Vinculin proteolysis unmasks an ActA homolog for actin-based Shigella motility.
To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1-3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 +/- 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that supplementing cells with p90 stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878-21885) and to vasodilator-stimulated phosphoprotein (VASP) (Brindle, N.P.J., M. R. Hold, J.E. Davies, C.J. Price, and D.R. Critchley. 1996. Biochem. J. 318:753-757). We now offer a working model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and profilin.
Laine
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Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Health Science Center, Gainesville, Florida 32610-0277, USA.
Zeile
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Kang
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Purich
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Southwick
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D000199:Actins / Q000502:physiology; D000818:Animals; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001792:Blood Platelets / Q000737:chemistry; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D003429:Cross Reactions; D004405:Dysentery, Bacillary / Q000378:metabolism / Q000382:microbiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007668:Kidney / Q000166:cytology; D007614:Macropodidae; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D008845:Microinjections; D010446:Peptide Fragments / Q000276:immunology / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D012763:Shigella flexneri / Q000737:chemistry / Q000166:cytology*; D016596:Vinculin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D001426:Bacterial Proteins; D015815:Cell Adhesion Molecules; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D016596:Vinculin; C072980:actA protein, Listeria monocytogenes; D011392:Proline