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A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis.

Poderoso C, Converso DP, Maloberti P, Duarte A, Neuman I, Galli S, Cornejo Maciel F, Paz C, Carreras MC, Poderoso JJ, Podestá EJ - PLoS ONE (2008)

Bottom Line: Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not.As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232).Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Moleculares de Enfermedades Hormonales, Neurodegenerativas y Oncológicas (IIMHNO), Department of Human Biochemistry, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.

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MEK1/2 activation in mitochondria and cytosol is entirely dependent on stimulus type and on PKA activity.(A) MA-10 cells were stimulated with 0.5 mM 8Br-cAMP or 10 ng/ml of EGF for the indicated times. Cytosolic and mitochondrial pMEK1/2 contents were analyzed by Western blot. A mitochondrial acyl-CoA thioesterase (Acot2), the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) and cytosolic β-tubulin detection were used as loading control. The immunoblots show a representative result of three independent experiments. Bars denote levels of pMEK1/2 (black bars) and total MEK1/2 (grey bars) relative to β-tubulin (cytosolic fractions), Acot2 (mitochondrial fractions- 8Br-cAMP treatments) or complex I (mitochondrial fractions – EGF treatments) in arbitrary units. Data are expressed as means±SD of three independent experiments. * p<0.05 vs. 0 min; **, p<0.01 vs. 0 min. (B) Mitochondrial pERK1/2 and pMEK1/2 contents were analyzed in mitochondria obtained from MA-10 cells stimulated with or without 0.5 mM 8Br-cAMP for varying times, in the presence or absence of 20 µM of H-89, an inhibitor of PKA activity. Acot2 detection was used as loading control in pMEK1/2 and total MEK1/2 western blots. The panel shows a representative immunoblot from three independent experiments. (C) MA-10 cells were transiently transfected with 100 nM siRNA against the α isoform of the PKA catalytic subunit, using Lipofectamine 2000 reagent. After transfection, the cells were incubated with or without 0.5 mM of 8Br-cAMP for varying times and the contents of mitochondrial pMEK1/2, total MEK1/2, α isoform of the PKA catalytic subunit and the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) were analyzed by western blot. This is a representative experiment from three separate experiments. (D) MA-10 cells were transiently transfected as described in (C) and stimulated with 8Br-cAMP for 15 and 30 minutes. pERK1/2 activity was measured using a pERK1/2 (pThr185/pTyr187) ELISA kit (Sigma Chemical Company, St. Louis, MO, USA), following the manufacturer's instructions. Bars represent the pERK1/2 activity as means±SD (n = 3). a, p<0.05, mock-transfected and 8Br-cAMP-treated cells (15 min) vs. mock-transfected and non-8Br-cAMP-treated cells (0 min); b, p<0.05, PKA catalytic subunit siRNA–transfected and 8Br-cAMP-treated (15 min) cells vs. mock-transfected and 8Br-cAMP-treated cells (15 min). (E) PKA activity in cytosolic and mitochondrial fractions isolated from MA-10 cells incubated with 0.5 mM 8Br-cAMP for varying times. Bars represent radioactivity incorporated into the kemptide-specific PKA synthetic substrate. The PKA assay was performed as previously described [61]. Data are expressed as means±SD (n = 3). **, p<0.01, * p<0.05 vs. 0 min.
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pone-0001443-g003: MEK1/2 activation in mitochondria and cytosol is entirely dependent on stimulus type and on PKA activity.(A) MA-10 cells were stimulated with 0.5 mM 8Br-cAMP or 10 ng/ml of EGF for the indicated times. Cytosolic and mitochondrial pMEK1/2 contents were analyzed by Western blot. A mitochondrial acyl-CoA thioesterase (Acot2), the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) and cytosolic β-tubulin detection were used as loading control. The immunoblots show a representative result of three independent experiments. Bars denote levels of pMEK1/2 (black bars) and total MEK1/2 (grey bars) relative to β-tubulin (cytosolic fractions), Acot2 (mitochondrial fractions- 8Br-cAMP treatments) or complex I (mitochondrial fractions – EGF treatments) in arbitrary units. Data are expressed as means±SD of three independent experiments. * p<0.05 vs. 0 min; **, p<0.01 vs. 0 min. (B) Mitochondrial pERK1/2 and pMEK1/2 contents were analyzed in mitochondria obtained from MA-10 cells stimulated with or without 0.5 mM 8Br-cAMP for varying times, in the presence or absence of 20 µM of H-89, an inhibitor of PKA activity. Acot2 detection was used as loading control in pMEK1/2 and total MEK1/2 western blots. The panel shows a representative immunoblot from three independent experiments. (C) MA-10 cells were transiently transfected with 100 nM siRNA against the α isoform of the PKA catalytic subunit, using Lipofectamine 2000 reagent. After transfection, the cells were incubated with or without 0.5 mM of 8Br-cAMP for varying times and the contents of mitochondrial pMEK1/2, total MEK1/2, α isoform of the PKA catalytic subunit and the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) were analyzed by western blot. This is a representative experiment from three separate experiments. (D) MA-10 cells were transiently transfected as described in (C) and stimulated with 8Br-cAMP for 15 and 30 minutes. pERK1/2 activity was measured using a pERK1/2 (pThr185/pTyr187) ELISA kit (Sigma Chemical Company, St. Louis, MO, USA), following the manufacturer's instructions. Bars represent the pERK1/2 activity as means±SD (n = 3). a, p<0.05, mock-transfected and 8Br-cAMP-treated cells (15 min) vs. mock-transfected and non-8Br-cAMP-treated cells (0 min); b, p<0.05, PKA catalytic subunit siRNA–transfected and 8Br-cAMP-treated (15 min) cells vs. mock-transfected and 8Br-cAMP-treated cells (15 min). (E) PKA activity in cytosolic and mitochondrial fractions isolated from MA-10 cells incubated with 0.5 mM 8Br-cAMP for varying times. Bars represent radioactivity incorporated into the kemptide-specific PKA synthetic substrate. The PKA assay was performed as previously described [61]. Data are expressed as means±SD (n = 3). **, p<0.01, * p<0.05 vs. 0 min.

Mentions: We found two different pools of MEK1/2 and phospho-MEK1/2 (pMEK1/2) constitutively present in the cytosol and mitochondria of MA-10 cells (Fig. 3). Interestingly, MEK1/2 responded differently to stimulation depending on distribution (Fig. 3A). 8Br-cAMP clearly induced prolonged MEK1/2 phosphorylation in mitochondria, but had a less significant effect on the cytosolic kinases. Conversely, EGF induced a sustained and robust MEK1/2 activation in the cytosol but only a modest phosphorylation in mitochondria. Interestingly, although both EGF and 8Br-cAMP increased total cytosolic MEK1/2, only EGF promoted its phosphorylation in this subcellular fraction. Due to the fact that total MEK1/2 changed during the treatments, in the experiments depicted in Fig. 3, we also evaluated the contents of an acyl-CoA thioesterase (Acot2), 39 kDa subunit of the NADH-cytochrome c reductase (complex I) and β-tubulin in the corresponding blots. In cells stimulated with 8Br-cAMP, Acot2 was used as mitochondrial loading control [41]. In cells treated with EGF, Acot2 detection was replaced by the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) since the content of the thioesterase changes with EGF treatment (unpublished observation). In both treatments, β-tubulin detection was used as cytosolic loading control. These loading controls indicated that the changes in total MEK1/2 signal are really due to a biological variation and not to different amount of proteins in each lane (15, 30 and 60 minutes of cAMP treatment in the cytosolic fraction and 60 minutes in the mitochondrial fraction; 30 minutes of EGF treatment in the cytosolic fraction). Therefore, we expressed pMEK1/2 relative to the loading control levels instead of relative to total MEK1/2 levels.


A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis.

Poderoso C, Converso DP, Maloberti P, Duarte A, Neuman I, Galli S, Cornejo Maciel F, Paz C, Carreras MC, Poderoso JJ, Podestá EJ - PLoS ONE (2008)

MEK1/2 activation in mitochondria and cytosol is entirely dependent on stimulus type and on PKA activity.(A) MA-10 cells were stimulated with 0.5 mM 8Br-cAMP or 10 ng/ml of EGF for the indicated times. Cytosolic and mitochondrial pMEK1/2 contents were analyzed by Western blot. A mitochondrial acyl-CoA thioesterase (Acot2), the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) and cytosolic β-tubulin detection were used as loading control. The immunoblots show a representative result of three independent experiments. Bars denote levels of pMEK1/2 (black bars) and total MEK1/2 (grey bars) relative to β-tubulin (cytosolic fractions), Acot2 (mitochondrial fractions- 8Br-cAMP treatments) or complex I (mitochondrial fractions – EGF treatments) in arbitrary units. Data are expressed as means±SD of three independent experiments. * p<0.05 vs. 0 min; **, p<0.01 vs. 0 min. (B) Mitochondrial pERK1/2 and pMEK1/2 contents were analyzed in mitochondria obtained from MA-10 cells stimulated with or without 0.5 mM 8Br-cAMP for varying times, in the presence or absence of 20 µM of H-89, an inhibitor of PKA activity. Acot2 detection was used as loading control in pMEK1/2 and total MEK1/2 western blots. The panel shows a representative immunoblot from three independent experiments. (C) MA-10 cells were transiently transfected with 100 nM siRNA against the α isoform of the PKA catalytic subunit, using Lipofectamine 2000 reagent. After transfection, the cells were incubated with or without 0.5 mM of 8Br-cAMP for varying times and the contents of mitochondrial pMEK1/2, total MEK1/2, α isoform of the PKA catalytic subunit and the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) were analyzed by western blot. This is a representative experiment from three separate experiments. (D) MA-10 cells were transiently transfected as described in (C) and stimulated with 8Br-cAMP for 15 and 30 minutes. pERK1/2 activity was measured using a pERK1/2 (pThr185/pTyr187) ELISA kit (Sigma Chemical Company, St. Louis, MO, USA), following the manufacturer's instructions. Bars represent the pERK1/2 activity as means±SD (n = 3). a, p<0.05, mock-transfected and 8Br-cAMP-treated cells (15 min) vs. mock-transfected and non-8Br-cAMP-treated cells (0 min); b, p<0.05, PKA catalytic subunit siRNA–transfected and 8Br-cAMP-treated (15 min) cells vs. mock-transfected and 8Br-cAMP-treated cells (15 min). (E) PKA activity in cytosolic and mitochondrial fractions isolated from MA-10 cells incubated with 0.5 mM 8Br-cAMP for varying times. Bars represent radioactivity incorporated into the kemptide-specific PKA synthetic substrate. The PKA assay was performed as previously described [61]. Data are expressed as means±SD (n = 3). **, p<0.01, * p<0.05 vs. 0 min.
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pone-0001443-g003: MEK1/2 activation in mitochondria and cytosol is entirely dependent on stimulus type and on PKA activity.(A) MA-10 cells were stimulated with 0.5 mM 8Br-cAMP or 10 ng/ml of EGF for the indicated times. Cytosolic and mitochondrial pMEK1/2 contents were analyzed by Western blot. A mitochondrial acyl-CoA thioesterase (Acot2), the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) and cytosolic β-tubulin detection were used as loading control. The immunoblots show a representative result of three independent experiments. Bars denote levels of pMEK1/2 (black bars) and total MEK1/2 (grey bars) relative to β-tubulin (cytosolic fractions), Acot2 (mitochondrial fractions- 8Br-cAMP treatments) or complex I (mitochondrial fractions – EGF treatments) in arbitrary units. Data are expressed as means±SD of three independent experiments. * p<0.05 vs. 0 min; **, p<0.01 vs. 0 min. (B) Mitochondrial pERK1/2 and pMEK1/2 contents were analyzed in mitochondria obtained from MA-10 cells stimulated with or without 0.5 mM 8Br-cAMP for varying times, in the presence or absence of 20 µM of H-89, an inhibitor of PKA activity. Acot2 detection was used as loading control in pMEK1/2 and total MEK1/2 western blots. The panel shows a representative immunoblot from three independent experiments. (C) MA-10 cells were transiently transfected with 100 nM siRNA against the α isoform of the PKA catalytic subunit, using Lipofectamine 2000 reagent. After transfection, the cells were incubated with or without 0.5 mM of 8Br-cAMP for varying times and the contents of mitochondrial pMEK1/2, total MEK1/2, α isoform of the PKA catalytic subunit and the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) were analyzed by western blot. This is a representative experiment from three separate experiments. (D) MA-10 cells were transiently transfected as described in (C) and stimulated with 8Br-cAMP for 15 and 30 minutes. pERK1/2 activity was measured using a pERK1/2 (pThr185/pTyr187) ELISA kit (Sigma Chemical Company, St. Louis, MO, USA), following the manufacturer's instructions. Bars represent the pERK1/2 activity as means±SD (n = 3). a, p<0.05, mock-transfected and 8Br-cAMP-treated cells (15 min) vs. mock-transfected and non-8Br-cAMP-treated cells (0 min); b, p<0.05, PKA catalytic subunit siRNA–transfected and 8Br-cAMP-treated (15 min) cells vs. mock-transfected and 8Br-cAMP-treated cells (15 min). (E) PKA activity in cytosolic and mitochondrial fractions isolated from MA-10 cells incubated with 0.5 mM 8Br-cAMP for varying times. Bars represent radioactivity incorporated into the kemptide-specific PKA synthetic substrate. The PKA assay was performed as previously described [61]. Data are expressed as means±SD (n = 3). **, p<0.01, * p<0.05 vs. 0 min.
Mentions: We found two different pools of MEK1/2 and phospho-MEK1/2 (pMEK1/2) constitutively present in the cytosol and mitochondria of MA-10 cells (Fig. 3). Interestingly, MEK1/2 responded differently to stimulation depending on distribution (Fig. 3A). 8Br-cAMP clearly induced prolonged MEK1/2 phosphorylation in mitochondria, but had a less significant effect on the cytosolic kinases. Conversely, EGF induced a sustained and robust MEK1/2 activation in the cytosol but only a modest phosphorylation in mitochondria. Interestingly, although both EGF and 8Br-cAMP increased total cytosolic MEK1/2, only EGF promoted its phosphorylation in this subcellular fraction. Due to the fact that total MEK1/2 changed during the treatments, in the experiments depicted in Fig. 3, we also evaluated the contents of an acyl-CoA thioesterase (Acot2), 39 kDa subunit of the NADH-cytochrome c reductase (complex I) and β-tubulin in the corresponding blots. In cells stimulated with 8Br-cAMP, Acot2 was used as mitochondrial loading control [41]. In cells treated with EGF, Acot2 detection was replaced by the 39 kDa subunit of the NADH-cytochrome c reductase (complex I) since the content of the thioesterase changes with EGF treatment (unpublished observation). In both treatments, β-tubulin detection was used as cytosolic loading control. These loading controls indicated that the changes in total MEK1/2 signal are really due to a biological variation and not to different amount of proteins in each lane (15, 30 and 60 minutes of cAMP treatment in the cytosolic fraction and 60 minutes in the mitochondrial fraction; 30 minutes of EGF treatment in the cytosolic fraction). Therefore, we expressed pMEK1/2 relative to the loading control levels instead of relative to total MEK1/2 levels.

Bottom Line: Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not.As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232).Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Moleculares de Enfermedades Hormonales, Neurodegenerativas y Oncológicas (IIMHNO), Department of Human Biochemistry, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.

Show MeSH
Related in: MedlinePlus