Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor

AbstractNFE2L2/NRF2 is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, and conversely NRF2 protein levels as well as activity is diminished with age and in neurodegenerative disorders. Though NRF2 activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here we used a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the understudied protein kinase Brain Specific Kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives AMPK activation and suppresses mTOR signaling. As a result, BRSK2 kinase activation suppressed ribosome-RNA complexes, global protein synthesis, and NRF2 protein levels. Collectively, our data establish the catalytically active BRSK2 kinase as a negative regulator of NRF2 via the AMPK/mTOR signaling. This signaling axis may prove useful for therapeutically targeting NRF2 in human diseases.Summary StatementBRSK2 suppresses NRF2 signaling by inhibiting protein synthesis through mTOR downregulation.

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Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor

Journal of Cell Science ◽

10.1242/jcs.241356 ◽

2020 ◽

Vol 133 (14) ◽

pp. jcs241356 ◽

Cited By ~ 5

Author(s):

Tigist Y. Tamir ◽

Brittany M. Bowman ◽

Megan J. Agajanian ◽

Dennis Goldfarb ◽

Travis P. Schrank ◽

...

Keyword(s):

Transcription Factor ◽

Protein Kinase ◽

Human Cancer ◽

Antioxidant Response ◽

Genetic Screen ◽

Related Factor ◽

Dependent Manner ◽

Gain Of Function ◽

Protein Levels ◽

Nrf2 Protein

ABSTRACTNuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5′-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.

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Platelet-Rich and Platelet-Poor Plasma Might Play Supportive Roles in Cancer Cell Culture: A Replacement for Fetal Bovine Serum?

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520621999210101225912 ◽

2021 ◽

Vol 21 ◽

Author(s):

Mehdi Talebi ◽

Mousa Vatanmakanian ◽

Ali Mirzaei ◽

Yaghoub Barfar ◽

Maryam Hemmatzadeh ◽

...

Keyword(s):

Cell Culture ◽

Cell Growth ◽

Cancer Cell ◽

Human Cancer ◽

High Price ◽

Dependent Manner ◽

Platelet Poor Plasma ◽

Protein Levels ◽

Healthy Donors ◽

Regulatory Functions

Background: Platelet-rich (PRP) and Platelet-poor plasma (PPP) are widely used in research and clinical platforms mainly due to their capacities to enhance cell growth. Although short half-life (5 days) and the high price of platelet products pose challenges regarding their usage, they maintain the growth regulatory functions for weeks. Thus, we aimed to assess the supplementary values of these products in human CCRF-CEM cancer cells. Mechanistically, we also checked if the PRP/PPP treatment enhances YKL-40 expression as a known protein regulating cell growth. Methods: The PRP/PPP was prepared from healthy donors using manual stepwise centrifugation and phase separation. The viability of the cells treated with gradient PRP/PPP concentrations (2, 5, 10, and 15%) was measured by the MTT assay. The YKL-40 mRNA and protein levels were assessed using qRT-PCR and western blotting. The data were compared to FBS-treated cells. Result: Our findings revealed that the cells treated by PRP/PPP not only were morphologically comparable to those treated by FBS but also, they showed greater viability at the concentrations of 10 and 15%. Moreover, it was shown that PRP/PPP induce cell culture support, at least in part, via inducing YKL-40 expression at both mRNA and protein levels in a time- and dose-dependent manner. Conclusion: Collectively, by showing cell culture support comparable to FBS, the PRP/PPP might be used as good candidates to supplement the cancer cell culture and overcome concerns regarding the use of FBS as a non-human source in human cancer research.

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Requirement of the E2F3 Transcription Factor for BCR/ABL Leukemogenesis.

Blood ◽

10.1182/blood.v110.11.33.33 ◽

2007 ◽

Vol 110 (11) ◽

pp. 33-33

Author(s):

Anna M. Eiring ◽

Paolo Neviani ◽

Ramasamy Santhanam ◽

Joshua J. Oaks ◽

Ji Suk Chang ◽

...

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Blast Crisis ◽

Scid Mice ◽

Myelogenous Leukemia ◽

Mrna Levels ◽

Dependent Manner ◽

Hnrnp A1 ◽

Protein Levels ◽

Abl Kinase

Abstract Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P<0.001). Furthermore, subcutaneous injection of shE2F3-expressing BCR/ABL+ cells into SCID mice markedly impaired in vivo tumorigenesis (>80% reduction in tumor burden, P<0.01). Accordingly, BCR/ABL leukemogenesis was strongly inhibited in SCID mice intravenously injected with E2F3 shRNA-expressing 32D-BCR/ABL cells and in mice transplanted with BCR/ABL-transduced Lin− bone marrow cells from E2F3−/− mice. Specifically, we demonstrate that reduced or absent levels of E2F3 resulted in dramatically decreased numbers of circulating BCR/ABL+ cells as determined by nested RT-PCR at 4 weeks post-injection (P=0.0001), normal splenic architecture and bone marrow cellularity and the absence of infiltrating myeloid blasts into non-hematopoietic compartments (i.e. liver). By contrast, SCID mice transplanted with vector-transduced 32D-BCR/ABL cells or BCR/ABL+ E2F3+/+ Lin− BM progenitors showed signs of an overt acute leukemia-like process with blast infiltration of hematopoietic and non-hematopoietic organs. Altogether, these data outline the importance of E2F3 expression for BCR/ABL leukemogenesis and characterize a new potential therapeutic target for the treatment of patients with advanced phase CML.

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Forkhead Transcription Factor FKHR-L1 Modulates Cytokine-Dependent Transcriptional Regulation of p27KIP1

Molecular and Cellular Biology ◽

10.1128/mcb.20.24.9138-9148.2000 ◽

2000 ◽

Vol 20 (24) ◽

pp. 9138-9148 ◽

Cited By ~ 435

Author(s):

Pascale F. Dijkers ◽

Rene H. Medema ◽

Cornelieke Pals ◽

Lolita Banerji ◽

N. Shaun B. Thomas ◽

...

Keyword(s):

Transcription Factor ◽

Transcriptional Regulation ◽

Cell Survival ◽

Kinase Inhibitor ◽

Ectopic Expression ◽

Dependent Manner ◽

Forkhead Transcription Factor ◽

Protein Levels ◽

Macrophage Colony ◽

Null Mutant Mice

ABSTRACT Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

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Protein Phosphatase 6C (PPP6C) Loss Significantly Raises Fetal Hemoglobin Levels and Reduces Cell Sickling

Blood ◽

10.1182/blood-2021-147275 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2031-2031

Author(s):

Scott A. Peslak ◽

Eugene Khandros ◽

Peng Huang ◽

Maryanne Kihiu ◽

Osheiza Abdulmalik ◽

...

Keyword(s):

Protein Phosphatase ◽

Fetal Hemoglobin ◽

Mrna Translation ◽

Genetic Screen ◽

Erythroid Cell ◽

Acute Chest Syndrome ◽

Dependent Manner ◽

Erythroid Cells ◽

Protein Levels ◽

Dose Dependent

Abstract Sickle cell disease (SCD) afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has remained an elusive goal. To identify additional potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen targeting all protein phosphatases (1308 independent sgRNA representing 218 phosphatases). The phosphatase sgRNA library was cloned into a lentivirus scaffold and introduced into the erythroid cell line HUDEP2 stably expressing Cas9, and the top and bottom 10% of HbF-expressing cells were sorted and the integrated sgRNAs were sequenced. This screen identified a single protein phosphatase - PPP6C - as an HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6, a serine/threonine cytosolic protein phosphatase that is widely expressed across tissues and throughout erythroid development to broadly regulate mRNA translation. PPP6C has been implicated in numerous cellular functions, including cell cycle regulation, autophagy, and innate immunity, but its role in HbF regulation has not previously been described. Depletion of PPP6C by 5 independent sgRNAs in HUDEP2 cells resulted in significant HbF enrichment. Importantly, PPP6C depletion did not affect cellular viability or differentiation, suggesting that PPP6C may serve as a targetable HbF regulator for the treatment of SCD. To validate the findings of this genetic screen in primary human erythroid cells, we performed CRISPR-Cas9 ribonuclear protein (RNP)-based genome editing of PPP6C in a three-phase in vitro culture of adult CD34+ hematopoietic cells. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We find that depletion of PPP6C protein levels by greater than 80% increases gamma-globin transcript levels in a dose-dependent manner to nearly 5 times basal levels. In addition, PPP6C loss leads to a greater than doubling in F-cell number and a 3-4-fold increase in HbF levels as measured by HPLC analysis. PPP6C depletion showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Mechanistically, loss of PPP6C leads to depletion of BCL11A protein levels by nearly 50% but unchanged levels of other key HbF regulators such as HRI and LRF, suggesting PPP6C-mediated HbF regulation may proceed at least in part via loss of BCL11A. However, additional studies are necessary to fully elucidate these underlying regulatory mechanisms. Importantly, depletion of PPP6C in SCD patient-derived cells was well tolerated, led to similar levels of HbF induction, and markedly reduced cell sickling by greater than 60%. Results from ongoing studies exploring the mechanism of PPP6C in HbF regulation will be discussed. Taken together, these data indicate that PPP6C functions in a dose-dependent manner to regulate HbF in primary erythroid cells and may serve as a therapeutic target in the treatment of SCD. Disclosures Blobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

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Oleanolic Acid Induces Differentiation of Neural Stem Cells to Neurons: An Involvement of Transcription Factor Nkx-2.5

Stem Cells International ◽

10.1155/2015/672312 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 7

Author(s):

You Ning ◽

Jianhua Huang ◽

Bill Kalionis ◽

Qin Bian ◽

Jingcheng Dong ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Neural Stem Cells ◽

Oleanolic Acid ◽

Maximum Effect ◽

Regulatory Sequences ◽

Specific Marker ◽

Dependent Manner ◽

Protein Levels ◽

Pentacyclic Triterpenoid

Neural stem cells (NSCs) harbor the potential to differentiate into neurons, astrocytes, and oligodendrocytes under normal conditions and/or in response to tissue damage. NSCs open a new way of treatment of the injured central nervous system and neurodegenerative disorders. Thus far, few drugs have been developed for controlling NSC functions. Here, the effect as well as mechanism of oleanolic acid (OA), a pentacyclic triterpenoid, on NSC function was investigated. We found OA significantly inhibited neurosphere formation in a dose-dependent manner and achieved a maximum effect at 10 nM. OA also reduced 5-ethynyl-2′-deoxyuridine (EdU) incorporation into NSCs, which was indicative of inhibited NSC proliferation. Western blotting analysis revealed the protein levels of neuron-specific marker tubulin-βIII (TuJ1) and Mash1 were increased whilst the astrocyte-specific marker glial fibrillary acidic protein (GFAP) decreased. Immunofluorescence analysis showed OA significantly elevated the percentage of TuJ1-positive cells and reduced GFAP-positive cells. Using DNA microarray analysis, 183 genes were differentially regulated by OA. Through transcription factor binding site analyses of the upstream regulatory sequences of these genes, 87 genes were predicted to share a common motif for Nkx-2.5 binding. Finally, small interfering RNA (siRNA) methodology was used to silence Nkx-2.5 expression and found silence of Nkx-2.5 alone did not change the expression of TuJ-1 and the percentage of TuJ-1-positive cells. But in combination of OA treatment and silence of Nkx-2.5, most effects of OA on NSCs were abolished. These results indicated that OA is an effective inducer for NSCs differentiation into neurons at least partially by Nkx-2.5-dependent mechanism.

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Alcohol impairs skeletal muscle protein synthesis and mTOR signaling in a time-dependent manner following electrically stimulated muscle contraction

Journal of Applied Physiology ◽

10.1152/japplphysiol.00180.2014 ◽

2014 ◽

Vol 117 (10) ◽

pp. 1170-1179 ◽

Cited By ~ 21

Author(s):

Jennifer L. Steiner ◽

Charles H. Lang

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Mammalian Target Of Rapamycin ◽

Mtor Signaling ◽

Saline Control ◽

Dependent Manner ◽

Skeletal Muscle Protein

Alcohol (EtOH) decreases protein synthesis and mammalian target of rapamycin (mTOR)-mediated signaling and blunts the anabolic response to growth factors in skeletal muscle. The purpose of the current investigation was to determine whether acute EtOH intoxication antagonizes the contraction-induced increase in protein synthesis and mTOR signaling in skeletal muscle. Fasted male mice were injected intraperitoneally with 3 g/kg EtOH or saline (control), and the right hindlimb was electrically stimulated (10 sets of 6 contractions). The gastrocnemius muscle complex was collected 30 min, 4 h, or 12 h after stimulation. EtOH decreased in vivo basal protein synthesis (PS) in the nonstimulated muscle compared with time-matched Controls at 30 min, 4 h, and 12 h. In Control, but not EtOH, PS was decreased 15% after 30 min. In contrast, PS was increased in Control 4 h poststimulation but remained unchanged in EtOH. Last, stimulation increased PS 10% in Control and EtOH at 12 h, even though the absolute rate remained reduced by EtOH. The stimulation-induced increase in the phosphorylation of S6K1 Thr421/Ser424 (20–52%), S6K1 Thr389 (45–57%), and its substrate rpS6 Ser240/244 (37–72%) was blunted by EtOH at 30 min, 4 h, and 12 h. Phosphorylation of 4E-BP1 Ser65 was also attenuated by EtOH (61%) at 4 h. Conversely, phosphorylation of extracellular signal-regulated kinase Thr202/Tyr204 was increased by stimulation in Control and EtOH mice at 30 min but only in Control at 4 h. Our data indicate that acute EtOH intoxication suppresses muscle protein synthesis for at least 12 h and greatly impairs contraction-induced changes in synthesis and mTOR signaling.

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Loss of function of bHLH transcription factor Nrd1 in tomato induces an arabinogalactan protein-encoding gene and enhances resistance to Pseudomonas syringae pv. tomato

10.1101/2021.11.08.467746 ◽

2021 ◽

Author(s):

Ning Zhang ◽

Chloe Hecht ◽

Xuepeng Sun ◽

Zhangjun Fei ◽

Gregory B Martin

Keyword(s):

Transcription Factor ◽

Pseudomonas Syringae ◽

Transcript Abundance ◽

Negative Regulator ◽

Arabinogalactan Protein ◽

Bhlh Transcription Factor ◽

Dependent Manner ◽

Rna Seq ◽

Loss Of Function ◽

Encoding Gene

Basic helix-loop-helix (bHLH) transcription factors constitute a superfamily in eukaryotes but their roles in plant immunity remain largely uncharacterized. We found that the transcript abundance in tomato leaves of one bHLH transcription factor-encoding gene, Nrd1 (negative regulator of resistance to DC3000 1), was significantly increased after treatment with the immunity-inducing flgII-28 peptide. Plants carrying a loss-of-function mutation in Nrd1 (Δnrd1) showed enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 although early pattern-triggered immunity responses such as generation of reactive oxygen species and activation of mitogen-activated protein kinases after treatment with flagellin-derived flg22 and flgII-28 peptides were unaltered compared to wild-type plants. An RNA-Seq analysis identified a gene, Agp1, whose expression is strongly suppressed in an Nrd1-dependent manner. Agp1 encodes an arabinogalactan protein and overexpression of the Agp1 gene in Nicotiana benthamiana led to ~10-fold less Pst growth compared to the control. These results suggest that the Nrd1 protein promotes tomato susceptibility to Pst by suppressing the defense gene Agp1. RNA-Seq also revealed that loss of Nrd1 function has no effect on the transcript abundance of immunity-associated genes including Bti9, Core, Fls2, Fls3 and Wak1 upon Pst inoculation, suggesting that the enhanced immunity observed in the Δnrd1 mutants is due to the activation of key PRR signaling components as well as loss of Nrd1-regulated suppression of Agp1.

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UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import

10.1101/2020.08.12.248161 ◽

2020 ◽

Author(s):

Tomer Shpilka ◽

YunGuang Du ◽

Qiyun Yang ◽

Andrew Melber ◽

Nandhitha U. Naresh ◽

...

Keyword(s):

Transcription Factor ◽

Protein Synthesis ◽

Protein Import ◽

Dependent Manner ◽

Mitochondrial Network ◽

Mitochondrial Import ◽

Network Expansion ◽

Unfolded Protein ◽

Protein Response ◽

Mitochondrial Unfolded Protein Response

AbstractAs organisms develop, individual cells generate mitochondria to fulfill physiologic requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth and impacted by environmental cues. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS)1. Here, we demonstrate that ATFS-1 mediates an adaptable mitochondrial expansion program that is active throughout normal development. Developmental mitochondrial network expansion required the relatively inefficient MTS2 in ATFS-1, which allowed the transcription factor to be responsive to parameters that impact protein import capacity of the entire mitochondrial network. Increasing the strength of the ATFS-1 MTS impaired UPRmt activity throughout development due to increased accumulation within mitochondria. The insulin-like signaling-TORC13 and AMPK pathways affected UPRmt activation4,5 in a manner that correlated with protein synthesis. Manipulation to increase protein synthesis caused UPRmt activation. Alternatively, S6 kinase inhibition had the opposite effect due to increased mitochondrial accumulation of ATFS-1. However, ATFS-1 with a dysfunctional MTS6 constitutively increased UPRmt activity independent of TORC1 function. Lastly, expression of a single protein with a strong MTS, was sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation. Mitochondrial network expansion is attenuated once ATFS-1 can be imported.

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