Publications by authors named "Manoj B Menon"

37 Publications

Cdc42-Borg4-Septin7 axis regulates HSC polarity and function.

EMBO Rep 2021 Dec 18;22(12):e52931. Epub 2021 Oct 18.

Institute of Molecular Medicine, Ulm University, Ulm, Germany.

Aging of hematopoietic stem cells (HSCs) is caused by the elevated activity of the small RhoGTPase Cdc42 and an apolar distribution of proteins. Mechanisms by which Cdc42 activity controls polarity of HSCs are not known. Binder of RhoGTPases proteins (Borgs) are known effector proteins of Cdc42 that are able to regulate the cytoskeletal Septin network. Here, we show that Cdc42 interacts with Borg4, which in turn interacts with Septin7 to regulate the polar distribution of Cdc42, Borg4, and Septin7 within HSCs. Genetic deletion of either Borg4 or Septin7 results in a reduced frequency of HSCs polar for Cdc42 or Borg4 or Septin7, a reduced engraftment potential and decreased lymphoid-primed multipotent progenitor (LMPP) frequency in the bone marrow. Taken together, our data identify a Cdc42-Borg4-Septin7 axis essential for the maintenance of polarity within HSCs and for HSC function and provide a rationale for further investigating the role of Borgs and Septins in the regulation of compartmentalization within stem cells.
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http://dx.doi.org/10.15252/embr.202152931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8647144PMC
December 2021

Measuring lncRNA Expression by Real-Time PCR.

Methods Mol Biol 2021 ;2348:93-111

Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India.

Long noncoding RNAs are defined as transcripts longer than 200 nt with no protein coding potential. Most lncRNAs are expressed in a tissue-specific manner and barring a few, their absolute expression is lower compared to most coding transcripts. Differential expression studies have contributed the most to the functional characterisation of the lncRNAs we know. Sensitive and specific quantification of lncRNA expression is crucial for such studies. SYBR Green dye based real time quantitative PCR is a simple and affordable method of quantitative PCR, wherein the specific binding of the dye to double stranded DNA amplicon emits fluorescence proportionate to the amount of PCR products. Here we describe a detailed protocol for successful lncRNA quantitation by reverse transcription followed by SYBR Green chemistry-based real-time PCR.
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http://dx.doi.org/10.1007/978-1-0716-1581-2_6DOI Listing
September 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Editorial: Autophagy and Related Transcription Factors in Liver and Gut Diseases.

Front Pharmacol 2019 30;10:1610. Epub 2020 Jan 30.

Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.

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http://dx.doi.org/10.3389/fphar.2019.01610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002321PMC
January 2020

SEPT7 Interacts with KIF20A and Regulates the Proliferative State of Neural Progenitor Cells During Cortical Development.

Cereb Cortex 2020 05;30(5):3030-3043

Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.

Balanced proliferation and differentiation of neural progenitor cells (NPCs) are critical for brain development, but how the process is regulated and what components of the cell division machinery is involved are not well understood. Here we report that SEPT7, a cell division regulator originally identified in Saccharomyces cerevisiae, interacts with KIF20A in the intercellular bridge of dividing NPCs and plays an essential role in maintaining the proliferative state of NPCs during cortical development. Knockdown of SEPT7 in NPCs results in displacement of KIF20A from the midbody and early neuronal differentiation. NPC-specific inducible knockout of Sept7 causes early cell cycle exit, precocious neuronal differentiation, and ventriculomegaly in the cortex, but surprisingly does not lead to noticeable cytokinesis defect. Our data uncover an interaction of SEPT7 and KIF20A during NPC divisions and demonstrate a crucial role of SEPT7 in cell fate determination. In addition, this study presents a functional approach for identifying additional cell fate regulators of the mammalian brain.
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http://dx.doi.org/10.1093/cercor/bhz292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197076PMC
May 2020

Alternative Translation Initiation Generates a Functionally Distinct Isoform of the Stress-Activated Protein Kinase MK2.

Cell Rep 2019 06;27(10):2859-2870.e6

Institute of Cell Biochemistry, Hannover Medical School (MHH), Carl-Neuberg-Str. 1, 30625 Hannover, Germany; Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. Electronic address:

Alternative translation is an important mechanism of post-transcriptional gene regulation leading to the expression of different protein isoforms originating from the same mRNA. Here, we describe an abundant long isoform of the stress/p38-activated protein kinase MK2. This isoform is constitutively translated from an alternative CUG translation initiation start site located in the 5' UTR of its mRNA. The RNA helicase eIF4A1 is needed to ensure translation of the long and the known short isoforms of MK2, of which the molecular properties were determined. Only the short isoform phosphorylated Hsp27 in vivo, supported migration and stress-induced immediate early gene (IEG) expression. Interaction profiling revealed short-isoform-specific binding partners that were associated with migration. In contrast, the long isoform contains at least one additional phosphorylatable serine in its unique N terminus. In sum, our data reveal a longer isoform of MK2 with distinct physiological properties.
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http://dx.doi.org/10.1016/j.celrep.2019.05.024DOI Listing
June 2019

Beclin 1 Phosphorylation - at the Center of Autophagy Regulation.

Front Cell Dev Biol 2018 12;6:137. Epub 2018 Oct 12.

Division of Cancer Research, Department of Thoracic Surgery, Medical Center - University of Freiburg, Freiburg, Germany.

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.
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http://dx.doi.org/10.3389/fcell.2018.00137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194997PMC
October 2018

Non-coding transcript variants of protein-coding genes - what are they good for?

RNA Biol 2018 10;15(8):1025-1031. Epub 2018 Sep 10.

c Institute of Cell Biochemistry , Hannover Medical School , Hannover , Germany.

The total number of protein-coding genes in the human genome is not significantly higher than those in much simpler eukaryotes, despite a general increase in genome size proportionate to the organismal complexity. The large non-coding transcriptome and extensive differential splicing, are increasingly being accepted as the factors contributing to the complex mammalian physiology and architecture. Recent studies reveal additional layers of functional complexity: some long non-coding RNAs have been re-defined as micropeptide or microprotein encoding transcripts, and in turn some protein-coding RNAs are bifunctional and display also non-coding functions. Moreover, several protein-coding genes express long non-coding RNA splice-forms and generate circular RNAs in addition to their canonical mRNA transcripts, revoking the strict definition of a gene as coding or non-coding. In this mini review, we discuss the current understanding of these hybrid genes and their possible roles and relevance.
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http://dx.doi.org/10.1080/15476286.2018.1511675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161691PMC
November 2018

Differentiated macrophages acquire a pro-inflammatory and cell death-resistant phenotype due to increasing XIAP and p38-mediated inhibition of RipK1.

J Biol Chem 2018 07 13;293(30):11913-11927. Epub 2018 Jun 13.

From the Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M8, Canada,

Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIP), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIP may allow inflammatory macrophages to participate in pathogen control for a longer duration.
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http://dx.doi.org/10.1074/jbc.RA118.003614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066316PMC
July 2018

Septins: Active GTPases or just GTP-binding proteins?

Cytoskeleton (Hoboken) 2019 01 30;76(1):55-62. Epub 2018 Aug 30.

Hannover Medical School, Institute of Cell Biochemistry, Hannover, 30625, Germany.

Septins are conserved cytoskeletal proteins with unique filament forming capabilities and roles in cytokinesis and cell morphogenesis. Septins undergo hetero-oligomerization and assemble into higher order structures including filaments, rings, and cages. Hetero- and homotypic interactions of septin isoforms involve alternating GTPase (G)-domain interfaces and those mediated by N- and C-terminal extensions. While most septins bind GTP, display weak GTP-hydrolysis activity and incorporate guanine nucleotides in their interaction interfaces, studies using GTPase-inactivating mutations have failed to conclusively establish a crucial role for GTPase activity in mediating septin functions. In this mini-review, we will critically assess the role of GTP-binding and -hydrolysis on septin assembly and function. The relevance of G-domain activity will also be discussed in the context of human septin mutations as well as the development of specific small-molecules targeting septin polymerization. As structural determinants of septin oligomer interfaces, G-domains are attractive targets for ligand-based inhibition of septin assembly. Whether such an intervention can predictably alter septin function is a major question for future research.
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http://dx.doi.org/10.1002/cm.21451DOI Listing
January 2019

To die or not to die: Regulatory feedback phosphorylation circuits determine receptor-interacting protein kinase-1 (RIPK1) function.

Mol Cell Oncol 2018 30;5(1):e1396389. Epub 2017 Nov 30.

Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany.

Complex posttranslational modifications determine the effects of receptor-interacting protein kinase-1 (RIPK1) on cell survival and death. Studies from us and others have revealed a p38/MK2-dependent checkpoint in RIPK1 signaling. MAPKAP kinase 2 (MK2) phosphorylates RIPK1 to suppress RIPK1-mediated apoptosis and necroptosis in response to diverse stimuli relevant to inflammation, infection, genotoxic stress and chemotherapy.
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http://dx.doi.org/10.1080/23723556.2017.1396389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791865PMC
November 2017

MK2-TNF-Signaling Comes Full Circle.

Trends Biochem Sci 2018 03 21;43(3):170-179. Epub 2017 Dec 21.

Institute of Cell Biochemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Electronic address:

MK2 (p38-activated protein kinase 2) is essential for tumor necrosis factor (TNF) biosynthesis, mainly operating by post-transcriptional regulation. Deletion of the gene encoding MK2 strongly reduced serum TNF and protected against endotoxic shock, demonstrating the positive role of p38/MK2 in TNF signaling at the level of ligand expression. Recent evidence indicates that MK2 directly phosphorylates the TNF receptor interactor RIPK1 and suppresses its activity, thereby limiting TNF-mediated apoptosis and necroptosis - pointing to a more complex, double-edged role of MK2 in TNF signaling. In addition, novel MK2 substrates have emerged in the DNA damage response, autophagy, and obesity, making MK2 a multifunctional kinase at the crossroads of stress response and cell death. We therefore propose a more general role of p38/MK2 signaling in the timely coordinated onset and resolution of inflammation and beyond.
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http://dx.doi.org/10.1016/j.tibs.2017.12.002DOI Listing
March 2018

Editorial: Emerging Functions of Septins.

Front Cell Dev Biol 2017 24;5:73. Epub 2017 Aug 24.

Institute for Cell Biochemistry, Hannover Medical SchoolHannover, Germany.

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http://dx.doi.org/10.3389/fcell.2017.00073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609633PMC
August 2017

p38/MK2-dependent phosphorylation controls cytotoxic RIPK1 signalling in inflammation and infection.

Nat Cell Biol 2017 Oct 18;19(10):1248-1259. Epub 2017 Sep 18.

Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Eppendorf, Hamburg 20246, Germany.

Receptor-interacting protein kinase-1 (RIPK1), a master regulator of cell fate decisions, was identified as a direct substrate of MAPKAP kinase-2 (MK2) by phosphoproteomic screens using LPS-treated macrophages and stress-stimulated embryonic fibroblasts. p38/MK2 interact with RIPK1 in a cytoplasmic complex and MK2 phosphorylates mouse RIPK1 at Ser321/336 in response to pro-inflammatory stimuli, such as TNF and LPS, and infection with the pathogen Yersinia enterocolitica. MK2 phosphorylation inhibits RIPK1 autophosphorylation, curtails RIPK1 integration into cytoplasmic cytotoxic complexes, and suppresses RIPK1-dependent apoptosis and necroptosis. In Yersinia-infected macrophages, RIPK1 phosphorylation by MK2 protects against infection-induced apoptosis, a process targeted by Yersinia outer protein P (YopP). YopP suppresses p38/MK2 activation to increase Yersinia-driven apoptosis. Hence, MK2 phosphorylation of RIPK1 is a crucial checkpoint for cell fate in inflammation and infection that determines the outcome of bacteria-host cell interaction.
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http://dx.doi.org/10.1038/ncb3614DOI Listing
October 2017

IL-1β-Induced Downregulation of the Multifunctional PDZ Adaptor PDZK1 Is Attenuated by ERK Inhibition, RXRα, or PPARα Stimulation in Enterocytes.

Front Physiol 2017 7;8:61. Epub 2017 Feb 7.

Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School Hannover, Germany.

The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients. We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1β, and analysing PDZK1 promotor activity, mRNA and protein expression. IL-1β was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1β-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-β downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1β mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1β-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1β-mediated inhibition. The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1β-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.
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http://dx.doi.org/10.3389/fphys.2017.00061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293818PMC
February 2017

Na /H exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells.

J Cell Physiol 2017 Jul 21;232(7):1669-1680. Epub 2017 Feb 21.

Departments of Gastroenterology, Hemostatsis, Oncology and Stem Cell Transplantation, Medical School of Hannover, Germany.

Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na /H exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1-dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1-dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady-state pH and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.
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http://dx.doi.org/10.1002/jcp.25758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396337PMC
July 2017

TPL2 meets p38MAPK: emergence of a novel positive feedback loop in inflammation.

Biochem J 2016 10;473(19):2995-9

Institute of Physiological Chemistry, Hannover Medical University, Carl-Neuberg-Str. 1, Hannover 30625, Germany.

The activation of p38(MAPK) by Toll-like receptor signalling is essential for the inflammatory response of innate immunity due to its role in post-transcriptional regulation of TNFα and cytokine biosynthesis. p38(MAPK) activation proceeds by the upstream MAP2Ks, MAPK kinase (MKK)3/6 as well as MKK4, which in turn are substrates for MAP3Ks, such as TGFβ-activated protein kinase-1 (TAK1). In contrast, TPL2 has been described as an exclusive MAP3K of MKK1/2-triggering activation of the classical ERKs, ERK1/2. In the recent issue of the Biochemical Journal, Pattison et al report their screening for TPL2 substrates in LPS-stimulated macrophages and the identification of MKK3/6. Using catalytic-dead TPL2 (Map3k8(D270A/D270A)) knockin macrophages, they demonstrated that activation of MKK3/6 by TPL2 significantly contributes to LPS-dependent TNFα biosynthesis and is also essential for TNF-receptor 1 signalling. Hence, a new signalling pathway from TAK1 via IκB kinase, p105 NFκB and TPL2 to MKK3/6 and p38(MAPK) is established in macrophages. Taking into account that some isoforms of p38(MAPK) are necessary for maintaining functional steady-state levels of TPL2, a positive feedback loop in inflammation emerges.
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http://dx.doi.org/10.1042/BCJ20160672CDOI Listing
October 2016

Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics.

Cancer Cell 2016 Feb;29(2):145-58

Cell Signaling & Cell Death and Cancer & Hematology Divisions, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia. Electronic address:

Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.
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http://dx.doi.org/10.1016/j.ccell.2016.01.006DOI Listing
February 2016

GTPase domain driven dimerization of SEPT7 is dispensable for the critical role of septins in fibroblast cytokinesis.

Sci Rep 2016 Jan 28;6:20007. Epub 2016 Jan 28.

Institute of Physiological Chemistry, Hannover Medical School, Hannover- 30625, Germany.

Septin 7 (SEPT7) has been described to be essential for successful completion of cytokinesis in mouse fibroblasts, and Sept7-deficiency in fibroblasts constitutively results in multinucleated cells which stop proliferation. Using Sept7(flox/flox)fibroblasts we generated a cellular system, where the cytokinetic defects of Cre-mediated deletion of the Sept7 gene can be rescued by ectopically expressed doxycycline-inducible wild type SEPT7. Using this system, we analyzed the ability of SEPT7-mutants with alterations in their GTPase domain-dependent dimerization to prevent multinucleation and rescue proliferation. Although biochemical analysis of the mutants demonstrates differences in homo- and/or hetero-polymerization, in GTP-binding and/or GTPase activities, all analyzed mutants were able to rescue the cytokinesis phenotype of Sept7(flox/flox)fibroblasts associated with Cre-mediated deletion of endogenous Sept7. These findings indicate that the ability of septins to assemble into well-defined SEPT7-dimerization dependent native filaments is dispensable for cytokinesis in fibroblasts and opens the way to search for other mechanisms of the involvement of SEPT7 in cytokinesis.
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http://dx.doi.org/10.1038/srep20007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730212PMC
January 2016

Comparative Analysis of Two Gene-Targeting Approaches Challenges the Tumor-Suppressive Role of the Protein Kinase MK5/PRAK.

PLoS One 2015 21;10(8):e0136138. Epub 2015 Aug 21.

Department of Biochemistry, Hannover Medical School, Hannover, Germany.

MK5 (MAPK-activated protein kinase 5) or PRAK (p38-regulated and -activated kinase) are alternative names for a serine/threonine protein kinase downstream to ERK3/4 and p38 MAPK. A previous gene targeting approach for MK5/PRAK (termed here MK5/PRAK-Δex8) revealed a seemingly tumor-suppressive role of MK5/PRAK in DMBA-induced one step skin carcinogenesis and Ras-induced transformation. Here we demonstrate that an alternative targeting strategy of MK5/PRAK (termed MK5/PRAK-Δex6) increased neither tumor incidence in the one step skin carcinogenesis model, nor Ras-induced transformation in primary cells. Interestingly, due to the targeting strategies and exon skipping both knockouts do not completely abolish the generation of MK5/PRAK protein, but express MK5/PRAK deletion mutants with different biochemical properties depending on the exon targeted: Targeting of exon 6 leads to expression of an unstable cytoplasmic catalytically inactive MK5/PRAK-Δex6 mutant while targeting of exon 8 results in a more stable nuclear MK5/PRAK-Δex8 mutant with residual catalytic activity. The different properties of the MK5/PRAK deletion mutants could be responsible for the observed discrepancy between the knockout strains and challenge the role of MK5/PRAK in p53-dependent tumor suppression. Further MK5/PRAK knockout and knock-in mouse strains will be necessary to assign a physiological function to MK5/PRAK in this model organism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136138PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546416PMC
May 2016

The problem of pyridinyl imidazole class inhibitors of MAPK14/p38α and MAPK11/p38β in autophagy research.

Autophagy 2015 ;11(8):1425-7

a Institute of Biochemistry; Medical School Hannover ; Hannover , Germany.

In addition to its established role in inflammation, the stress-activated p38 MAP kinase pathway plays major roles in the regulation of cell cycle, senescence, and autophagy. Robust studies could establish mechanistic links between MAPK11-MAPK14/p38 signaling and macroautophagy converging at ATG9-trafficking and BECN1 phosphorylation. However, several reports seem to monitor MAPK11-MAPK14/p38-dependence of autophagy exclusively by the use of the SB203580/SB202190 class of MAPK14/MAPK11/p38α/β inhibitors. In this "Letter to the editor" we present data to support our claim that these inhibitors interfere with autophagic flux in a MAPK11-MAPK14/p38-independent manner and hence should no longer be used as pharmacological tools in the analysis of MAPK11-MAPK14/p38-dependence of autophagy. We propose a general guideline from Autophagy with regard to this issue to avoid such misinterpretations in the future.
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http://dx.doi.org/10.1080/15548627.2015.1059562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4590591PMC
June 2016

Sep(t)arate or not – how some cells take septin-independent routes through cytokinesis.

J Cell Sci 2015 May 17;128(10):1877-86. Epub 2015 Feb 17.

Institute of Physiological Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

Cytokinesis is the final step of cell division, and is a process that requires a precisely coordinated molecular machinery to fully separate the cytoplasm of the parent cell and to establish the intact outer cell barrier of the daughter cells. Among various cytoskeletal proteins involved, septins are known to be essential mediators of cytokinesis. In this Commentary, we present recent observations that specific cell divisions can proceed in the absence of the core mammalian septin SEPT7 and its Drosophila homolog Peanut (Pnut) and that thus challenge the view that septins have an essential role in cytokinesis. In the pnut mutant neuroepithelium, orthogonal cell divisions are successfully completed. Similarly, in the mouse, Sept7-null mutant early embryonic cells and, more importantly, planktonically growing adult hematopoietic cells undergo productive proliferation. Hence, as discussed here, mechanisms must exist that compensate for the lack of SEPT7 and the other core septins in a cell-type-specific manner. Despite there being crucial non-canonical immune-relevant functions of septins, septin depletion is well tolerated by the hematopoietic system. Thus differential targeting of cytokinesis could form the basis for more specific anti-proliferative therapies to combat malignancies arising from cell types that require septins for cytokinesis, such as carcinomas and sarcomas, without impairing hematopoiesis that is less dependent on septin.
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http://dx.doi.org/10.1242/jcs.164830DOI Listing
May 2015

p38MAPK/MK2-mediated phosphorylation of RBM7 regulates the human nuclear exosome targeting complex.

RNA 2015 Feb 18;21(2):262-78. Epub 2014 Dec 18.

Institute of Physiological Chemistry, Hannover Medical School, 30625 Hannover, Germany

The nuclear exosome targeting complex (NEXT) directs a major 3'-5' exonuclease, the RNA exosome, for degradation of nuclear noncoding (nc) RNAs. We identified the RNA-binding component of the NEXT complex, RBM7, as a substrate of p38(MAPK)/MK2-mediated phosphorylation at residue S136. As a result of this phosphorylation, RBM7 displays a strongly decreased RNA-binding capacity, while inhibition of p38(MAPK) or mutation of S136A in RBM7 increases its RNA association. Interestingly, promoter-upstream transcripts (PROMPTs), such as proRBM39, proEXT1, proDNAJB4, accumulated upon stress stimulation in a p38(MAPK)/MK2-dependent manner, a process inhibited by overexpression of RBM7(S136A). While there are no stress-dependent changes in RNA-polymerase II (RNAPII) occupation of PROMPT regions representing unchanged transcription, stability of PROMPTs is increased. Hence, we propose that phosphorylation of RBM7 by the p38(MAPK)/MK2 axis increases nuclear ncRNA stability by blocking their RBM7-binding and subsequent RNA exosome targeting to allow stress-dependent modulations of the noncoding transcriptome.
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http://dx.doi.org/10.1261/rna.048090.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338353PMC
February 2015

Resident CD4+ T cells accumulate in lymphoid organs after prolonged antigen exposure.

Nat Commun 2014 Sep 5;5:4821. Epub 2014 Sep 5.

1] Institute of Immunology, Hannover Medical School, D-30625 Hannover, Germany [2] Institute of Molecular Medicine, RWTH Aachen University, D-52074 Aachen, Germany.

Effector and memory CD4(+) T cells acquire distinct migratory properties depending on the type and location of the immune responses. Due to the highly dynamic nature of T cell circulation, the comprehensive analysis of these migratory routes requires dedicated experimental approaches. Here, we analyse the migration of effector/memory CD4(+) T cells by long-term in vivo cell tracking. We identify a resident population of antigen-experienced CD4(+) T cells that resides in lymph nodes and Peyer's patches without circulation or proliferation. Resident CD4(+) T cells constitute up to 50% of all effector/memory cells, including, but not limited to, follicular helper T cells. Furthermore, these non-circulating T cells possess a distinct T cell receptor repertoire and accumulate in Peyer's patches after continuous oral antigen exposure. Our results provide the first direct evidence for a resident population of effector/memory CD4(+) T cells that is retained in lymphoid tissues.
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http://dx.doi.org/10.1038/ncomms5821DOI Listing
September 2014

Genetic deletion of SEPT7 reveals a cell type-specific role of septins in microtubule destabilization for the completion of cytokinesis.

PLoS Genet 2014 Aug 14;10(8):e1004558. Epub 2014 Aug 14.

Institute of Physiological Chemistry, Hannover Medical School, Hannover, Germany.

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.
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http://dx.doi.org/10.1371/journal.pgen.1004558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133155PMC
August 2014

Expression of fibulin-6 in failing hearts and its role for cardiac fibroblast migration.

Cardiovasc Res 2014 Sep 20;103(4):509-20. Epub 2014 Jun 20.

Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany

Aims: The cardiac extracellular matrix (ECM) undergoes a dynamic transition following myocardial infarction. Fibulin-6 is expressed in cell junctions particularly in tissues subjected to significant mechanical stress. Fibulin-6 deficiency results in defective cell migration in nematodes and early embryonic lethality in mice. The role of fibulin-6 in healthy and failing myocardium is unknown. We have examined the expression and distribution pattern of fibulin-6 during myocardial remodelling (MR) and detailed its effect on the migratory function of cardiac fibroblasts (CFs) in response to TGF-β1.

Methods And Results: In healthy murine myocardium, fibulin-6 expression is largely confined to larger coronary arteries. It is induced during the early and the late phase of remodelling after infarction in murine hearts predominantly in the scar-muscle junction. Similar results are obtained in human ischaemic cardiomyopathy. Fibulin-6 is mostly expressed in close vicinity to vimentin-positive cells and is also abundantly expressed in vitro in cultured neonatal CF. TGF-β1 does not induce smooth muscle actin in fibroblasts deficient of fibulin-6, which also compromised their migration. Cells that had migrated expressed more fibulin-6 compared with stationary cells. Plated on fibulin-6-depleted matrix, stress fibre induction in fibroblast in response to TGF-β1 was impaired. In ex vivo explant cultures from post-infarct myocardium, the number of emigrating fibroblasts was also significantly reduced by fibulin-6 siRNA knockdown.

Conclusion: Fibulin-6, a fibroblast-released ECM protein, may play an important role during MR by imparting an effect on CF migration in close and complementary interplay with TGF-β1 signalling.
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http://dx.doi.org/10.1093/cvr/cvu161DOI Listing
September 2014

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity.

Proc Natl Acad Sci U S A 2013 Oct 30;110(42):16856-61. Epub 2013 Sep 30.

Institute of Molecular Oncology and Göttingen Centre of Molecular Biosciences, Faculty of Medicine, University of Göttingen, 37077 Göttingen, Germany.

DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.
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http://dx.doi.org/10.1073/pnas.1304355110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3801042PMC
October 2013

Endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 is a novel substrate of MK2 (MAPKAP kinase-2) involved in MK2-mediated TNFα production.

Biochem J 2013 Dec;456(2):163-72

*Institute of Physiological Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.

The p38 MAPK (mitogen-activated protein kinase)/MK2 [MAPKAP (MAPK-activated protein) kinase-2] signalling pathway is a major regulator of stress- and cytokine-induced gene expression at the transcriptional and post-transcriptional level. Using phosphoproteomics we identified the ER (endoplasmic reticulum)-associated ubiquitin-conjugating enzyme Ube2j1 as a potential substrate of MK2. We demonstrate that Ube2j1 is phosphorylated in a cytokine-, cytosolic stress- and LPS (lipopolysaccharide)-induced manner. The cytosolic stress-induced phosphorylation of Ube2j1 proceeds at Ser(184), a site described previously to be phosphorylated in response to ER stress, which is located in a perfect MK2 consensus motif. The cytosolic stress-induced phosphorylation of Ube2j1, but not its ER-stress-induced phosphorylation is sensitive to p38/MK2 inhibitors and abrogated in MK2/MK3-deficient cells. In a pull-down assay we demonstrate the interaction of MK2 with Ube2j1 in HEK (human embryonic kidney)-293T cells. Furthermore, MK2 is able to phosphorylate recombinant Ube2j1, but not the S184A mutant in an in vitro kinase assay. These findings strongly suggest that MK2 directly phosphorylates Ube2j1 at Ser(184) upon p38-activating stress in vivo. However, ectopically expressed Ube2j1-S184A mutant displays ubiquitinating activity towards the model substrate ER-synthesized T-cell receptor-α similar to that of the wild-type protein. Interestingly, Ube2j1 is phosphorylated in response to LPS also in macrophages and contributes to MK2-dependent TNFα biosynthesis by a so far unknown mechanism.
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http://dx.doi.org/10.1042/BJ20130755DOI Listing
December 2013
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