Activation of a Metabolic Gene Regulatory Network Downstream of mTOR Complex 1

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1α) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease

Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2

The PTEN and TSC2 tumor suppressors inhibit mammalian target of rapamycin (mTOR) signaling and are defective in distinct hamartoma syndromes. Using mouse genetics, we find that Pten and Tsc2 act synergistically to suppress the severity of a subset of tumors specific to loss of each of these genes. Interestingly, we find that the slow-growing tumors specific to Tsc2(+/-) mice exhibit defects in signaling downstream of Akt. However, Pten haploinsufficiency restores Akt signaling in these tumors and dramatically enhances their severity. This study demonstrates that attenuation of the PI3K-Akt pathway in tumors lacking TSC2 contributes to their benign nature

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

<div><p>DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for <i>D</i>NA<i>J</i>-<i>a</i>ssociated <i>n</i>uclear <i>g</i>l<i>o</i>bular <i>s</i>tructures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity.</p></div

DJANGOS formation requires DNAJ/Hsc70 activity.

<p><b>A.</b> RIPA buffer extracts were prepared from unmodified HeLa cells (H) or cells transduced with genes encoding wild-type B12-HA (B12) or the H138Q-HA mutant (12Q). Extracts were immunoprecipitated with anti-B12 and subjected to SDS-polyacrylamide gel electrophoresis, or electrophoresed without immunoprecipitation (lane 1). Top and bottom panels show anti-HA and Hsc70 immunoblots, respectively. <b>B.</b> HeLa cells were infected with retroviruses expressing the indicated wild-type or mutant B12-HA (top panels) or B14-HA (bottom panels) gene and stained with anti-HA to detect B12 or B14. Images are individual confocal slices. The left panels show cells expressing wild-type B12 or B14, the middle panels the J-domain mutants and the right panel the carboxy- truncated B12ΔC.</p

Live-cell imaging of DJANGOS.

<p>Static images retrieved from live cell imaging of HeLaM or CV1 cells co-transfected with plasmids expressing B12-mCherry and LBR-eGFP. <b>A.</b> Top panels show two images of a HeLaM cell separated by 15 minutes acquired shortly before mitosis. Bottom panels shows images acquired one hour apart of the daughter cells of the cell shown in the top panels. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094322#pone.0094322.s006" target="_blank">Movie S3</a>. <b>B.</b> Left panel shows cytoplasmic billowing of B12 structures in CV1 cells; right panel shows cytoplasmic B12 “balloons on strings” in CV1 cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094322#pone.0094322.s007" target="_blank">Movies S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094322#pone.0094322.s008" target="_blank">S5</a>.</p

Co-localization of B12 with Hsc70 and other proteins in DJANGOS.

<p>HeLa cells over-expressing B12-HA were stained with anti-B12 (A–F) or anti-HA (G) to detect B12 (in green) and with antibodies specific for the indicated cellular protein (in red). Overlapping signals in the merged images on the right are shown in yellow. The same confocal slice is shown in each row of panels. <b>A.</b> Hsc70. <b>B.</b> BiP. <b>C.</b> Sec61γ. <b>D.</b> Emerin. This section is from the same nucleus shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094322#pone-0094322-g001" target="_blank">Figure 1B</a>. <b>E.</b> Lamin A. <b>F.</b> Lamin B. <b>G.</b> NPC. This slice is near the bottom of the nucleus.</p

DNAJB12 induces formation of DJANGOS.

<p>Immunofluorescent staining of DJANGOS in cells over-expressing HA-tagged DNAJB12 (A–F) or DNAJB12 fused to tGFP (G–I), detected with anti-HA or anti-tGFP antibodies (both green), respectively. All images except (F) are from confocal stacks compressed along the Z-axis to create a single image. <b>A.</b> HeLa cells were also stained with anti-PDI (red) and DAPI (blue, to visualize nuclei). <b>B–E.</b> HeLa cell nuclei displaying different varieties of DJANGOS. <b>F.</b> Single confocal slice midway up the nucleus showing regular distribution of DJANGOS under the nuclear envelope in a HeLa cell. <b>G and H.</b> Nuclei of human foreskin fibroblasts and CV1 monkey cells, respectively. <b>I.</b> Rare cytoplasmic form of DJANGOS, seen here at lower magnification in a HeLa cell lacking the more common nuclear forms.</p

Electron tomography of DJANGOS.

<p><b>A.</b> Tomographic slice of an EM thick section showing a nuclear pore-associated structure formed in HeLa cells expressing B12-HA. Cytoplasm and nucleus are labeled with “C” and “N,” respectively. The thick arrow points to an atypical nuclear pore structure in cross-section at the junction with the nuclear envelope; the thin arrow points to a classic nuclear pore in the double membrane inside the nucleus. <b>B.</b> End-on view of the atypical nuclear pore at the neck of the double-membrane structure. <b>C.</b> 3D rendering of the tomogram shows the NPCs in blue. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094322#pone.0094322.s004" target="_blank">Movie S1</a>.</p

Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm

Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm