title: MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB.
abstract: The facultative intracellular pathogen Listeria monocytogenes causes listeriosis, a rare but life-threatening disease. Host cell entry begins with activation of the human receptor tyrosine kinase MET through the bacterial invasion protein InlB, which contains an internalin domain, a B-repeat, and three GW domains. The internalin domain is known to bind MET, but no interaction partner is known for the B-repeat. Adding the B-repeat to the internalin domain potentiates MET activation and is required to stimulate Madin-Darby canine kidney (MDCK) cell scatter. Therefore, it has been hypothesized that the B-repeat may bind a co-receptor on host cells. To test this hypothesis, we mutated residues that might be important for binding an interaction partner. We identified two adjacent residues in strand β2 of the β-grasp fold whose mutation abrogated induction of MDCK cell scatter. Biophysical analysis indicated that these mutations do not alter protein structure. We then tested these mutants in human HT-29 cells that, in contrast to the MDCK cells, were responsive to the internalin domain alone. These assays revealed a dominant negative effect, reducing the activity of a construct of the internalin domain and mutated B-repeat below that of the individual internalin domain. Phosphorylation assays of MET and its downstream targets AKT and ERK confirmed the dominant negative effect. Attempts to identify a host cell receptor for the B-repeat were not successful. We conclude that there is limited support for a co-receptor hypothesis and instead suggest that the B-repeat contributes to MET activation through low affinity homodimerization.
authors: From the Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
authors: Willem M
authors: WM
authors: Bleymüller
authors: From the Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
authors: Nina
authors: N
authors: Lämmermann
authors: From the Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
authors: Maria
authors: M
authors: Ebbes
authors: From the Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
authors: Daniel
authors: D
authors: Maynard
authors: From the Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
authors: Christina
authors: C
authors: Geerds
authors: From the Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany Hartmut.Niemann@uni-bielefeld.de.
authors: Hartmut H
authors: HH
authors: Niemann
mesh_terms: D000072283:A549 Cells; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004285:Dogs; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D020935:MAP Kinase Signaling System; D061985:Madin Darby Canine Kidney Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D014709:Vero Cells
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; C095768:inlB protein, Listeria monocytogenes; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met; D051057:Proto-Oncogene Proteins c-akt
keywords: cell signaling; cell surface receptor; dimerization; growth factor; protein domain; protein structure; protein-protein interaction; receptor tyrosine kinase; signal transduction; structure-function