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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 and active ERK1/2 are strictly required for steroidogenesis.(A) MA-10 cells were pre-incubated for 30 minutes with 50 µM of PD98059 or 10 µM of U0126. Then, they were treated with or without 0.5 mM of 8Br-cAMP for 15 minutes. Progesterone (P4) was measured by radioimmunoassay (RIA) as previously described [41], [60] (a, p<0.05 vs. control; b, p<0.05 vs. 8Br-cAMP alone). MA-10 cells were also transfected by electroporation using two plasmids, bearing the sequence of wild-type or a mutated form of ERK2 (H230R). Data are expressed as means±SD of three independent experiments. c, p<0.05 vs. control transfection pRc/CMVi-GFP; d, p<0.05 vs. transfected cells+8Br-cAMP; e, p<0.05 vs. wild-type ERK2). After the incubation with or without 10 µM of U0126, MA-10 cells were stimulated with 0.5 mM of 8Br-cAMP (B) or 10 ng/ml of EGF (C) for the indicated times. Cell lysates were subjected to SDS-PAGE and Western blot as previously described [41], with specific antibodies against pERK1/2 and total ERK1/2 sequentially. The immunoblots show a representative result of three independent experiments. The intensity of the bands was quantitated using total ERK1/2 as loading control. Bars denote relative levels of pERK1/2 presence in arbitrary units. Data are expressed as means±SD of three independent experiments. **, p<0.01 vs. 0 min.
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pone-0001443-g001: MEK1/2 activation and active ERK1/2 are strictly required for steroidogenesis.(A) MA-10 cells were pre-incubated for 30 minutes with 50 µM of PD98059 or 10 µM of U0126. Then, they were treated with or without 0.5 mM of 8Br-cAMP for 15 minutes. Progesterone (P4) was measured by radioimmunoassay (RIA) as previously described [41], [60] (a, p<0.05 vs. control; b, p<0.05 vs. 8Br-cAMP alone). MA-10 cells were also transfected by electroporation using two plasmids, bearing the sequence of wild-type or a mutated form of ERK2 (H230R). Data are expressed as means±SD of three independent experiments. c, p<0.05 vs. control transfection pRc/CMVi-GFP; d, p<0.05 vs. transfected cells+8Br-cAMP; e, p<0.05 vs. wild-type ERK2). After the incubation with or without 10 µM of U0126, MA-10 cells were stimulated with 0.5 mM of 8Br-cAMP (B) or 10 ng/ml of EGF (C) for the indicated times. Cell lysates were subjected to SDS-PAGE and Western blot as previously described [41], with specific antibodies against pERK1/2 and total ERK1/2 sequentially. The immunoblots show a representative result of three independent experiments. The intensity of the bands was quantitated using total ERK1/2 as loading control. Bars denote relative levels of pERK1/2 presence in arbitrary units. Data are expressed as means±SD of three independent experiments. **, p<0.01 vs. 0 min.

Mentions: Fig. 1A shows that treatment with U0126 or PD98095 (inhibitors of the activation of MEK1/2) inhibited production of progesterone (P4) by MA-10 Leydig cells stimulated with 8Br-cAMP during 15 minutes. These findings confirm those by Gyles et al. and Manna and Stocco [27], [29]. However, the results do not agree with those by Seger et al. which show that ERK signaling cascade inhibited gonadotropin-stimulated steroidogenesis [31]. It is worth to mention that in this paper, there are several methodological differences, being the main of them that the effect of MEK/ERK inhibitors are tested after 24 and 48 hs of stimulation of rLHR4 granulosa cells with gonadotropin [31].


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 and active ERK1/2 are strictly required for steroidogenesis.(A) MA-10 cells were pre-incubated for 30 minutes with 50 µM of PD98059 or 10 µM of U0126. Then, they were treated with or without 0.5 mM of 8Br-cAMP for 15 minutes. Progesterone (P4) was measured by radioimmunoassay (RIA) as previously described [41], [60] (a, p<0.05 vs. control; b, p<0.05 vs. 8Br-cAMP alone). MA-10 cells were also transfected by electroporation using two plasmids, bearing the sequence of wild-type or a mutated form of ERK2 (H230R). Data are expressed as means±SD of three independent experiments. c, p<0.05 vs. control transfection pRc/CMVi-GFP; d, p<0.05 vs. transfected cells+8Br-cAMP; e, p<0.05 vs. wild-type ERK2). After the incubation with or without 10 µM of U0126, MA-10 cells were stimulated with 0.5 mM of 8Br-cAMP (B) or 10 ng/ml of EGF (C) for the indicated times. Cell lysates were subjected to SDS-PAGE and Western blot as previously described [41], with specific antibodies against pERK1/2 and total ERK1/2 sequentially. The immunoblots show a representative result of three independent experiments. The intensity of the bands was quantitated using total ERK1/2 as loading control. Bars denote relative levels of pERK1/2 presence in arbitrary units. Data are expressed as means±SD of three independent experiments. **, p<0.01 vs. 0 min.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2175533&req=5

pone-0001443-g001: MEK1/2 activation and active ERK1/2 are strictly required for steroidogenesis.(A) MA-10 cells were pre-incubated for 30 minutes with 50 µM of PD98059 or 10 µM of U0126. Then, they were treated with or without 0.5 mM of 8Br-cAMP for 15 minutes. Progesterone (P4) was measured by radioimmunoassay (RIA) as previously described [41], [60] (a, p<0.05 vs. control; b, p<0.05 vs. 8Br-cAMP alone). MA-10 cells were also transfected by electroporation using two plasmids, bearing the sequence of wild-type or a mutated form of ERK2 (H230R). Data are expressed as means±SD of three independent experiments. c, p<0.05 vs. control transfection pRc/CMVi-GFP; d, p<0.05 vs. transfected cells+8Br-cAMP; e, p<0.05 vs. wild-type ERK2). After the incubation with or without 10 µM of U0126, MA-10 cells were stimulated with 0.5 mM of 8Br-cAMP (B) or 10 ng/ml of EGF (C) for the indicated times. Cell lysates were subjected to SDS-PAGE and Western blot as previously described [41], with specific antibodies against pERK1/2 and total ERK1/2 sequentially. The immunoblots show a representative result of three independent experiments. The intensity of the bands was quantitated using total ERK1/2 as loading control. Bars denote relative levels of pERK1/2 presence in arbitrary units. Data are expressed as means±SD of three independent experiments. **, p<0.01 vs. 0 min.
Mentions: Fig. 1A shows that treatment with U0126 or PD98095 (inhibitors of the activation of MEK1/2) inhibited production of progesterone (P4) by MA-10 Leydig cells stimulated with 8Br-cAMP during 15 minutes. These findings confirm those by Gyles et al. and Manna and Stocco [27], [29]. However, the results do not agree with those by Seger et al. which show that ERK signaling cascade inhibited gonadotropin-stimulated steroidogenesis [31]. It is worth to mention that in this paper, there are several methodological differences, being the main of them that the effect of MEK/ERK inhibitors are tested after 24 and 48 hs of stimulation of rLHR4 granulosa cells with gonadotropin [31].

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