ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

Machner, Matthias P.; Urbanke, Claus; Barzik, Melanie; Otten, Sonja; Sechi, Antonio; Wehland, Jürgen; Heinz, Dirk W.

doi:10.1074/jbc.m104279200

Cited by 32 publications

(25 citation statements)

References 49 publications

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“…Much of what we know about the effectors of VASP in motile processes has come from the Listeria system. The N-terminal EVH1 domain of VASP binds four FPPPP proline-rich repeats on ActA (4,5,26,28,32,42,43). In eukaryotic cells, VASP is thought to regulate filament branching by some unidentified cellular machinery using the Arp2/3 complex, since filaments at the leading edge are less densely branched in the presence of VASP than in its absence (3).…”

Section: Sh2-b␤ (Src Homology 2 B␤)mentioning

confidence: 99%

Adapter Protein SH2-Bβ Stimulates Actin-Based Motility of Listeria monocytogenes in a Vasodilator-Stimulated Phosphoprotein (VASP)-Dependent Fashion

Diakonova¹,

Helfer

Seveau

et al. 2007

Infect Immun

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SH2-B␤ (Src homology 2 B␤)is an adapter protein that is required for maximal growth hormone-dependent actin reorganization in membrane ruffling and cell motility. Here we show that SH2-B␤ is also required for maximal actin-based motility of Listeria monocytogenes. SH2-B␤ localizes to Listeria-induced actin tails and increases the rate of bacterial propulsion in infected cells and in cell extracts. Furthermore, Listeria motility is decreased in mouse embryo fibroblasts from SH2-B ؊/؊ mice. Both recruitment of SH2-B␤ to Listeria and SH2-B␤ stimulation of actin-based propulsion require the vasodilator-stimulated phosphoprotein (VASP), which binds ActA at the surfaces of Listeria cells and enhances bacterial actin-based motility. SH2-B␤ enhances actin-based movement of ActA-coated beads in a biomimetic actin-based motility assay, provided that VASP is present. In vitro binding assays show that SH2-B␤ binds ActA but not VASP; however, binding to ActA is greater in the presence of VASP. Because VASP also plays an essential regulatory role in actin-based processes in eukaryotic cells, the present results provide mechanistic insight into the functions of both SH2-B␤ and VASP in motility and also increase our understanding of the fundamental mechanism by which Listeria spreads.Listeria monocytogenes is a facultative intracellular grampositive bacterial pathogen that can invade a broad range of cell types and cause a variety of syndromes in humans and animals. Bacterial invasion of host cells starts by the interaction of Listeria with the plasma membrane, which progressively enwraps the bacterium. The bacterium escapes from the phagosome and freely proliferates within the host cell cytosol. Concomitant with bacterial replication, Listeria induces polymerization of host actin at its surface, leading to the formation of an actin tail. This tail propels the bacterium through the cytosol to neighboring cells (27,45,47). Actin and a limited subset of actin-binding proteins reconstitute bacterial motility in a purified system (25). However, beads covered with the listerial protein ActA move several times slower in this assay than in cells. This lower movement rate suggests that other proteins may also stimulate Listeria movement in cells. In this study, we introduce SH2-B␤ (Src homology 2 B␤) as a protein that increases the efficiency of Listeria actin-based motility.SH2-B␤ is a member of a widely expressed conserved family of adapter proteins containing several proline-rich regions, a central pleckstrin homology domain, and a C-terminal SH2 domain. SH2-B␤ binds, via its SH2 domain, to growth hormone-activated Janus tyrosine kinase (JAK2) (39) and to the activated receptor tyrosine kinases for insulin, insulin-like growth factor I, nerve growth factor, platelet-derived growth factor, and fibroblast growth factor (20,33,36,37,48). Several lines of evidence show that SH2-B␤ is involved in signaling to the actin cytoskeleton. First, SH2-B␤ increases membrane ruffling and pinocytosis induced by growth hormone, and a dominant-negati...

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Section: Sh2-b␤ (Src Homology 2 B␤)mentioning

confidence: 99%

Adapter Protein SH2-Bβ Stimulates Actin-Based Motility of Listeria monocytogenes in a Vasodilator-Stimulated Phosphoprotein (VASP)-Dependent Fashion

Diakonova¹,

Helfer

Seveau

et al. 2007

Infect Immun

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“…Wavelength scans from 190 to 260 nanometers were collected sequentially at 4,20,37,65,90,65,37,20, and ActAs using a 1-nm bandwidth and 1-nm step size. For melts at 222 nm, the temperature was increased in 5°steps with a 4.5-min equilibration time between steps.…”

Section: ϫ9mentioning

confidence: 99%

“…Analytical ultracentrifugation has demonstrated that ActA exists as a monomer in solution (20,32) with ambiguous results regarding higher order multimers (32). ActA has been shown to behave as an anomalously large species by gel filtration, suggesting that it may be an elongated molecule (32,34).…”

mentioning

confidence: 99%

Close Packing of Listeria monocytogenes ActA, a Natively Unfolded Protein, Enhances F-actin Assembly without Dimerization

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Lyo

Theriot

2008

Journal of Biological Chemistry

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Studies of the biochemistry of Listeria monocytogenes virulence protein ActA have typically focused on the behavior of bacteria in complex systems or on the characterization of the protein after expression and purification. Although prior in vivo work has proposed that ActA forms dimers on the surface of L. monocytogenes, dimerization has not been demonstrated in vitro, and little consideration has been given to the surface environment where ActA performs its pivotal role in bacterial actinbased motility. We have synthesized and characterized an ActA dimer and provide evidence that the two ActA molecules do not interact with each other even when tethered together. However, we also demonstrate that artificial dimers provide superior activation of actin nucleation by the Arp2/3 complex compared with monomers and that increased activation of the Arp2/3 complex by dimers may be a general property of Arp2/3 activators. It appears that the close packing (ϳ19 nm) of ActA molecules on the surface of L. monocytogenes is so dense that the kinetics of actin nucleation mimic that of synthetic ActA dimers. We also present observations indicating that ActA is a natively unfolded protein, largely random coil that is responsible for many of the unique physical properties of ActA including its extended structure, aberrant mobility during SDS-PAGE, and ability to resist irreversible denaturation upon heating.

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“…The G-actin-binding site is not required for intracellular bacterial motility, although it plays a role in the Arp2/3-mediated actin filament nucleation in vitro (Pistor et al, 2000;Skoble et al, 2000Skoble et al, , 2001. The central proline-rich domain, which includes three to four copies of the E/DFPPPPXD/E motif (Pistor et al, 1995;Smith et al, 1996;Niebuhr et al, 1997), binds to proteins of the Ena/VASP family (Pistor et al, 1995;Smith et al, 1996;Niebuhr et al, 1997;Machner et al, 2001), which includes the mammalian proteins vasodilator-stimulated phosphoprotein (VASP), mammalian Enabled (Mena), Ena-VASP-like (EVL), and the Drosophila protein Ena (Gertler et al, 1990(Gertler et al, , 1996Halbrugge et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Contribution of Ena/VASP Proteins to Intracellular Motility ofListeriaRequires Phosphorylation and Proline-rich Core but Not F-Actin Binding or Multimerization

Geese

Loureiro

Bear

et al. 2002

MBoC

Self Cite

101

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The Listeria model system has been essential for the identification and characterization of key regulators of the actin cytoskeleton such as the Arp2/3 complex and Ena/vasodilator-stimulated phosphoprotein (VASP) proteins. Although the role of Ena/VASP proteins in Listeria motility has been extensively studied, little is known about the contributions of their domains and phosphorylation state to bacterial motility. To address these issues, we have generated a panel of Ena/ VASP mutants and, upon expression in Ena/VASP-deficient cells, evaluated their contribution to Ena/VASP function in Listeria motility. The proline-rich region, the putative G-actin binding site, and the Ser/Thr phosphorylation of Ena/VASP proteins are all required for efficient Listeria motility. Surprisingly, the interaction of Ena/VASP proteins with F-actin and their potential ability to form multimers are both dispensable for their involvement in this process. Our data suggest that Ena/VASP proteins contribute to Listeria motility by regulating both the nucleation and elongation of actin filaments at the bacterial surface.

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ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

Cited by 32 publications

References 49 publications

Adapter Protein SH2-Bβ Stimulates Actin-Based Motility of Listeria monocytogenes in a Vasodilator-Stimulated Phosphoprotein (VASP)-Dependent Fashion

Adapter Protein SH2-Bβ Stimulates Actin-Based Motility of Listeria monocytogenes in a Vasodilator-Stimulated Phosphoprotein (VASP)-Dependent Fashion

Close Packing of Listeria monocytogenes ActA, a Natively Unfolded Protein, Enhances F-actin Assembly without Dimerization

Contribution of Ena/VASP Proteins to Intracellular Motility ofListeriaRequires Phosphorylation and Proline-rich Core but Not F-Actin Binding or Multimerization

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