ERK Dephosphorylation through MKP1 Deacetylation by SIRT1 Attenuates RAS-Driven Tumorigenesis

The role of Situin 1 (SIRT1) in tumorigenesis is still controversial due to its wide range of substrates, including both oncoproteins and tumor suppressors. A recent study has demonstrated that SIRT1 interferes in the Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven activation of the Raf-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway, thereby inhibiting tumorigenesis. However, the molecular mechanism of SIRT1 as a tumor suppressor in RAS-driven tumorigenesis has been less clearly determined. This study presents evidence that the ectopic expression of SIRT1 attenuates RAS- or MEK-driven ERK activation and reduces cellular proliferation and transformation in vitro. The attenuation of ERK activation by SIRT1 results from prompt dephosphorylation of ERK, while MEK activity remains unchanged. We identified that MKP1, a dual specific phosphatase for MAPK, was deacetylated by SIRT1. Deacetylation of MKP1 by direct interaction with SIRT1 increased the binding affinity to ERK which in turn facilitated inactivation of ERK. Taken together, these results suggest that SIRT1 would act as a tumor suppressor by modulating RAS-driven ERK activity through MKP1 deacetylation.

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Transcriptional Repressor ERF Is a Ras/Mitogen-Activated Protein Kinase Target That Regulates Cellular Proliferation

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4121 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4121-4133 ◽

Cited By ~ 62

Author(s):

Lionel le Gallic ◽

Dionyssios Sgouras ◽

Gregory Beal ◽

George Mavrothalassitis

Keyword(s):

Protein Kinase ◽

Signaling Pathway ◽

Cellular Proliferation ◽

Transcriptional Repressor ◽

Mitogen Activated Protein Kinase ◽

Ras Signaling ◽

Mitogen Activated Protein ◽

Ets Family

ABSTRACT A limited number of transcription factors have been suggested to be regulated directly by Erks within the Ras/mitogen-activated protein kinase signaling pathway. In this paper we demonstrate that ERF, a ubiquitously expressed transcriptional repressor that belongs to the Ets family, is physically associated with and phosphorylated in vitro and in vivo by Erks. This phosphorylation determines the ERF subcellular localization. Upon mitogenic stimulation, ERF is immediately phosphorylated and exported to the cytoplasm. The export is blocked by specific Erk inhibitors and is abolished when residues undergoing phosphorylation are mutated to alanine. Upon growth factor deprivation, ERF is rapidly dephosphorylated and transported back into the nucleus. Phosphorylation-defective ERF mutations suppress Ras-induced tumorigenicity and arrest the cells at the G0/G1 phase of the cell cycle. Our findings strongly suggest that ERF may be important in the control of cellular proliferation during the G0/G1 transition and that it may be one of the effectors in the mammalian Ras signaling pathway.

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Reciprocal Role of ERK and Nf-κb Pathways in Survival and Activation of Osteoclasts

The Journal of Cell Biology ◽

10.1083/jcb.148.2.333 ◽

2000 ◽

Vol 148 (2) ◽

pp. 333-342 ◽

Cited By ~ 296

Author(s):

Tsuyoshi Miyazaki ◽

Hideki Katagiri ◽

Yumi Kanegae ◽

Hiroshi Takayanagi ◽

Yasuhiro Sawada ◽

...

Keyword(s):

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Erk Activation ◽

Iκb Kinase ◽

Mitogen Activated Protein ◽

Osteoclast Activation ◽

Osteoclast Survival ◽

Constitutively Active

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-κB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (RasDN), constitutively active MEK1 (MEKCA), dominant negative IκB kinase 2 (IKKDN), and constitutively active IKK2 (IKKCA). Inhibiting ERK activity by RasDN overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEKCA remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-κB pathway with IKKDN virus suppressed the pit-forming activity of OCLs and NF-κB activation by IKKCA expression upregulated it without affecting their survival. Interleukin 1α (IL-1α) strongly induced ERK activation as well as NF-κB activation. RasDN virus partially inhibited ERK activation, and OCL survival promoted by IL-1α. Inhibiting NF-κB activation by IKKDN virus significantly suppressed the pit-forming activity enhanced by IL-1α. These results indicate that ERK and NF-κB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-κB regulates osteoclast activation for bone resorption.

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Relative importance of phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK3/1) signaling during maturational steroid-induced meiotic G2–M1 transition in zebrafish oocytes

Zygote ◽

10.1017/s0967199417000545 ◽

2017 ◽

Vol 26 (1) ◽

pp. 62-75 ◽

Cited By ~ 2

Author(s):

Debabrata Das ◽

Poulomi Nath ◽

Soumojit Pal ◽

Sudip Hajra ◽

Pritha Ghosh ◽

...

Keyword(s):

Protein Kinase ◽

Mitogen Activated Protein Kinase ◽

Relative Importance ◽

Phosphatidylinositol 3 Kinase ◽

Kinase Activation ◽

Erk Activation ◽

Mapk Signalling ◽

Mitogen Activated Protein ◽

Phosphatidylinositol 3

SummaryParticipation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20β-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2−M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30–120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.

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Optimizing photoswitchable MEK

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912320116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 25756-25763 ◽

Cited By ~ 3

Author(s):

Aleena L. Patel ◽

Eyan Yeung ◽

Sarah E. McGuire ◽

Andrew Y. Wu ◽

Jared E. Toettcher ◽

...

Keyword(s):

Mitogen Activated Protein Kinase ◽

Kinase Activation ◽

Erk Activation ◽

Signaling Systems ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Morphogenetic Event ◽

Zebrafish Embryogenesis

Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in bothDrosophilaand zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.

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The regulation of ATF3 gene expression by mitogen-activated protein kinases

Biochemical Journal ◽

10.1042/bj20061081 ◽

2006 ◽

Vol 401 (2) ◽

pp. 559-567 ◽

Cited By ~ 107

Author(s):

Dan Lu ◽

Jingchun Chen ◽

Tsonwin Hai

Keyword(s):

Transcription Factor ◽

Protein Kinase ◽

Critical Role ◽

Ectopic Expression ◽

Signalling Pathway ◽

Mitogen Activated Protein Kinase ◽

Wide Range ◽

Mitogen Activated Protein ◽

Stress Signals ◽

P38 Pathway

ATF3 (activating transcription factor 3) gene encodes a member of the ATF/CREB (cAMP-response-element-binding protein) family of transcription factors. Its expression is induced by a wide range of signals, including stress signals and signals that promote cell proliferation and motility. Thus the ATF3 gene can be characterized as an ‘adaptive response’ gene for the cells to cope with extra- and/or intra-cellular changes. In the present study, we demonstrate that the p38 signalling pathway is involved in the induction of ATF3 by stress signals. Ectopic expression of CA (constitutively active) MKK6 [MAPK (mitogen-activated protein kinase) kinase 6], a kinase upstream of p38, indicated that activation of the p38 pathway is sufficient to induce the expression of the ATF3 gene. Inhibition of the pathway indicated that the p38 pathway is necessary for various signals to induce ATF3, including anisomycin, IL-1β (interleukin 1β), TNFα (tumour necrosis factor α) and H2O2. Analysis of the endogenous ATF3 gene indicates that the regulation is at least in part at the transcription level. Specifically, CREB, a transcription factor known to be phosphorylated by p38, plays a role in this induction. Interestingly, the ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase)/SAPK (stress-activated protein kinase) signalling pathways are neither necessary nor sufficient to induce ATF3 in the anisomycin stress paradigm. Furthermore, analysis of caspase 3 activation indicated that knocking down ATF3 reduced the ability of MKK6(CA) to exert its pro-apoptotic effect. Taken together, our results indicate that a major signalling pathway, the p38 pathway, plays a critical role in the induction of ATF3 by stress signals, and that ATF3 is functionally important to mediate the pro-apoptotic effects of p38.

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Lyn tyrosine kinase regulates thrombopoietin-induced proliferation of hematopoietic cell lines and primary megakaryocytic progenitors

Blood ◽

10.1182/blood-2003-10-3566 ◽

2004 ◽

Vol 103 (10) ◽

pp. 3736-3743 ◽

Cited By ~ 42

Author(s):

Brian J. Lannutti ◽

Jonathan G. Drachman

Keyword(s):

Cellular Proliferation ◽

Kinase Inhibitors ◽

Dominant Negative ◽

Janus Kinase ◽

Mitogen Activated Protein Kinase ◽

Mapk Activity ◽

Targeted Mutation ◽

Mitogen Activated Protein ◽

Nuclear Ploidy

Abstract In this study we demonstrate that thrombopoietin (TPO)–stimulated Src family kinases (SFKs) inhibit cellular proliferation and megakaryocyte differentiation. Using the Src kinase inhibitors pyrolopyrimidine 1 and 2 (PP1, PP2), we show that TPO-dependent proliferation of BaF3/Mpl cells was enhanced at concentrations that are specific for SFKs. Similarly, proliferation is increased after introducing a dominant-negative form of Lyn into BaF3/Mpl cells. Murine marrow cells from Lyn-deficient mice or wild-type mice cultured in the presence of the Src inhibitor, PP1, yielded a greater number of mature megakaryocytes and increased nuclear ploidy. Truncation and targeted mutation of the Mpl cytoplasmic domain indicate that Y112 is critical for Lyn activation. Examining the molecular mechanism for this antiproliferative effect, we determined that SFK inhibitors did not affect tyrosine phosphorylation of Janus kinase 2 (JAK2), Shc, signal transducer and activator of transcription (STAT)5, or STAT3. In contrast, pretreatment of cells with PP2 increased Erk1/2 (mitogen-activated protein kinase [MAPK]) phosphorylation and in vitro kinase activity, particularly after prolonged TPO stimulation. Taken together, our results show that Mpl stimulation results in the activation of Lyn kinase, which appears to limit the proliferative response through a signaling cascade that regulates MAPK activity. These data suggest that SFKs modify the rate of TPO-induced proliferation and are likely to affect cell cycle regulation during megakaryocytopoiesis.

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Ras/Mitogen-Activated Protein Kinase Signaling Activates Ets-1 and Ets-2 by CBP/p300 Recruitment

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10954-10964.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10954-10964 ◽

Cited By ~ 138

Author(s):

Charles E. Foulds ◽

Mary L. Nelson ◽

Adam G. Blaszczak ◽

Barbara J. Graves

Keyword(s):

Transcription Factors ◽

Protein Kinase ◽

Direct Interaction ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Creb Binding Protein ◽

Mitogen Activated Protein ◽

P300 Binding

ABSTRACT Cell signaling affects gene expression by regulating the activity of transcription factors. Here, we report that mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2, at a conserved site N terminal to their Pointed (PNT) domains, resulted in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. We discovered this phosphorylation-augmented interaction in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain were required for the interaction. Minimal regions that were competent for induced CBP/p300 binding in vitro also supported MAPK-enhanced transcription in vivo. CBP coexpression potentiated MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interacted in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, our findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis.

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The Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Cascade Activation Is a Key Signalling Pathway Involved in the Regulation of G1 Phase Progression in Proliferating Hepatocytes

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6003 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6003-6011 ◽

Cited By ~ 197

Author(s):

Hélène Talarmin ◽

Claude Rescan ◽

Sandrine Cariou ◽

Denise Glaise ◽

Giuliana Zanninelli ◽

...

Keyword(s):

Cyclin D1 ◽

Partial Hepatectomy ◽

Mek Inhibitor ◽

Signalling Pathway ◽

Mitogen Activated Protein Kinase ◽

Mrna Accumulation ◽

Erk Activation ◽

Mitogen Activated Protein

ABSTRACT In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G1 phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G1 (30 min to 4 h), and the other occurred in mid-late G1, peaking at around 10.5 h. Interestingly, the mid-late G1 activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G1/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G1 MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G1 MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G1 phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.

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MiR-17/20/93/106 promote hematopoietic cell expansion by targeting sequestosome 1–regulated pathways in mice

Blood ◽

10.1182/blood-2011-02-336487 ◽

2011 ◽

Vol 118 (4) ◽

pp. 916-925 ◽

Cited By ~ 103

Author(s):

Annemarie Meenhuis ◽

Peter A. van Veelen ◽

Hans de Looper ◽

Nicole van Boxtel ◽

Iris J. van den Berge ◽

...

Keyword(s):

Cell Expansion ◽

Ectopic Expression ◽

Inverse Correlation ◽

Mitogen Activated Protein Kinase ◽

Protein Levels ◽

Sequestosome 1 ◽

Mitogen Activated Protein ◽

Neutrophil Differentiation ◽

Myeloid Progenitors

Abstract MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways.

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c-Jun N-terminal kinase–mediated Rad18 phosphorylation facilitates Polη recruitment to stalled replication forks

Molecular Biology of the Cell ◽

10.1091/mbc.e11-10-0829 ◽

2012 ◽

Vol 23 (10) ◽

pp. 1943-1954 ◽

Cited By ~ 17

Author(s):

Laura R. Barkley ◽

Komaraiah Palle ◽

Michael Durando ◽

Tovah A. Day ◽

Aditi Gurkar ◽

...

Keyword(s):

Dna Damage ◽

Ectopic Expression ◽

Mitogen Activated Protein Kinase ◽

Nuclear Antigen ◽

Binding Motif ◽

Dependent Manner ◽

Replication Forks ◽

Mitogen Activated Protein ◽

Stalled Replication Forks

The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1–dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage–independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη−PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance.

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