Beclin 1 Phosphorylation – at the Center of Autophagy Regulation

Autophagy is a tightly regulated catabolic process wherein cells under stress sequester cytosolic constituents like damaged proteins and organelles in double-membrane vesicles called autophagosomes. The autophagosomes degrade their cargo by lysosomal proteolysis generating raw materials for the biosynthesis of vital macromolecules. One of the initial steps in the assembly of autophagosomes from pre-autophagic structures is the recruitment and activation of the class III phosphatidylinositol 3-kinase complex consisting of Beclin 1 (BECN1), VPS34, VPS15, and ATG14 proteins. Several pieces of evidence indicate that the phosphorylation and ubiquitination of BECN1 at an array of residues fine-tune the responses to diverse autophagy modulating stimuli and helps in maintaining the balance between pro-survival autophagy and pro-apoptotic responses. In this mini-review, we will discuss the importance of distinct BECN1 phosphorylation events, the diverse signaling pathways and kinases involved and their role in the regulation of autophagy

LINC00261 and the Adjacent Gene FOXA2 Are Epithelial Markers and Are Suppressed during Lung Cancer Tumorigenesis and Progression

Lung cancer continues to be the leading cause of cancer-related deaths worldwide, with little improvement in patient survival rates in the past decade. Long non-coding RNAs (lncRNAs) are gaining importance as possible biomarkers with prognostic potential. By large-scale data mining, we identified LINC00261 as a lncRNA which was significantly downregulated in lung cancer. Low expression of LINC00261 was associated with recurrence and poor patient survival in lung adenocarcinoma. Moreover, the gene pair of LINC00261 and its neighbor FOXA2 were significantly co-regulated. LINC00261 as well as FOXA2 negatively correlated with markers for epithelial-to-mesenchymal transition (EMT) and were suppressed by the EMT inducer TGFβ. Hierarchical clustering of gene expression data from lung cancer cell lines could further verify the association of high LINC00261/FOXA2 expression to an epithelial gene signature. Furthermore, higher expression of the LINC00261/FOXA2 locus was associated with lung cancer cell lines with lower migratory capacity. All these data establish LINC00261 and FOXA2 as an epithelial-specific marker pair, downregulated during EMT and lung cancer progression, and associated with lower cell migration potential in lung cancer cells

Functional Analysis of KSRP Interaction with the AU-Rich Element of Interleukin-8 and Identification of Inflammatory mRNA Targets▿ †

mRNA stability is a major determinant of inflammatory gene expression. Rapid degradation of interleukin-8 (IL-8) mRNA is imposed by a bipartite AU-rich element (ARE) in the 3′ untranslated region (R. Winzen et al., Mol. Cell. Biol. 24:4835-4847, 2004). Small interfering RNA-mediated knockdown of the ARE-binding protein KSRP resulted in stabilization of IL-8 mRNA or of a β-globin reporter mRNA containing the IL-8 ARE. Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression

The p38/MK2-Driven Exchange between Tristetraprolin and HuR Regulates AU–Rich Element–Dependent Translation

<div><p>TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU–rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE–binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP's own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.</p> </div

Lyz2-Cre-Mediated Genetic Deletion of Septin7 Reveals a Role of Septins in Macrophage Cytokinesis and Kras-Driven Tumorigenesis

By crossing septin7-floxed mice with Lyz2-Cre mice carrying the Cre recombinase inserted in the Lysozyme-M (Lyz2) gene locus we aimed the specific deletion of septin7 in myeloid cells, such as monocytes, macrophages and granulocytes. Septin7flox/flox:Lyz2-Cre mice show no alterations in the myeloid compartment. Septin7-deleted macrophages (BMDMs) were isolated and analyzed. The lack of Septin7 expression was confirmed and a constitutive double-nucleation was detected in Septin7-deficient BMDMs indicating a defect in macrophage cytokinesis. However, phagocytic function of macrophages as judged by uptake of labelled E. coli particles and LPS-stimulated macrophage activation as judged by induction of TNF mRNA expression and TNF secretion were not compromised. In addition to myeloid cells, Lyz2-Cre is also active in type II pneumocytes (AT2 cells). We monitored lung adenocarcinoma formation in these mice by crossing them with the conditional knock-in Kras-LSL-G12D allele. Interestingly, we found that control mice without septin7 depletion die after 3–5 weeks, while the Septin7-deficient animals survived 11 weeks or even longer. Control mice sacrificed in the age of 4 weeks display a bronchiolo-alveolar hyperplasia with multiple adenomas, whereas the Septin7-deficient animals of the same age are normal or show only a weak multifocal brochiolo-alveolar hyperplasia. Our findings indicate an essential role of Septin7 in macrophage cytokinesis but not in macrophage function. Furthermore, septin7 seems absolutely essential for oncogenic Kras-driven lung tumorigenesis making it a potential target for anti-tumor interventions

A comprehensive model of the regulation of TNF mRNA and TNF release by p38 and MK2/3.

<p>LPS induces expression of TTP and activates p38 and MK2/3. TTP and PABP1 are MK2 substrates <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002977#pgen.1002977-Chrestensen1" target="_blank">[11]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002977#pgen.1002977-Bollig1" target="_blank">[14]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002977#pgen.1002977-Stoecklin1" target="_blank">[15]</a>, while HuR is a direct substrate for both p38 and MK2 and is shifted in its subcellular localization from the nuclear (n) to the cytoplasmic (c) compartment after phosphorylation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002977#pgen.1002977-Lafarga1" target="_blank">[47]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002977#pgen.1002977-Tran1" target="_blank">[74]</a>. HuR binds to the ARE and stimulates initiation of translation. Phosphorylation of TTP by MK2/3 impairs its capacity to compete with cytoplasmic HuR at the ARE in the 3′ UTR of translational controlled mRNAs (right part of scheme). Without phosphorylation at its MK2/3 sites TTP replaces HuR and interferes with translational initiation by competition with eIF4G binding to PABP1 or by inhibition of binding of the 43S PIC. Thereby TTP limits expression of ARE-containing mRNAs, including its own mRNA, at later stages in the response (left part of scheme).</p

Translational regulation of TNF reconstituted by macrophage cell lines.

<p>MK2/3-deficient macrophages were immortalized as described and the immortalized cell line was transduced with retroviral constructs coding for MK2 and GFP (+MK2) or GFP alone (+GFP), respectively. A) Western blot analysis of LPS (1 µg/ml)-induced protein expression and phosphorylation of the protein kinases p38 and MK2. Compared with MK2-rescued “wild type” cells, GFP-transduced “knockout” cells display absence of MK2, NOGO-B phosphorylation and a reduced TTP concentration. B) Relative TNF mRNA level in MK2- and GFP-rescued cells 1 h after LPS treatment as determined by RT-qPCR of TNF and ß-actin mRNA does not differ significantly. C) Comparison of pro-TNF protein expression between MK2- rescued and GFP-transduced cells 1 h after LPS-treatment. Pro-TNF level was calculated from a quantitative evaluation of the chemoluminescence of specific Western blot signals of total cell lysates (cf. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002977#pgen-1002977-g003" target="_blank">Figure 3D</a>). D) Comparison of the amount of secreted TNF between MK2- rescued and GFP-transduced cells measured by ELISA 1 h after LPS stimulation. P values of the two-tailed student's t-test are given.</p