Mhairi A. Skinner

V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway.

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.

Dynamic life and death interactions between Mycobacterium smegmatis and J774 macrophages

After internalization into macrophages non‐pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phago‐lysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill ‘non‐pathogenic’Mycobacterium smegmatis. This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V‐ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.

Abelson Tyrosine Kinase Controls Phagosomal Acidification Required for Killing of Mycobacterium tuberculosis in Human Macrophages

The mechanisms that regulate the acidification of intracellular compartments are key to host defense against pathogens. In this paper, we demonstrate that Abl tyrosine kinase, a master switch for cell growth and trafficking of intracellular organelles, controls the acidification of lysosomes in human macrophages. Pharmacological inhibition by imatinib and gene silencing of Abelson (Abl) tyrosine kinase reduced the lysosomal pH in human macrophages by increasing the transcription and expression of the proton pumping enzyme vacuolar-type H+-adenosine triphosphatase. Because lysosomal acidification is required for antimicrobial activity against intracellular bacteria, we determined the effect of imatinib on the growth of the major human pathogen Mycobacterium tuberculosis. Imatinib limited the multiplication of M. tuberculosis, and growth restriction was dependent on acidification of the mycobacterial compartment. The effects of imatinib were also active in vivo because circulating monocytes from imatinib-treated leukemia patients were more acidic than monocytes from control donors. Importantly, sera from imatinib-treated patients triggered acidification and growth restriction of M. tuberculosis in macrophages. In summary, our results identify the control of phagosomal acidification as a novel function of Abl tyrosine kinase and provide evidence that the regulation occurs on the level of the vacuolar-type H+-adenosine triphosphatase. Given the efficacy of imatinib in a mouse model of tuberculosis and our finding that orally administered imatinib increased the ability of human serum to trigger growth reduction of intracellular M. tuberculosis, clinical evaluation of imatinib as a complementary therapy of tuberculosis, in particular multidrug or extremely drug-resistant disease, is warranted.

β1 Integrin Binds the 16-kDa Subunit of Vacuolar H+-ATPase at a Site Important for Human Papillomavirus E5 and Platelet-derived Growth Factor Signaling

Integrins mediate adhesive interactions between cells and the extracellular matrix, and play a role in cell migration, proliferation, differentiation, cytoskeletal organization, and signal transduction. We have identified an interaction between the β1 integrin and the 16-kDa subunit of vacuolar H+-ATPase (16K). This interaction was first isolated in a yeast two-hybrid screen and confirmed by coimmunoprecipitation and inin vitro binding assays using bacterially expressed proteins. Immunofluorescent studies performed in L6 myoblasts expressing both native and epitope-tagged 16K demonstrate co-localization with β1 integrin in focal adhesions. Deletion of the fourth of four transmembrane helices in 16K results in loss of interaction with β1 integrin in vitroand in the two-hybrid system, and less prominent staining in focal adhesions. This helix is also required for ligand-independent activation of platelet-derived growth factor-β receptor signaling by the human papillomavirus E5 oncoprotein. Overexpression of 16K or expression of 16K lacking this helix alters the morphology of myoblasts and fibroblasts, suggesting that the interaction of 16K with integrins could be important for cell growth control. We also discuss the possible role 16K might play in integrin movement.

Stage-dependent Redistribution of the V-ATPase During Bovine Implantation

The 16-kD subunit of the vacuolar H+ -ATPase (V-ATPase), or ductin, is essential for the activity of this proton pump and has roles in intercellular communication and control of cell growth and differentiation. The V-ATPase is important for acidification-dependent degradation of tissue matrices through which some cell types move, and for pH regulation across some epithelial cell layers. Placentation involves intricate signaling, cell proliferation, and controlled invasion. We examined the distribution of three subunits of the V-ATPase in bovine trophoblast and endometrium at the time of implantation to determine the relationship of ductin expression to that of two other subunits, A (approximately 73 kD) and B (approximately 58 kD). Epithelial expression of all three subunits was observed, and in nonpregnant animals this expression was apical. As pregnancy proceeded, expression of all subunits became pericellular in luminal but not glandular epithelium, suggesting a redistribution of V-ATPase activity. The trophoblast expressed all three subunits during initial contact with the epithelium. In the stroma, ductin expression was reduced after implantation, and we discuss the possibility that ductin plays a role in the shifting communication between stromal and epithelial cells induced by embryo attachment.

Suppression of Tumor-related Glycosylation of Cell Surface Receptors by the 16-kDa Membrane Subunit of Vacuolar H+-ATPase

The glycosylation of integrins and other cell surface receptors is altered in many transformed cells. Notably, an increase in the number of β1,6-branched N-linked oligosaccharides correlates strongly with invasive growth of cells. An ectopic expression of the Golgi enzymeN-acetylglucosaminyltransferase V (GlcNAc-TV), which forms β1,6 linkages, promotes metastasis of a number of cell types. It is shown here that the 16-kDa transmembrane subunit (16K) of vacuolar H+-ATPase suppresses β1,6 branching of β1integrin and the epidermal growth factor receptor. Overexpression of 16K inhibits cell adhesion and invasion. 16K contains four hydrophobic membrane-spanning α-helices, and its ability to influence glycosylation is localized primarily within the second and fourth membrane-spanning α-helices. 16K also interacts directly with the transmembrane domain of β1 integrin, but its effects on glycosylation were independent of its binding to β1integrin. These data link cell surface tumor-related glycosylation to a component of the enzyme responsible for acidification of the exocytic pathway.

Abstract 2885: The NCI-Nature Pathway Interaction Database: A comprehensive resource for cell signaling information

The NCI-Nature Pathway Interaction Database (PID, http://pid.nci.nih.gov) is a freely available collection of professionally curated and expert-reviewed signaling and regulatory pathways composed of human molecular interactions and cellular processes extracted from the primary literature. As of November 2010, the database contains 116 pathways and networks encompassing 7810 interactions, 3531 proteins, 137 small molecules, 3101 complexes, 5930 peer-reviewed publications and more than 10500 uses of evidence codes, and includes recent additions to the Notch and Wnt pathways along with the AP1 network. Created in a collaboration between the U.S. National Cancer Institute and Nature Publishing Group, the PID offers a range of tools to facilitate pathway exploration. Users can browse pathways and create network maps based on molecule or biological process queries; interactive network maps are displayed in JPG, SVG and Silverlight formats. The Batch query tool allows users to overlay molecule lists, such as those derived from microarray data, onto pathways. Users can also download a list of references used to create the pathway, pathway molecule lists, and complete database content in extensible markup language (XML) or Biological Pathways Exchange (BioPAX) Level 2 or Level 3 formats. The database is updated every month and supplemented by a concise editorial section that provides synopses of recent noteworthy papers in cell signaling and specially commissioned articles on the practical uses of other relevant Biomedical Informatics tools. Users can sign up for email alerts or RSS feeds to receive database content updates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2885. doi:10.1158/1538-7445.AM2011-2885

Abstract LB-130: The NCI-Nature Pathway Interaction Database: A cell signaling resource

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The NCI-Nature Pathway Interaction Database (PID, http://pid.nci.nih.gov) is a freely available collection of professionally curated and expert-reviewed signaling and regulatory pathways composed of human molecular interactions, signaling events and cellular processes extracted from primary literature. Pathways selected for curation are based on potential drug targets, suggestions made by our users and reviewers, and other prominent cell signaling molecules. As of February 2010, the database contains 106 pathways encompassing 6696 interactions, 3231 proteins, 142 small molecules, 2622 complexes and 4843 peer-reviewed publications, and includes recent additions to both the p53 and EGFR pathways. Created in a collaboration between the U.S. National Cancer Institute and Nature Publishing Group, the PID is aimed at researchers interested in cell signaling pathways, such as molecular cell biologists, and bioinformaticians. The database offers a range of tools to facilitate pathway exploration. Users can browse the pre-defined set of pathways and create network maps centered on a single molecule or biological process of interest. The Batch query tool allows users to upload molecule lists, such as those derived from microarray data, and visualize the resulting molecular connectivity map. In addition, users can download lists of proteins, references used to create the pathway and complete database content in extensible markup language (XML) or Biological Pathways Exchange (BioPAX) format. The database is updated every month and supplemented by a concise editorial section that provides synopses of recent noteworthy papers in cell signaling and specially commissioned articles on the practical uses of other relevant Bioinformatics tools. Users can sign up for email alerts or RSS feeds to receive database updates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-130.