Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own.
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Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners. (+info)
An early stage in the establishment of cell polarity during chemotaxis of Dictyostelium dicoideum has been identified by a recent study; the new results also show that the development of cell polarity does not rely upon cytoskeletal rearrangement, and may use a spatial sensing mechanism. (+info)
The hematopoietic-specific adaptor protein gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors.
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BACKGROUND: The adaptor protein Gads is a Grb2-related protein originally identified on the basis of its interaction with the tyrosine-phosphorylated form of the docking protein Shc. Gads protein expression is restricted to hematopoietic tissues and cell lines. Gads contains a Src homology 2 (SH2) domain, which has previously been shown to have a similar binding specificity to that of Grb2. Gads also possesses two SH3 domains, but these have a distinct binding specificity to those of Grb2, as Gads does not bind to known Grb2 SH3 domain targets. Here, we investigated whether Gads is involved in T-cell signaling. RESULTS: We found that Gads is highly expressed in T cells and that the SLP-76 adaptor protein is a major Gads-associated protein in vivo. The constitutive interaction between Gads and SLP-76 was mediated by the carboxy-terminal SH3 domain of Gads and a 20 amino-acid proline-rich region in SLP-76. Gads also coimmunoprecipitated the tyrosine-phosphorylated form of the linker for activated T cells (LAT) adaptor protein following cross-linking of the T-cell receptor; this interaction was mediated by the Gads SH2 domain. Overexpression of Gads and SLP-76 resulted in a synergistic augmentation of T-cell signaling, as measured by activation of nuclear factor of activated T cells (NFAT), and this cooperation required a functional Gads SH2 domain. CONCLUSIONS: These results demonstrate that Gads plays an important role in T-cell signaling via its association with SLP-76 and LAT. Gads may promote cross-talk between the LAT and SLP-76 signaling complexes, thereby coupling membrane-proximal events to downstream signaling pathways. (+info)
Tyrosine phosphorylation is required for actin-based motility of vaccinia but not Listeria or Shigella.
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Studies of the actin-based motility of pathogens have provided important insights into the events occurring at the leading edge of motile cells [1] [2] [3]. To date, several actin-cytoskeleton-associated proteins have been implicated in the motility of Listeria or Shigella: vasodilator-stimulated phosphoprotein (VASP), vinculin and the actin-related protein complex of Arp2 and Arp3 [4] [5] [6] [7]. To further investigate the underlying mechanism of actin-tail assembly, we examined the localization of components of the actin cytoskeleton including Arp3, VASP, vinculin and zyxin during vaccinia, Listeria and Shigella infections. The most striking difference between the systems was that a phosphotyrosine signal was observed only at the site of vaccinia actin-tail assembly. Micro-injection experiments demonstrated that a phosphotyrosine protein plays an important role in vaccinia actin-tail formation. In addition, we observed a phosphotyrosine signal on clathrin-coated vesicles that have associated actin-tail-like structures and on endogenous vesicles in Xenopus egg extracts which are able to nucleate actin tails [8] [9]. Our observations indicate that a host phosphotyrosine protein is required for the nucleation of actin filaments by vaccinia and suggest that this phosphoprotein might be associated with cellular membranes that can nucleate actin. (+info)
Intracellular signalling: PDK1--a kinase at the hub of things.
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Phosphoinositide-dependent kinase 1 (PDK1) is at the hub of many signalling pathways, activating PKB and PKC isoenzymes, as well as p70 S6 kinase and perhaps PKA. PDK1 action is determined by colocalization with substrate and by target site availability, features that may enable it to operate in both resting and stimulated cells. (+info)
Prodigious substrate specificity of AAC(6')-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci.
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BACKGROUND: High-level gentamicin resistance in enterococci and staphylococci is conferred by AAC(6')-APH(2"), an enzyme with 6'-N-acetyltransferase and 2"-O-phosphotransferase activities. The presence of this enzyme in pathogenic gram-positive bacteria prevents the successful use of gentamicin C and most other aminoglycosides as therapeutic agents. RESULTS: In an effort to understand the mechanism of aminoglycoside modification, we expressed AAC(6')-APH(2") in Bacillus subtilis. The purified enzyme is monomeric with a molecular mass of 57 kDa and displays both the expected aminoglycoside N-acetyltransferase and O-phosphotransferase activities. Structure-function analysis with various aminoglycosides substrates reveals an enzyme with broad specificity in both enzymatic activities, accounting for AAC(6')-APH(2")'s dramatic negative impact on clinical aminoglycoside therapy. Both lividomycin A and paromomycin, aminoglycosides lacking a 6'-amino group, were acetylated by AAC(6')-APH(2"). The infrared spectrum of the product of paromomycin acetylation yielded a signal consistent with O-acetylation. Mass spectral and nuclear magnetic resonance analysis of the products of neomycin phosphorylation indicated that phosphoryl transfer occurred primarily at the 3'-OH of the 6-aminohexose ring A, and that some diphosphorylated material was also present with phosphates at the 3'-OH and the 3"'-OH of ring D, both unprecedented observations for this enzyme. Furthermore, the phosphorylation site of lividomycin A was determined to be the 5"-OH of the pentose ring C. CONCLUSIONS: The bifunctional AAC(6')-APH(2") has the capacity to inactivate virtually all clinically important aminoglycosides through N- and O-acetylation and phosphorylation of hydroxyl groups. The extremely broad substrate specificity of this enzyme will impact on future development of aminoglycosides and presents a significant challenge for antibiotic design. (+info)
High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.
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BACKGROUND: Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. RESULTS: We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. CONCLUSIONS: We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications. (+info)
Tyrosine phosphorylation and complex formation of Cbl-b upon T cell receptor stimulation.
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Cbl-b, a mammalian homolog of Cbl, consists of an N-terminal region (Cbl-b-N) highly homologous to oncogenic v-Cbl, a Ring finger, and a C-terminal region containing multiple proline-rich stretches and potential tyrosine phosphorylation sites. In the present study, we demonstrate that upon engagement of the T cell receptor (TCR), endogenous Cbl-b becomes rapidly tyrosine-phosphorylated. In heterogeneous COS-1 cells, Cbl-b was phosphorylated on tyrosine residues by both Syk- (Syk/Zap-70) and Src- (Fyn/Lck) family kinases, with Syk kinase inducing the most prominent effect. Syk associates and phosphorylates Cbl-b in Jurkat T cells. A Tyr-316 Cbl-binding site in Syk was required for the association with and for the maximal tyrosine phosphorylation of Cbl-b. Mutation at a loss-of-function site (Gly-298) in Cbl-b-N disrupts its interaction with Syk. Cbl-b constitutively binds Grb2 and becomes associated with Crk-L upon TCR stimulation. The Grb2- and the Crk-L-binding regions were mapped to the C-terminus of Cbl-b. The Crk-L-binding sites were further determined to be Y655DVP and Y709KIP, with the latter being the primary binding site. Taken together, these results implicate that Cbl-b is involved in TCR-mediated intracellular signaling pathways. (+info)