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J Biol Chem. 2016 Dec 2;291(49):25567-25577. Epub 2016 Oct 27.

MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB.

Author information

1
From the Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
2
From the Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany Hartmut.Niemann@uni-bielefeld.de.

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.

KEYWORDS:

cell signaling; cell surface receptor; dimerization; growth factor; protein domain; protein structure; protein-protein interaction; receptor tyrosine kinase; signal transduction; structure-function

PMID:
27789707
PMCID:
PMC5207255
DOI:
10.1074/jbc.M116.746685
[Indexed for MEDLINE]
Free PMC Article

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