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Lallet-Daher H.,French Institute of Health and Medical Research | Lallet-Daher H.,French National Center for Scientific Research | Lallet-Daher H.,Center LeonBerard | Lallet-Daher H.,University of Lyon | And 29 more authors.
Cancer Research | Year: 2013

Oncogene-induced senescence (OIS) constitutes a failsafe program that restricts tumor development. However, the mechanisms that link oncogenesis to senescence are not completely understood. We carried out a loss-of-function genetic screen that identified the potassium channel KCNA1 as a determinant of OIS escape that can license tumor growth. Oncogenic stress triggers an increase in KCNA1 expression and its relocation from the cytoplasm to the membrane. Mechanistically, this relocation is due to a loss of protein kinase A(PKA)-induced phosphorylation at residue S446 of KCNA1. Accordingly, sustaining PKA activity or expressing a KCNA1 phosphomimetic mutant maintained KCNA1 in the cytoplasm and caused escape from OIS. KCNA1 relocation to the membrane induced a change in membrane potential that invariably resulted in cellular senescence. Restoring KCNA1 expression in transformation-competent cells triggered variation in membrane potential and blocked RAS-induced transformation, and PKA activation suppressed both effects. Furthermore, KCNA1 expression was reduced in human cancers, and this decrease correlated with an increase in breast cancer aggressiveness. Taken together, our results identify a novel pathway that restricts oncogenesis through a potassium channel-dependent senescence pathway. © 2013 American Association for Cancer Research.


Chennell G.,Cellular Stress Group | Chennell G.,Imperial College London | Willows R.J.W.,Cellular Stress Group | Warren S.C.,University of New South Wales | And 6 more authors.
Sensors (Switzerland) | Year: 2016

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. © 2016 by the authors; licensee MDPI, Basel, Switzerland.


Thornton C.,Cellular Stress Group | Bright N.J.,Cellular Stress Group | Sastre M.,Imperial College London | Muckett P.J.,Cellular Stress Group | Carling D.,Cellular Stress Group
Biochemical Journal | Year: 2011

Hyperphosphorylation of tau is a hallmark of Alzheimer's disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK (AMP-activated protein kinase)-related kinases MARK (microtubule-associated protein-regulating kinase/microtubule affinity-regulating kinase) and BRSK (brain-specific kinase) have been implicated in tau phosphorylation, but are insensitive to activation by cellular stress. In the present study, we show that AMPK itself phosphorylates tau on a number of sites, including Ser 262 and Ser 396, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKKβ (Ca 2+/calmodulin-dependent protein kinase kinase β) activation of AMPK in response to Aβ (amyloid-β peptide)-(1-42) leads to increased phosphorylation of tau at Ser 262/Ser 356 and Ser 396. Activation of AMPK by Aβ-(1-42) is inhibited by memantine, a partial antagonist of the NMDA (N-methyl-D-aspartate) receptor and currently licensed for the treatment of Alzheimer's disease. These findings identify a pathway in which Aβ-(1-42) activates CaMKKβ and AMPK via the NMDA receptor, suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau. © The Authors Journal compilation © 2011 Biochemical Society.


PubMed | Cellular Stress Group
Type: Journal Article | Journal: The Biochemical journal | Year: 2011

Hyperphosphorylation of tau is a hallmark of Alzheimers disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK (AMP-activated protein kinase)-related kinases MARK (microtubule-associated protein-regulating kinase/microtubule affinity-regulating kinase) and BRSK (brain-specific kinase) have been implicated in tau phosphorylation, but are insensitive to activation by cellular stress. In the present study, we show that AMPK itself phosphorylates tau on a number of sites, including Ser and Ser, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKK (Ca+/calmodulin-dependent protein kinase kinase ) activation of AMPK in response to A (amyloid- peptide)-(1-42) leads to increased phosphorylation of tau at Ser/Ser and Ser3. Activation of AMPK by A-(1-42) is inhibited by memantine, a partial antagonist of the NMDA (N-methyl-D-aspartate) receptor and currently licensed for the treatment of Alzheimers disease. These findings identify a pathway in which A-(1-42) activates CaMKK and AMPK via the NMDA receptor, suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau.

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