Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function.

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular "traffic sign" for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission

Dlg1 in the ternary complex also recruits PI3K to the plasma membrane.

<p>Indirect immunofluorescence assays were performed and analyzed as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004102#ppat-1004102-g008" target="_blank">Figure 8</a>, except the indicated MCF10A lines were dually stained with antibodies to p85α/β (green) and Dlg1 (red). White scale bar = 20 µm.</p

E4-ORF1 activates PI3K and upregulates PI3K protein levels in a PBM-dependent manner but does not activate the MAP kinases ERK1 and ERK2.

<p>MCF10A lines transduced with empty retroviral vector (vector cells) or the vector encoding <i>wt</i> Ad9 E4-ORF1 (<i>wt</i>ORF1 cells), PBM mutant Ad9 E4-ORF1 V125A (V125A cells), or rasV12 (rasV12 cells) were treated with either DMSO vehicle (−) or 100 µM LY294002 (LY) (+) for 30 min. Cell extracts were analyzed in an immunoblot assay with antibodies to the indicated proteins.</p

E4-ORF1 also assembles the ternary complex in adenovirus-infected cells.

<p>(<b>A</b>) The ternary complex forms in cells infected with <i>wt</i> Ad9 virus but not with mutant Ad9-V125A virus that expresses PBM mutant V125A. MCF10A cells were mock infected or infected with the indicated virus at an approximate multiplicity of infection of 1. Extracts (375 µg of protein) prepared at 24 h post infection (p.i.) were immunoprecipitated with Dlg1 antibody. Recovered proteins, as well as cell extract (input), were analyzed in an immunoblot assay with antibodies to the indicated proteins. (<b>B</b>) Cells that are infected with <i>wt</i> Ad9 or mutant Ad9-V125A virus accumulate similar amounts of adenovirus major capsid protein hexon. Cell extracts were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. (<b>C</b>) Cells that are infected with <i>wt</i> Ad9 or mutant Ad9-V125A virus display similar cytopathic effects. Cells were visualized by phase contrast microscopy (40× magnification).</p

E4-ORF1 activates PI3K and upregulates PI3K protein levels in a Dlg1-dependent manner.

<p>(<b>A</b>) Depletion of Dlg1 with an shRNA diminishes PI3K activation and PI3K protein elevation in <i>wt</i>ORF1 cells. Extracts from vector or <i>wt</i>ORF1 cells stably transduced with pSUPER-Dlg1 shRNA (+) or pSUPER-Dlg1 scrambled control shRNA (−) were analyzed in an immunoblot assay with antibodies to the indicated proteins. (<b>B</b>) Depletion of Dlg1 has no effect on either PI3K activation or PI3K protein elevation in rasV12 cells. Extracts of rasV12 cells transduced with pSUPER-Dlg1 shRNA (+) or pSUPER-Dlg1 scrambled control shRNA (−) were analyzed in an immunoblot assay with antibodies to the indicated proteins.</p

The Human Adenovirus E4-ORF1 Protein Subverts Discs Large 1 to Mediate Membrane Recruitment and Dysregulation of Phosphatidylinositol 3-Kinase

<div><p>Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus <i>E4-ORF1</i> gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K). While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1), a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells.</p></div

Sites of E4-ORF1-induced activation of PI3K effector Akt co-localize with Dlg1 at the plasma membrane.

<p>Indirect immunofluorescence assays were performed and analyzed as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004102#ppat-1004102-g008" target="_blank">Figure 8</a>, except the indicated MCF10A lines were dually stained with antibodies to P-Akt (Ser473) (green) and Dlg1 (red). White scale bar = 20 µm.</p

E4-ORF1 binds endogenous cellular PI3K and tethers it to Dlg1 to form a ternary complex in cells.

<p>Extracts (300 µg of protein) of vector, <i>wt</i>ORF1, V125A, and T123D cells were immunoprecipitated with either p110α antibody or Dlg1 antibody as indicated. Recovered proteins, as well as cell extract (input), were analyzed in an immunoblot assay with antibodies to the indicated proteins. Note that V125A and T123D cells express Ad9 E4-ORF1 PBM mutants unable to bind PDZ proteins.</p