorylated band of 175 kD identified as EGFR. These interactions were dependent on the intrinsic ki- nase activity of ErbB2 or EGFR, since it was prevented by AG1478 treatment. These observations confirmed our con- clusion that, although EGF induced EGFR/ErbB2 het- erodimerization, bacteria induced the clustering of ErbB2 as homodimers. Since cortactin is known to interact with actin, we as- sessed whether cortactin was recruited at the site of actin po- lymerization. Although cortactin displayed a generalized cy- toplasmic staining with some enrichment in membrane ruffles in uninfected cells, it was recruited in cortical plaques in cells infected for 4 h with a the 2C43 wild-type strain of N. meningitidis (Fig. 7). AG1478 and PP2 only slightly af- fected cortactin recruitment into cortical plaques, which Figure 6. Localized adhesion of N. meningitidis leads to src association with phosphorylated ErbB2. HBMECs (starved for 18 h) were either uninfected ( ) or infected with the wild-type (WT), or the PilT-defective mutant (PilT ) of the 2C43 strain of N. meningiti- dis for 4 h before lysis. As control, cells pretreated the same way were stimulated with 10 ng/ml EGF for 3 min before lysis. (A) ErbB2 was immunoprecipitated and immunoblotted with an anti-src anti- body (top). Blot was reprobed with an anti-ErbB2 antibody (bottom) to show that similar protein levels were immunoprecipitated. (B and C) Whole cell extracts were incubated with 5 g of a GST-tagged peptide containing the SH2 domain of src and coupled to glutathione-Sepharose. The complexes were immunoblotted with an antiphosphotyrosine antibody (4G10) (top). Blots were reprobed with an anti-ErbB2 antibody (middle) and anti-EGFR antibody (bot- tom). Three independent experiments were carried out with similar results and representative results from one experiment are shown. was instead totally prevented by Y27632 treatment. Thus, ErbB2 activation promotes cortactin phosphorylation, but this event is not required for cortactin recruitment within cortical plaques, a process dependent on cortactin interac- tion with the actin cytoskeleton. ErbB2/Src signaling pathway activation is involved in bacterial internalization by human endothelial cells The internalization of N. meningitidis in endothelial cells, as shown previously, is maximal during localized adhesion, i.e., when the ErbB2/src pathway is fully activated, as shown here. The potential role of the ErbB2/src signaling pathway on bacterial internalization into endothelial cells was there- fore investigated. We observed that the 2C43 PilT mutant and the ROU strain, which activate the ErbB2/src signaling pathway more efficiently than the 2C43 wild-type strain, were also more effectively internalized (2.5 and 10 , respec- tively) (Fig. 8 A). As expected, no internalization of the non- adherent PilE mutant was observed. Furthermore, treat- ment with 5 M AG1478 or 5 M PP2 decreased bacterial internalization by 50 or 70%, respectively (Fig. 8 B). In agreement with our conclusion that src is a downstream sig- naling effector of ErbB2, the addition of both inhibitors did not further reduce bacterial entry. By contrast, AG1296, the specific inhibitor of the PDGF receptor which failed to af-