scholarly journals Metformin Inhibits Tumor Metastasis through Suppressing Hsp90α Secretion in an AMPKα1-PKCγ Dependent Manner

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 144 ◽  
Author(s):  
Yuanchao Gong ◽  
Caihong Wang ◽  
Yi Jiang ◽  
Shaosen Zhang ◽  
Shi Feng ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tumor Metastasis ◽  
Epidemiological Studies ◽  
Secreted Proteins ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Amp Activated Protein Kinase ◽  
Mcf 7

Metformin has been documented in epidemiological studies to mitigate tumor progression. Previous reports show that metformin inhibits tumor migration in several cell lines, such as MCF-7 and H1299, but the mechanisms whereby metformin exerts its inhibitory effects on tumor metastasis remain largely unknown. The secreted proteins in cancer cell-derived secretome have been reported to play important roles in tumor metastasis, but whether metformin has an effect on tumor secretome remains unclear. Here we show that metformin inhibits tumor metastasis by suppressing Hsp90α (heat shock protein 90α) secretion. Mass spectrometry (MS) analysis and functional validation identify that eHsp90α (extracellular Hsp90α) is one of the most important secreted proteins for metformin to inhibit tumor cells migration, invasion and metastasis both in vitro and in vivo. Moreover, we find that metformin inhibits Hsp90α secretion in an AMPKα1 dependent manner. Our data elucidate that AMPKα1 (AMP-activated protein kinase α1) decreases the phosphorylation level of Hsp90α by inhibiting the kinase activity of PKCγ (protein kinase Cγ), which suppresses the membrane translocation and secretion of Hsp90α. Collectively, our results illuminate that metformin inhibits tumor metastasis by suppressing Hsp90α secretion in an AMPKα1 dependent manner.

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle

2001 ◽  
Vol 280 (5) ◽  
pp. E677-E684 ◽  
Author(s):  
Nicolas Musi ◽  
Tatsuya Hayashi ◽  
Nobuharu Fujii ◽  
Michael F. Hirshman ◽  
Lee A. Witters ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Muscle Contractions ◽  
Treadmill Running ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Amp Activated Protein Kinase ◽  
Dose Dependent

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-β-d-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPKα1 and AMPKα2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-β-d-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3- O-methyl-d-glucose (3-MG) uptake. There were dose-dependent increases in AMPKα2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPKα1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPKα2 activity and 3-MG uptake but had little effect on AMPKα1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPKα1 and -α2 activity and 3-MG uptake. Although the AMPKα1 and -α2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPKα2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.

14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation

2000 ◽  
Vol 345 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Paulus C. J. VAN DER HOEVEN ◽  
José C. M. VAN DER WAL ◽  
Paula RUURS ◽  
Marc C. M. VAN DIJK ◽  
Wim J. VAN BLITTERSWIJK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Dependent Manner ◽  
Cos Cells ◽  
Kinase C ◽  
Pkc Ζ ◽  
Co Precipitation

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

2005 ◽  
Vol 288 (5) ◽  
pp. H2412-H2421 ◽  
Author(s):  
Markus Frederich ◽  
Li Zhang ◽  
James A. Balschi
Keyword(s):  
Protein Kinase ◽  
Metabolic Inhibitors ◽  
Protein Kinase Activity ◽  
Α Subunit ◽  
Rat Hearts ◽  
Upstream Kinase ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1 in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5 for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2 content, α-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

5′-AMP-activated protein kinase is inactivated by adrenergic signalling in adult cardiac myocytes

2011 ◽  
Vol 32 (2) ◽  
pp. 197-209 ◽  
Author(s):  
Yugo Tsuchiya ◽  
Fiona C. Denison ◽  
Richard B. Heath ◽  
David Carling ◽  
David Saggerson
Keyword(s):  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Calcium Signalling ◽  
Metabolic Stress ◽  
Protein Kinase Activity ◽  
Adult Rat ◽  
Protein Kinase C Inhibitor ◽  
Amp Activated Protein Kinase

In adult rat cardiac myocytes adrenaline decreased AMPK (AMP-activated protein kinase) activity with a half-time of approximately 4 min, decreased phosphorylation of AMPK (α-Thr172) and decreased phosphorylation of ACC (acetyl-CoA carboxylase). Inactivation of AMPK by adrenaline was through both α1- and β-ARs (adrenergic receptors), but did not involve cAMP or calcium signalling, was not blocked by the PKC (protein kinase C) inhibitor BIM I (bisindoylmaleimide I), by the ERK (extracellular-signal-regulated kinase) cascade inhibitor U0126 or by PTX (pertussis toxin). Adrenaline caused no measurable change in LKB1 activity. Adrenaline decreased AMPK activity through a process that was distinct from AMPK inactivation in response to insulin or PMA. Neither adrenaline nor PMA altered the myocyte AMP:ATP ratio although the adrenaline effect was attenuated by oligomycin and by AICAR (5-amino-4-imidazolecarboxamide-1-β-D-ribofuranoside), agents that mimic ‘metabolic stress’. Inactivation of AMPK by adrenaline was abolished by 1 μM okadaic acid suggesting that activation of PP2A (phosphoprotein phosphatase 2A) might mediate the adrenaline effect. However, no change in PP2A activity was detected in myocyte extracts. Adrenaline increased phosphorylation of the AMPK β-subunit in vitro but there was no detectable change in vivo in phosphorylation of previously identified AMPK sites (β-Ser24, β-Ser108 or β-Ser182) suggesting that another site(s) is targeted.

scholarly journals Statins Activate AMP-Activated Protein Kinase In Vitro and In Vivo

Circulation ◽  
2006 ◽  
Vol 114 (24) ◽  
pp. 2655-2662 ◽  
Author(s):  
Wei Sun ◽  
Tzong-Shyuan Lee ◽  
Minjia Zhu ◽  
Chunang Gu ◽  
Yinsheng Wang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Amp Activated Protein Kinase

Possible involvement of the α1 isoform of 5′AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle

2004 ◽  
Vol 287 (1) ◽  
pp. E166-E173 ◽  
Author(s):  
Taro Toyoda ◽  
Tatsuya Hayashi ◽  
Licht Miyamoto ◽  
Shin Yonemitsu ◽  
Masako Nakano ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Activation Process ◽  
Dependent Manner ◽  
Upstream Kinase ◽  
Metabolic Stresses ◽  
Amp Activated Protein Kinase

Recent studies have suggested that 5′AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H2O2, AMPKα1 activity increased in a time- and dose-dependent manner, whereas AMPKα2 activity remained unchanged. The activation of AMPKα1 was associated with phosphorylation of AMPK Thr172, suggesting that an upstream kinase is involved in the activation process. H2O2-induced AMPKα1 activation was blocked in the presence of the antioxidant N-acetyl-l-cysteine (NAC), and H2O2 significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H2O2 did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H2O2 increased 3- O-methyl-d-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKα1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKα1-mediated mechanism.

Adiponectin promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-27b through a CaMKII/AMPK/p38 signaling pathway in human chondrosarcoma cells

2016 ◽  
Vol 130 (17) ◽  
pp. 1523-1533 ◽  
Author(s):  
Chun-Yin Huang ◽  
An-Chen Chang ◽  
Hsien-Te Chen ◽  
Shih-Wei Wang ◽  
Yuan-Shun Lo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Tube Formation ◽  
Human Chondrosarcoma ◽  
Dependent Protein ◽  
And Migration ◽  
Factor C ◽  
Amp Activated Protein Kinase

Chondrosarcoma is the second most frequently occurring type of bone malignancy characterized by distant metastatic propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumour lymphangiogenesis and lymphatic metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. In recent years, adiponectin has also been indicated as facilitating tumorigenesis, angiogenesis and metastasis. However, the effect of adiponectin on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has remained largely a mystery. In the present study, we have shown a clinical correlation between adiponectin and VEGF-C, as well as tumour stage, in human chondrosarcoma tissues. We further demonstrated that adiponectin promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium from adiponectin-treated cells significantly induced tube formation and migration of human lymphatic endothelial cells. In addition, adiponectin knock down inhibited lymphangiogenesis in vitro and in vivo. We also found that adiponectin-induced VEGF-C is mediated by the calmodulin-dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK) and p38 signaling pathway. Furthermore, the expression of miR-27b was negatively regulated by adiponectin via the CaMKII, AMPK and p38 cascade. The present study is the first to describe the mechanism of adiponectin-promoted lymphangiogenesis by up-regulating VEGF-C expression in chondrosarcomas. Thus, adiponectin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis.

scholarly journals An Intramolecular Association between Two Domains of the Protein Kinase Fused Is Necessary for Hedgehog Signaling

2004 ◽  
Vol 24 (23) ◽  
pp. 10397-10405 ◽  
Author(s):  
Manuel Ascano ◽  
David J. Robbins
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Hedgehog Signaling ◽  
Membrane Vesicles ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Intramolecular Association

ABSTRACT The protein kinase Fused (Fu) is an integral member of the Hedgehog (Hh) signaling pathway. Although genetic studies demonstrate that Fu is required for the regulation of the Hh pathway, the mechanistic role that it plays remains largely unknown. Given our difficulty in developing an in vitro kinase assay for Fu, we reasoned that the catalytic activity of Fu might be highly regulated. Several mechanisms are known to regulate protein kinases, including self-association in either an intra- or an intermolecular fashion. Here, we provide evidence that Hh regulates Fu through intramolecular association between its kinase domain (ΔFu) and its carboxyl-terminal domain (Fu-tail). We show that ΔFu and Fu-tail can interact in trans, with or without the kinesin-related protein Costal 2 (Cos2). However, since the majority of Fu is found associated with Cos2 in vivo, we hypothesized that Fu-tail, which binds Cos2 directly, would be able to tether ΔFu to Cos2. We demonstrate that ΔFu colocalizes with Cos2 in the presence of Fu-tail and that this colocalization occurs on a subset of membrane vesicles previously characterized to be important for Hh signal transduction. Additionally, expression of Fu-tail in fu mutant flies that normally express only the kinase domain rescues the fu wing phenotype. Therefore, reestablishing the association between these two domains of Fu in trans is sufficient to restore Hh signal transduction in vivo. In such a manner we validate our hypothesis, demonstrating that Fu self-associates and is functional in an Hh-dependent manner. Our results here enhance our understanding of one of the least characterized, yet critical, components of Hh signal transduction.

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