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A new paradigm for MAPK: structural interactions of hERK1 with mitochondria in HeLa cells.

ABSTRACT: Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.

SUBMITTER: Galli S

PROVIDER: S-EPMC2760858 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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A new paradigm for MAPK: structural interactions of hERK1 with mitochondria in HeLa cells.

Galli Soledad S Jahn Olaf O Hitt Reiner R Hesse Doerte D Opitz Lennart L Plessmann Uwe U Urlaub Henning H Poderoso Juan Jose JJ Jares-Erijman Elizabeth A EA Jovin Thomas M TM

PloS one 20091022 10

Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochon ...[more]

PMID: 19847302

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Project description:Hydrogen peroxide (H2O2) promotes a range of phenotypes depending on its intracellular concentration and dosing kinetics, including cell death. While this qualitative relationship has been well established, the quantitative and mechanistic aspects of H2O2 signaling are still being elucidated. Mitochondria, a putative source of intracellular H2O2, have recently been demonstrated to be particularly vulnerable to localized H2O2 perturbations, eliciting a dramatic cell death response in comparison to similar cytosolic perturbations. We sought to improve our dynamic and mechanistic understanding of the mitochondrial H2O2 reaction network in HeLa cells by creating a kinetic model of this system and using it to explore basal and perturbed conditions. The model uses the most current quantitative proteomic and kinetic data available to predict reaction rates and steady-state concentrations of H2O2 and its reaction partners within individual mitochondria. Time scales ranging from milliseconds to one hour were simulated. We predict that basal, steady-state mitochondrial H2O2 will be in the low nM range (2-4 nM) and will be inversely dependent on the total pool of peroxiredoxin-3 (Prx3). Neglecting efflux of H2O2 to the cytosol, the mitochondrial reaction network is expected to control perturbations well up to H2O2 generation rates ~50 μM/s (0.25 nmol/mg-protein/s), above which point the Prx3 system would be expected to collapse. Comparison of these results with redox Western blots of Prx3 and Prx2 oxidation states demonstrated reasonable trend agreement at short times (≤ 15 min) for a range of experimentally perturbed H2O2 generation rates. At longer times, substantial efflux of H2O2 from the mitochondria to the cytosol was evidenced by peroxiredoxin-2 (Prx2) oxidation, and Prx3 collapse was not observed. A refined model using Monte Carlo parameter sampling was used to explore rates of H2O2 efflux that could reconcile model predictions of Prx3 oxidation states with the experimental observations.

Project description:Subcellular organelles communicate with each other to regulate function and coordinate responses to changing cellular conditions. The physical-functional coupling of the endoplasmic reticulum (ER) with mitochondria allows for the direct transfer of Ca2+ between organelles and is an important avenue for rapidly increasing mitochondrial metabolic activity. As such, increasing ER-mitochondrial coupling can boost the generation of ATP that is needed to restore homeostasis in the face of cellular stress. The mitochondrial unfolded protein response (mtUPR) is activated by the accumulation of unfolded proteins in mitochondria. Retrograde signaling from mitochondria to the nucleus promotes mtUPR transcriptional responses aimed at restoring protein homeostasis. It is currently unknown whether the changes in mitochondrial-ER coupling also play a role during mtUPR stress. We hypothesized that mitochondrial stress favors an expansion of functional contacts between mitochondria and ER, thereby increasing mitochondrial metabolism as part of a protective response. Hela cells were treated with doxycycline, an antibiotic that inhibits the translation of mitochondrial-encoded proteins to create protein disequilibrium. Treatment with doxycycline decreased the abundance of mitochondrial encoded proteins while increasing expression of CHOP, C/EBPβ, ClpP, and mtHsp60, markers of the mtUPR. There was no change in either mitophagic activity or cell viability. Furthermore, ER UPR was not activated, suggesting focused activation of the mtUPR. Within 2 h of doxycycline treatment, there was a significant increase in physical contacts between mitochondria and ER that was distributed throughout the cell, along with an increase in the kinetics of mitochondrial Ca2+ uptake. This was followed by the rise in the rate of oxygen consumption at 4 h, indicating a boost in mitochondrial metabolic activity. In conclusion, an early phase of the response to doxycycline-induced mitochondrial stress is an increase in mitochondrial-ER coupling that potentiates mitochondrial metabolic activity as a means to support subsequent steps in the mtUPR pathway and sustain cellular adaptation.

Project description:The genesis and progression of cervical cancer involve the mutation or deviant expression of numerous genes, including the activation of oncogenes (Ha-ras, C-myc, C-erbB2 and Bcl-2) and inactivation of tumor-suppressor genes (p53 and Rb). Previous studies showed that small-interfering RNAs (siRNAs) targeting the MAPK p42 gene partly inhibit proliferation and increase apoptosis in human cervical carcinoma HeLa cells. Results of a microarray analysis showed that MAPK p42 siRNA inhibited cell growth through the regulation of cell cycle control and apoptosis and induced interferon-like response in HeLa cells. In order to confirm the dual effects of MAPK p42 siRNA, we compared the roles of siRNA and U0126, an inhibitor of MAPK p42, in HeLa cells. Short 21-mer double-stranded/siRNAs were synthesized to target MAPK p42 mRNA in HeLa cells. The siRNAs were transfected into HeLa cells using Lipofectamine. The cells were treated with siRNA or U0126 at different concentrations for a period of 48 h. The biological effect of siRNA and U0126 on HeLa cells was measured by MTT and flow cytometry. MAPK1, NUP188, P38, STAT1, STAT2, PML and OAS1 were analyzed by real-time quantitative PCR. HeLa cell growth was inhibited by siRNA or U0126, and the effect of siRNA inhibition was greater than that of U0126. Cell cycle phases were different for siRNA or U0126, but HeLa cell growth was arrested at the S phase by siRNA and at G1 phase by U0126. A down-regulation in MAPK p42 expression by siRNA and up-regulation by U0126 were noted. The results of real-time quantitative PCR showed that P38 was up-regulated and NUP188 was down-regulated by siRNA in comparison with the control groups, and the results were consistent with those of U0126. Expression levels of STAT1, STAT2, PML and OAS1 induced by siRNA differed from those induced by U0126. siRNA-mediated silencing and deactivation induced by U0126 in MAPK p42 led to growth inhibition in the HeLa cells. The effects of siRNA on HeLa cell growth were different from those of U0126. Dual effects of MAPK p42 siRNA-2 on HeLa cell growth were noted: one consisted of a specific effect induced by siRNA-mediated p42 MAPK silencing and the other exhibited a non-specific interferon-like response.

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A new paradigm for MAPK: structural interactions of hERK1 with mitochondria in HeLa cells.

Publications

A new paradigm for MAPK: structural interactions of hERK1 with mitochondria in HeLa cells.

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