Multiple Regions of Internalin B Contribute to Its Ability to Turn on the Ras-Mitogen-activated Protein Kinase Pathway

Jeremy Copp, Michael Marino, Manidipa Banerjee, Partho Ghosh, Peter van der Geer
2002 Journal of Biological Chemistry  
Internalin B (InlB) is a protein present on the surface of Listeria monocytogenes that mediates bacterial entry into mammalian cells. It is thought that InlB acts by binding directly to the hepatocyte growth factor (HGF) receptor, present on the surface of host cells. Binding of InlB to the HGF receptor results in mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase activation, followed by changes in the organization of the actin cytoskeleton. Here we have compared signaling by
more » ... GF and InlB. Whereas stimulation with equivalent concentrations of HGF and InlB elicits similar activation of the HGF receptor, we observed striking differences in downstream activation of MAP kinase. InlB leads to a greater activation of the Ras-MAP kinase pathway than does HGF. The leucine-rich repeat region, which was previously shown to be sufficient for binding and activation of the HGF receptor, lacks the ability to super-activate the Ras-MAP kinase pathway. Analysis of a series of deletion mutants suggests that it is the B repeat region between the leucine-rich repeat and GW domains that endows InlB with an increased ability to turn on the Ras-MAP kinase pathway. These unexpected observations suggest that HGF and InlB use alternative mechanisms to turn on cellular signaling pathways. The hepatocyte growth factor (HGF) 1 receptor is a proteintyrosine kinase that is expressed predominantly on the surface of epithelial cells. Its natural ligand, HGF or scatter factor, is expressed in fibroblast cells (1). In vivo, HGF and the HGF receptor play key roles in the development of the liver, kidneys, muscle, and neuronal precursors (2). In vitro activation of the HGF receptor stimulates cell division, cell scattering, and the formation of tubular structures in a three-dimensional matrix (3). Whereas normal HGF receptor signaling plays a crucial role in embryonic development, abnormal HGF receptor signaling has been implicated in both tumor development and metastasis (4). Activation of the HGF receptor leads to autophosphorylation on several tyrosine residues. Tyrosine phosphorylation sites regulate kinase activity and act as binding sites for cellular signaling proteins. The activated HGF receptor interacts either directly or indirectly with several signaling molecules including Grb2, Gab1, Shc, phosphoinositide 3 (PI 3)-kinase, Crk, CrkL, and Cbl (5-11). Gab1 is a docking protein that provides the receptor with binding sites for a variety of other proteins (12). Grb2 is a cytoplasmic adaptor protein that is involved in linking Gab1 to the HGF receptor (13). Grb2 also works together with Sos to link activated receptor protein-tyrosine kinases to Ras activation (14, 15). Shc is a docking protein that is thought to collaborate with Grb2 upstream of Ras (16 -19). PI 3-kinase is a lipid kinase that is composed of an 85-kDa regulatory subunit, which contains two SH2 domains and one SH3 domain, and a 110-kDa catalytic subunit (20 -22). Activation of PI 3-kinase affects cytoskeletal dynamics and cell survival. Crk and CrkL are related adaptor proteins that are also involved in regulating cytoskeletal changes (23). Cbl is a ubiquitin ligase that regulates receptor internalization and degradation (24). The Listeria monocytogenes surface-protein internalin B (InlB) binds to and activates the HGF receptor. Initiation of host cell invasion by this intracellular pathogen occurs through the interaction of InlB with the HGF receptor, leading to host cell protein-tyrosine phosphorylation and cytoskeletal rearrangements. It has been established that the activation of both the Ras-MAP kinase pathway and PI 3-kinase are essential for uptake of L. monocytogenes into host cells (25, 26) . InlB is composed of an amino-terminal receptor-binding domain followed by a B repeat region (B) and three GW domains. The receptor-binding domain contains three motifs: an aminoterminal cap, a leucine-rich repeat (LRR) segment, and an immunoglobin-like region (IR) (27, 28) . The amino-terminal cap and the LRR domain have been shown to be sufficient to bind and activate the HGF receptor (29). The GW domains attach InlB non-covalently to the bacterial cell wall and have been shown recently to bind to host cell components upon release of InlB from the bacterium (30, 31). Both HGF and InlB bind and activate the HGF receptor. Interestingly, whereas HGF stimulates mitogenesis, cell migration, and tubulogenesis, InlB induces phagocytosis. This prompted us to compare signaling downstream of these two HGF receptor ligands. Our results show that HGF and InlB are very similar in their ability to activate the HGF receptor, whereas InlB is a stronger activator of the Ras-MAP kinase pathway. We found that InlB induces tyrosine phosphorylation of a 90-kDa PI 3-kinase-associated protein, whereas HGF did not. Analysis of InlB deletion mutants showed that, whereas both the IR and B repeat are necessary for maximal signaling
doi:10.1074/jbc.m211666200 pmid:12488439 fatcat:5xsav5vznbdrjizaueprpuy5im