IQGAP1 binds AMPK and is required for maximum AMPK activation

AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.

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Oxidative stress inhibits MEKK1 by site-specific glutathionylation in the ATP-binding domain

Biochemical Journal ◽

10.1042/bj20040591 ◽

2004 ◽

Vol 381 (3) ◽

pp. 675-683 ◽

Cited By ~ 145

Author(s):

Janet V. CROSS ◽

Dennis J. TEMPLETON

Keyword(s):

Oxidative Stress ◽

Protein Kinase ◽

Cysteine Residue ◽

Binding Domain ◽

Mitogen Activated Protein Kinase ◽

Atp Binding ◽

Cysteine Oxidation ◽

Mitogen Activated Protein ◽

In Cells

Many intracellular signalling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. We have found that MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1], an upstream activator of the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, is directly inhibited by cysteine alkylation using NEM (N-ethylmaleimide). The related kinase, ASK1 (apoptosis signal-regulating kinase 1), was not inhibited, but was instead activated by NEM. Inhibition of MEKK1 requires a single unique cysteine residue (Cys1238) in the ATP-binding domain of MEKK1. Oxidative stress induced by menadione (2-methyl-1,4-naphthoquinone) also inhibited MEKK1, but activated ASK1, in cells. MEKK1 inhibition by menadione also required Cys1238. Oxidant-inhibited MEKK1 was re-activated by dithiothreitol and glutathione, supporting reversible cysteine oxidation as a mechanism. Using various chemical probes, we excluded modification by S-nitrosylation or oxidation of cysteine to sulphenic acid. Oxidant-inhibited MEKK1 migrated normally on non-reducing gels, excluding the possibility of intra- or inter-molecular disulphide bond formation. MEKK1 was inhibited by glutathionylation in vitro, and MEKK1 isolated from menadione-treated cells was shown by MS to be modified by glutathione on Cys1238. Our results support a model whereby the redox environment within the cell selectively regulates stress signalling through MEKK1 versus ASK1, and may thereby participate in the induction of apoptosis by oxidative stress.

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AMP-activated protein kinase pathway and bone metabolism

Journal of Endocrinology ◽

10.1530/joe-11-0306 ◽

2011 ◽

Vol 212 (3) ◽

pp. 277-290 ◽

Cited By ~ 58

Author(s):

J Jeyabalan ◽

M Shah ◽

B Viollet ◽

C Chenu

Keyword(s):

Protein Kinase ◽

Energy Homeostasis ◽

Bone Cells ◽

Peripheral Tissues ◽

Ampk Signaling ◽

Obesity And Diabetes ◽

Regulate Food Intake ◽

Ampk Activity ◽

Amp Activated Protein Kinase

There is increasing evidence that osteoporosis, similarly to obesity and diabetes, could be another disorder of energy metabolism. AMP-activated protein kinase (AMPK) has emerged over the last decade as a key sensing mechanism in the regulation of cellular energy homeostasis and is an essential mediator of the central and peripheral effects of many hormones on the metabolism of appetite, fat and glucose. Novel work demonstrates that the AMPK signaling pathway also plays a role in bone physiology. Activation of AMPK promotes bone formationin vitroand the deletion of α or β subunit of AMPK decreases bone mass in mice. Furthermore, AMPK activity in bone cells is regulated by the same hormones that regulate food intake and energy expenditure through AMPK activation in the brain and peripheral tissues. AMPK is also activated by antidiabetic drugs such as metformin and thiazolidinediones (TZDs), which also impact on skeletal metabolism. Interestingly, TZDs have detrimental skeletal side effects, causing bone loss and increasing the risk of fractures, although the role of AMPK mediation is still unclear. These data are presented in this review that also discusses the potential roles of AMPK in bone as well as the possibility for AMPK to be a future therapeutic target for intervention in osteoporosis.

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MSK1 activity is controlled by multiple phosphorylation sites

Biochemical Journal ◽

10.1042/bj20041501 ◽

2005 ◽

Vol 387 (2) ◽

pp. 507-517 ◽

Cited By ~ 119

Author(s):

Claire E. McCOY ◽

David G. CAMPBELL ◽

Maria DEAK ◽

Graham B. BLOOMBERG ◽

J. Simon C. ARTHUR

Keyword(s):

Protein Kinase ◽

P38 Mapk ◽

Kinase Domain ◽

Mitogen Activated Protein Kinase ◽

Mapk Cascades ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Ribosomal S6 Kinase ◽

In Cells

MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.

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Abstract 11302: Sevoflurane Pre-Conditioning Effectively Ameliorates Diabetic Myocardial Ischemia/Reperfusion Injury via Differential Regulation of p38 and ERK

Circulation ◽

10.1161/circ.130.suppl_2.11302 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Jianli Zhao ◽

Jingjing Li ◽

Rui Li ◽

Liyuan Jiao ◽

Yanqing Zhang ◽

...

Keyword(s):

Protein Kinase ◽

Myocardial Ischemia ◽

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Inhibitory Effect ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Ampk Signaling ◽

Myocardial Ischemia Reperfusion

Diabetes (DB) significantly exacerbates myocardial ischemia/reperfusion (MI/R) injury. Unfortunately, conventional pre-conditioning (PreCon) provides diminished cardioprotection during DB, due partially to impaired AMP-activated protein kinase (AMPK) signaling. The current study investigated whether PreCon by inhaled anesthetic sevoflurane (SF-PreCon) remains protective in DB, and if so, to dissect the involved mechanisms. Non-diabetic (ND) or high-fat diet-induced DB mice were subjected to MI/R and randomized into control and SF-PreCon (3 cycles of 15 minute-exposures to 2% sevoflurane prior to MI) groups. As expected, SF-PreCon significantly reduced MI/R injury in ND mice. Importantly, SF-PreCon also significantly reduced MI/R injury in DB mice, as evidenced by reduced apoptosis (-23.1±1.6, P<0.01), decreased infarct size (-21.2±2.3%, P<0.01), and augmented cardiac function (+24±3.0%, P<0.01). To determine the role of AMPK in SF-PreCon-mediated cardioprotection, the effect of SF-PreCon upon MI/R injury was determined in cardiac specific AMPKα2 dominant negative overexpression mice (AMPK-DN). We demonstrate SF-PreCon remained cardioprotective in AMPK-DN mice. To explore the responsible molecular mechanisms, multiple cell-survival signaling molecules were screened. Interestingly, SF-PreCon differentially regulated mitogen-activated protein kinase (MAPK) family members in the MI/R heart. In ND mice, SF-PreCon dramatically reduced (-81±7.2%) MI/R-induced activation of p38, a pro-death MAPK, without significantly altering ERK and JNK. In DB and AMPK-DN mice, the inhibitory effect of SF-PreCon upon p38 activation was significantly blunted (DB: -8±2.1%) or virtually abolished (AMPK-DN). However, SF-PreCon significantly increased (P<0.05) phosphorylation of ERK1/2, a pro-survival MAPK. Collectively, we demonstrate SF-PreCon protected the heart via AMPK-dependent inhibition of pro-death MAPK in ND mice. However, SF-PreCon exerts its cardioprotective actions via AMPK-independent activation of pro-survival MAPK in DB mice. These results suggest SF-PreCon may be a superior intervention over conventional PreCon in DB patients, where AMPK signaling is impaired.

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Nur77 is phosphorylated in cells by RSK in response to mitogenic stimulation

Biochemical Journal ◽

10.1042/bj20050967 ◽

2006 ◽

Vol 393 (3) ◽

pp. 715-724 ◽

Cited By ~ 64

Author(s):

Andrew D. Wingate ◽

David G. Campbell ◽

Mark Peggie ◽

J. Simon C. Arthur

Keyword(s):

Protein Kinase ◽

Orphan Receptor ◽

Mitogen Activated Protein Kinase ◽

Mitogenic Stimulation ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Ribosomal S6 Kinase ◽

In Cells

Nur77 is a nuclear orphan receptor that is able to activate transcription independently of exogenous ligand, and has also been shown to promote apoptosis on its localization to mitochondria. Phosphorylation of Nur77 on Ser354 has been suggested to reduce ability of Nur77 to bind DNA; however, the kinase responsible for this phosphorylation in cells has not been clearly established. In the present study, we show that Nur77 is phosphorylated on this site by RSK (ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase), but not by PKB (protein kinase B) or PKA (protein kinase A), in vitro. In cells, phosphorylation of Nur77 in vivo is catalysed by RSK, which is activated downstream of the classical MAPK (mitogen-activated protein kinase) cascade. Phosphorylation of Nur77 by RSK is able to promote the binding of Nur77 to 14-3-3 proteins in vitro, however, no evidence could be seen for this interaction in cells. We have established that two related proteins, Nurr1 and Nor1, are also phosphorylated on the equivalent site by RSK in cells in response to mitogenic stimulation.

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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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IQGAP1 binds the Axl receptor kinase and inhibits its signaling

Biochemical Journal ◽

10.1042/bcj20180594 ◽

2018 ◽

Vol 475 (19) ◽

pp. 3073-3086

Author(s):

Laëtitia Gorisse ◽

Zhigang Li ◽

Andrew C. Hedman ◽

David B. Sacks

Keyword(s):

Growth Factor ◽

Intracellular Signaling ◽

Growth Factor Receptor ◽

Direct Interaction ◽

Specific Protein ◽

Tyrosine Kinase Receptor ◽

Proximity Ligation ◽

Vitro Analysis ◽

In Cells

Axl is a tyrosine kinase receptor that is important for hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival. Binding of growth arrest-specific protein 6 (Gas6) activates Axl signaling, but the mechanism of inactivation of the Axl receptor is poorly understood. In the present study, we show that IQGAP1 modulates Axl signaling. IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules. Our in vitro analysis revealed a direct interaction between the IQ domain of IQGAP1 and Axl. Analysis by both immunoprecipitation and proximity ligation assays demonstrated an association between Axl and IQGAP1 in cells and this interaction was decreased by Gas6. Unexpectedly, reducing IQGAP1 levels in cells significantly enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, IQGAP1 regulates the interaction of Axl with the epidermal growth factor receptor. Our data identify IQGAP1 as a previously undescribed suppressor of Axl and provide insight into regulation of Axl function.

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Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

Molecular and Cellular Biology ◽

10.1128/mcb.23.20.7177-7188.2003 ◽

2003 ◽

Vol 23 (20) ◽

pp. 7177-7188 ◽

Cited By ~ 126

Author(s):

Hoanh Tran ◽

Fabienne Maurer ◽

Yoshikuni Nagamine

Keyword(s):

Hydrogen Peroxide ◽

Protein Kinase ◽

Active Form ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Hydrogen Peroxide Treatment ◽

Mitogen Activated Protein ◽

Cytoplasmic Accumulation ◽

In Cells

ABSTRACT The mRNAs of urokinase plasminogen activator (uPA) and its receptor, uPAR, contain instability-determining AU-rich elements (AREs) in their 3′ untranslated regions. The cellular proteins binding to these RNA sequences (AREuPA/uPAR) are not known. We show here that the mRNA-stabilizing factor HuR functionally interacts with these sequences. HuR stabilized an AREuPA-containing RNA substrate in vitro and stabilized in HeLa Tet-off cells both endogenous uPA and uPAR mRNAs and a β-globin reporter mRNA containing the AREuPA. RNAi-mediated depletion of HuR in BT-549 and MDA-MB-231 cells significantly reduced the steady-state levels of endogenous uPA and uPAR mRNAs. Furthermore, we show that a constitutively active form of mitogen-activated protein kinase-activated protein kinase 2 (MK2), MK2-EE, has an ARE-mRNA-stabilizing effect that correlates with its ability to enhance the cytoplasmic accumulation of endogenous HuR, but not in cells cotransfected with a dominant negative version of MK2, MK2-K76R. These effects were mimicked by hydrogen peroxide treatment (oxidative stress), which resulted in the phosphorylation of endogenous MK2. In addition, hydrogen peroxide treatment enhanced the cytoplasmic binding of HuR to the AREuPA, which was abrogated in cells transfected with MK2-K76R. These results indicate a role for HuR and MK2 in regulating the expression of uPA and uPAR genes at the posttranscriptional level.

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The Keap1-Nrf2 System: A Mediator between Oxidative Stress and Aging

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/6635460 ◽

2021 ◽

Vol 2021 ◽

pp. 1-16

Author(s):

Chao Yu ◽

Jian-Hui Xiao

Keyword(s):

Oxidative Stress ◽

Protein Kinase ◽

Molecular Mechanisms ◽

Glycogen Synthase ◽

Mammalian Target Of Rapamycin ◽

Direct Interaction ◽

Mitogen Activated Protein Kinase ◽

Related Factor ◽

Sequestosome 1 ◽

Molecular Damage

Oxidative stress, a term that describes the imbalance between oxidants and antioxidants, leads to the disruption of redox signals and causes molecular damage. Increased oxidative stress from diverse sources has been implicated in most senescence-related diseases and in aging itself. The Kelch-like ECH-associated protein 1- (Keap1-) nuclear factor-erythroid 2-related factor 2 (Nrf2) system can be used to monitor oxidative stress; Keap1-Nrf2 is closely associated with aging and controls the transcription of multiple antioxidant enzymes. Simultaneously, Keap1-Nrf2 signaling is also modulated by a more complex regulatory network, including phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C, and mitogen-activated protein kinase. This review presents more information on aging-related molecular mechanisms involving Keap1-Nrf2. Furthermore, we highlight several major signals involved in Nrf2 unbinding from Keap1, including cysteine modification of Keap1 and phosphorylation of Nrf2, PI3K/Akt/glycogen synthase kinase 3β, sequestosome 1, Bach1, and c-Myc. Additionally, we discuss the direct interaction between Keap1-Nrf2 and the mammalian target of rapamycin pathway. In summary, we focus on recent progress in research on the Keap1-Nrf2 system involving oxidative stress and aging, providing an empirical basis for the development of antiaging drugs.

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Opioid Effects on Mitogen-activated Protein Kinase Signaling Cascades

Anesthesiology ◽

10.1097/00000542-199711000-00016 ◽

1997 ◽

Vol 87 (5) ◽

pp. 1118-1126 ◽

Cited By ~ 65

Author(s):

Howard B. Gutstein ◽

Elizabeth A. Rubie ◽

Alfred Mansour ◽

Huda Akil ◽

James R. Woodgett

Keyword(s):

Protein Kinase ◽

Molecular Mechanisms ◽

Mitogen Activated Protein Kinase ◽

C6 Glioma Cells ◽

Kinase Activation ◽

Extracellular Signal ◽

Activation Assay ◽

Mitogen Activated Protein ◽

Delta Receptor

Background The molecular mechanisms underlying both beneficial and undesirable opioid actions are poorly understood. Recently, the three currently known mammalian mitogen-activated protein kinase (MAPK) signaling cascades (extracellular signal-related kinase [ERK], stress-activated protein kinase, and p38 kinase) were shown to play important roles in transducing receptor-mediated signaling processes. Methods To determine whether any of these kinase cascades were activated by opioids, mu, delta, or kappa opioid receptors were transiently introduced into COS-7 cells together with MAPKs tagged to allow recognition by specific antibodies, and then exposed to opioids. Mitogen-activated protein kinase activation was determined by an in vitro MAPK activation assay. In addition, C6 glioma cells with either mu, delta, or kappa receptors stably introduced were exposed to opioids and MAPK activation determined by in vitro activation assay or antibody detection of activated forms. Results Transient experiments in COS cells revealed potent stimulation of ERK by mu and delta receptor activation, weak stimulation of stress-activated protein kinase by all receptor types, and no activation of p38. In stably transfected C6 glioma cells, only ERK activation was observed. Extracellular signal-related kinase induction was rapid, peaking 5 min after stimulation, and its activation was receptor-type specific. Mu and delta receptor stimulation activated ERK, but kappa stimulation did not. Conclusions These results show that acute opioid signaling is not only inhibitory, but can strongly activate an important signaling cascade. Extracellular signal-related kinase activation may contribute to desirable responses to opioids, such as analgesia and sedation, and also to undesirable adaptive responses, such as tolerance, physical dependence, and possibly addiction. Further study of this system could provide greater insight into the molecular mechanisms underlying these clinical problems.

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