The TRANSPATH signal transduction database: a knowledge base on signal transduction networks.

UNLABELLED: TRANSPATH is an information system on gene-regulatory pathways, and an extension module to the TRANSFAC database system (Wingender et al., Nucleic Acids Res., 28, 316-319, 2000). It focuses on pathways involved in the regulation of transcription factors in different species, mainly human, mouse and rat. Elements of the relevant signal transduction pathways like complexes, signaling molecules, and their states are stored together with information about their interaction in an object-oriented database. The database interface provides clickable maps and automatically generated pathway cascades as additional ways to explore the data. All information is validated with references to the original publications. Also, references to other databases are provided (TRANSFAC, SWISS-PROT, EMBL, PubMed and others). AVAILABILITY: The database is available over (http://transpath.gbf.de) for interactive perusal. As an exchange format for the data, eXtensible Markup Language (XML) flatfiles and a Document Type Definition (DTD) are provided.

Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex.

The recruitment of actin to the surface of intracellular Listeria monocytogenes and subsequent tail formation is dependent on the expression of the bacterial surface protein ActA. Of the different functional domains of ActA identified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility. Mutational analysis of this domain which abolished actin recruitment by intracellular Listeria monocytogenes identified two arginine residues within the 146-KKRRK-150 motif that are essential for its activity. More specifically, recruitment of the Arp2/3 complex to the bacterial surface, as assessed by immunofluorescence staining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells with latrunculin B, which abrogated actin filament formation, did not affect association of ActA with p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and precedes, actin polymerisation. Our data suggest that binding of the Arp2/3 complex is mediated by specific interactions dependent on arginine residues within the 146-KKRRK-150 motif present in ActA.

Cloning and functional expression of a 'fast' fungal kinesin.

Conventional kinesins are molecular motors that move towards the plus end of microtubules. In animal species, they have been shown to be remarkably conserved in terms of both their primary sequence and several physiological properties, including their velocity of movement. Here we report the cloning of Synkin, a homologue of conventional kinesin from the zygomycete fungus Syncephalastrum racemosum [Steinberg, Eur. J. Cell Biol. 73 (1997) 124-131] that is 4-5 times faster than its animal counterparts. Expression in bacteria yields a fully functional motor that moves at the same speed as the native motor isolated from fungal hyphae and has similar hydrodynamic properties. Its sequence is most closely related to that of two other fungal kinesins from Neurospora and Ustilago, and shares several biochemical properties with the Neurospora motor. Fungal kinesins therefore seem to form a conserved subfamily of conventional kinesins distantly related to animal kinesins. They may help to identify sequence features important for determining motor velocity.

Ligand recruitment by vinculin domains in transfected cells.

Vinculin, a prominent protein component of microfilament-membrane attachment sites, consists of three major domains: an N-terminal, compact head and a C-terminal rod-like tail that are connected by a flexible, proline-rich hinge. In vitro, the protein has been shown to interact with numerous ligands, including other components of the microfilament system. To characterize the ligand recruitment ability of the different vinculin domains in a cellular environment, we used a novel approach of comprising chimeric proteins of either the vinculin head, hinge or tail regions, fused to the membrane anchor sequence of ActA, a surface protein of the intracellular bacterial pathogen Listeria monocytogenes. When PtK2 cells were transfected with the corresponding constructs, the ActA membrane anchor directed the chimeric polypeptides to mitochondrial membranes. In this position, they accumulated microfilament proteins, as seen by immunofluorescence analysis. A chimera comprising the full length vinculin clone recruited a substantial amount of the cellular F-actin, the vasodilator stimulated phosphoprotein (VASP) and paxillin, but little alpha-actinin and talin. The presence of only the vinculin head directed some of the fusion protein to focal contacts, and alpha-actinin recruitment was still ineffective. Prominent recruitment of F-actin and of VASP required the presence of the tail and proline-rich hinge, respectively. Reducing the vinculin tail to short pieces harboring only one of the two F-actin binding sequences, which were defined by in vitro experiments, resulted in loss of activity, possibly by incorrect polypeptide folding. The proline-rich hinge domain could be exchanged for the analogous region of the ActA protein, and the number of such proline-clusters, containing an FPPPP motif, correlated with the extent of VASP recruitment. The results show that this system can be used to analyze in vivo the activity of vinculin domains responsible for the assembly of various cytoskeletal ligands.

The ActA polypeptides of Listeria ivanovii and Listeria monocytogenes harbor related binding sites for host microfilament proteins.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes acts as a nucleator protein, generating the actin cytoskeleton around intracellularly motile bacteria. In this work, we examined the functional similarity of ActA from Listeria ivanovii (iActA) ATCC 19119 to its L. monocytogenes counterpart. The amino acid sequence of iActA predicts a molecular mass of 123 kDa and harbors eight proline-rich repeats. For functional analysis, various iActA derivatives and hybrid constructs of L. ivanovii and L. monocytogenes ActA polypeptides were transiently expressed in epithelial cells and examined for recruitment of host microfilament proteins by a mitochondrial targeting assay. As has been demonstrated with ActA, iActA also spontaneously inserted into the surface of mitochondria and induced recruitment of actin, alpha-actinin, and the vasodilator-stimulated phosphoprotein (VASP) to these subcellular organelles. By comparison of amino-terminally truncated iActA derivatives for their ability to recruit cytoskeletal proteins, a region essential for actin filament accumulation was identified between amino acid residues 290 and 325. Such derivatives, however, retained their ability to bind VASP. Replacement of the proline-rich repeats in ActA with those of iActA also resulted in VASP recruitment. Hence, despite the limited overall sequence homology between ActA and iActA, the two molecules consist of at least two similar domains: a highly positively charged N-terminal domain that is directly involved in actin filament recruitment and a proline-rich repeat region required for VASP binding.

The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins.

BACKGROUND: Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein, VASP. Only VASP is known to bind directly to ActA. We sought to determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system. RESULTS: We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP. CONCLUSIONS: Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton.

Listeria monocytogenes, a facultative intracellular pathogen, employs actin and other microfilament-associated proteins to move through the host cell cytoplasm. Isogenic mutants of L. monocytogenes lacking the surface-bound ActA polypeptide no longer interact with cytoskeletal elements and are, as a consequence, non-motile (Domann et al., 1992, EMBO J., 11, 1981-1990; Kocks et al., 1992, Cell, 68, 521-531). To investigate the interaction of ActA with the microfilament system in the absence of other bacterial factors, the listerial actA gene was expressed in eukaryotic cells. Immunofluorescence studies revealed that the complete ActA, including its C-terminally located bacterial membrane anchor, colocalized with mitochondria in transfected cells. When targeted to mitochondria, the ActA polypeptide recruited actin and alpha-actinin to these cellular organelles with concomitant reorganization of the microfilament system. Removal of the internal proline-rich repeat region of ActA completely abrogated interaction with cytoskeletal components. Our results identify the ActA polypeptide as a nucleator of the actin cytoskeleton and provide the first insights into the molecular nature of such controlling elements in microfilament organization.

A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin.

The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.

Expression of viral hemagglutinin on the surface of E. coli.

Expression of a foreign protein molecule on the E. coli bacterial surface has been achieved through hybrid plasmid construction of fusion proteins using outer membrane protein ompA as a carrier system. Influenza virus hemagglutinin fusion proteins of this character have been shown to become integrated into the bacterial outer membrane and to expose their hemagglutinin moiety at the exterior surface in a conformation which is at least similar to the authentic viral antigen structure.