OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination

Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development

OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination

Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer developmen

Omkeerbare monoubiquitinatie betrokken bij de regulatie van kleine GTPasen.

status: publishe

BMMCs with NTAL KD exhibit enhanced actin depolymerization after stimulation with Ag or Ag + SCF.

<p>Cells were activated with Ag (250 ng/ml TNP-BSA; A), SCF (40 ng/ml; B) or Ag + SCF (C). At the indicated times, the cells were fixed, stained for F-actin with Alexa Fluor 488-phalloidin and analyzed by flow cytometry. Data were normalized to fluorescence of resting cells (similar in all cell types). Values indicate mean ± SE (n = 6). *^,+<i>p</i><0.05; **^,++<i>p</i><0.01; ***^,+++<i>p</i><0.001; significant differences between NTAL KOs and WTs (asterisks) and NTAL KDs vs WT pLKOs (crosslets) are shown.</p

Multiple Regulatory Roles of the Mouse Transmembrane Adaptor Protein NTAL in Gene Transcription and Mast Cell Physiology

<div><p>Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.</p></div

BMMCs with NTAL KD exhibit increased degranulation and calcium response.

<p>(A) IgE-sensitized BMMCs (WT, NTAL KO, NTAL KD, and WT pLKO) were stimulated for 30 minutes with various concentrations of Ag (TNP-BSA), and β-glucuronidase released into supernatant was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105539#s2" target="_blank">Materials and Methods</a>. Data represent means ± SE from 7–17 independent experiments performed in duplicates or triplicates). (B) IgE-sensitized BMMCs were stimulated with Ag [TNP-BSA at a concentration 100 ng/ml (Ag-100) or 500 ng/ml (Ag-500)], SCF (40 ng/ml), or both activators together. Data represent means ± SE from 6–20 independent experiments). (C–E) BMMCs were sensitized with IgE, loaded with Fura-2-AM (1 µg/ml), then stimulated with Ag (100 ng/ml TNP-BSA; C), SCF (40 ng/ml; D) or both activators together (E) and free intracellular Ca²⁺ was monitored by measuring fluorescence emission at 510 nm after excitation at 340 and 380 nm. Arrows indicate addition of Ag and/or SCF. Data are means ± SE from 11 (C), 7 (D) or 6 (E) independent experiments performed in duplicates. All data presented in A–E were obtained with BMMCs isolated from 3–5 mice. *^,+<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; in A, C and E, significant differences between NTAL KOs and WTs (asterisks) and NTAL KDs and WT pLKOs (crosslets) are shown.</p

Decreased NTAL expression after shRNA silencing.

<p>(A) BMMCs were infected with five lentiviral shRNA constructs (NTAL KD 1–5) or empty pLKO.1 construct (WT pLKO). After selection in puromycin, the amount of NTAL was assessed by immunoblotting. For comparison, NTAL in noninfected WT and NTAL KO cells was also evaluated. Actin was used as a loading control. (B) Densitometry analysis of NTAL immunoblots. The data were normalized to the amount of NTAL in WT pLKO cells and that of actin. Means ± SD were calculated from 3–7 independent experiments. ***<i>p</i><0.001.</p

Primers used in quantitative RT-PCR.

<p>Primers used in quantitative RT-PCR.</p

Length-Dependent Translation Efficiency of ER-Destined Proteins

Gene expression is a fundamental process that enables cells to produce specific proteins in a timely and spatially dependent manner. In eukaryotic cells, the complex organization of the cell body requires precise control of protein synthesis and localization. Certain mRNAs encode proteins with an N-terminal signal sequences that direct the translation apparatus toward a specific organelle. Here, we focus on the mechanisms governing the translation of mRNAs, which encode proteins with an endoplasmic reticulum (ER) signal in human cells. The binding of a signal-recognition particle (SRP) to the translation machinery halts protein synthesis until the mRNA–ribosome complex reaches the ER membrane. The commonly accepted model suggests that mRNA that encodes a protein that contains an ER signal peptide continuously repeats the cycle of SRP binding followed by association and dissociation with the ER. In contrast to the current view, we show that the long mRNAs remain on the ER while being translated. On the other hand, due to low ribosome occupancy, the short mRNAs continue the cycle, always facing a translation pause. Ultimately, this leads to a significant drop in the translation efficiency of small, ER-targeted proteins. The proposed mechanism advances our understanding of selective protein synthesis in eukaryotic cells and provides new avenues to enhance protein production in biotechnological settings

A hypothetical model on the role of NTAL in mast cell activation and transcriptional regulation.

<p>(A) In nonactivated WT cells both adaptor proteins, LAT and NTAL, and FcεRI β and γ subunits only exhibit weak phosphorylation, low [Ca²⁺]_i and transcription corresponding to nonactivated cells (Transcription profile 1). (B) After Ag-mediated aggregation of the FcεRI-IgE complex, β and γ subunits of the FcεRI are tyrosine phosphorylated by LYN and SYK. SYK then phosphorylates NTAL and LAT and this leads to enhanced Ca²⁺ uptake and further propagation of the signal, including dramatic changes in transcriptional regulation (Transcription profile 2). (C) In nonactivated NTAL-deficient cells, LAT and FcεRI subunits are only weakly tyrosine phosphorylated and the cells exhibit slightly different transcriptional regulation when compared to WT cells (Transcription profile 3). (D) After FcεRI triggering of NTAL-deficient cells, β and γ subunits of the FcεRI are tyrosine phosphorylated as in B, but because of the absence of NTAL, LAT is more phosphorylated by SYK. This leads to enhanced mobilization of Ca²⁺ and other signaling events and transcriptional regulation which differs from the one in activated WT cells (Transcription profile 4).</p