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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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Comparison of subcellular distribution of hormone- or EGF-stimulated pERK1/2 activation.MA-10 cells were incubated with or without 0.5 mM 8Br-cAMP (upper panels of A, B and C), 10 ng/ml of EGF (middle panels of A, B and C) or 20 ng/ml of hCG (lower panels of A, B and C) for the indicated times. Next, subcellular fractions were obtained and 40 µg of the total protein of each fraction were subjected to SDS-PAGE and Western blot to detect pERK1/2 (indicated with pERK1/2 in A, B, and C). After stripping, total ERK1/2 (indicated with ERK1/2 in A, B, and C) was detected in the same membrane. The Western blots show the results of a representative experiment performed three times. D shows the quantification, performed as described in Figure 1. **, p<0.01, *, p<0.05 vs. time 0. (E) Immunofluorescent staining for pERK1/2 (green) and mitochondria (red) in MA-10 cells after treatment with or without 0.5 mM of 8Br-cAMP or 10 ng/ml of EGF for the indicated times. Merged images are shown in the right panel.
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pone-0001443-g002: Comparison of subcellular distribution of hormone- or EGF-stimulated pERK1/2 activation.MA-10 cells were incubated with or without 0.5 mM 8Br-cAMP (upper panels of A, B and C), 10 ng/ml of EGF (middle panels of A, B and C) or 20 ng/ml of hCG (lower panels of A, B and C) for the indicated times. Next, subcellular fractions were obtained and 40 µg of the total protein of each fraction were subjected to SDS-PAGE and Western blot to detect pERK1/2 (indicated with pERK1/2 in A, B, and C). After stripping, total ERK1/2 (indicated with ERK1/2 in A, B, and C) was detected in the same membrane. The Western blots show the results of a representative experiment performed three times. D shows the quantification, performed as described in Figure 1. **, p<0.01, *, p<0.05 vs. time 0. (E) Immunofluorescent staining for pERK1/2 (green) and mitochondria (red) in MA-10 cells after treatment with or without 0.5 mM of 8Br-cAMP or 10 ng/ml of EGF for the indicated times. Merged images are shown in the right panel.

Mentions: The immunoblots showed that after stimulation with 8Br-cAMP, phospho-ERK1/2 (pERK1/2) was located in the cytosol and mitochondria and, to a much lesser extent, in the nuclear fraction (Fig. 2, upper panels of A, B and C). In both the mitochondria and cytosol, an early peak of ERK1/2 phosphorylation was followed by a slow progressive decrease of the signal during the first hour of 8Br-cAMP action. On the contrary, after EGF stimulation, pERK1/2 is mainly localized in the cytosol and nucleus (Fig. 2, middle panels of A, B, and C). In mitochondria, activation peaked early (5 minutes) and then decayed. hCG stimulation led to ERK1/2 activation with a similar profile to that induced by 8Br-cAMP (Fig. 2, lower panels of A, B, and C). This later result validates the use of 8Br-cAMP to replace the hormone.


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)

Comparison of subcellular distribution of hormone- or EGF-stimulated pERK1/2 activation.MA-10 cells were incubated with or without 0.5 mM 8Br-cAMP (upper panels of A, B and C), 10 ng/ml of EGF (middle panels of A, B and C) or 20 ng/ml of hCG (lower panels of A, B and C) for the indicated times. Next, subcellular fractions were obtained and 40 µg of the total protein of each fraction were subjected to SDS-PAGE and Western blot to detect pERK1/2 (indicated with pERK1/2 in A, B, and C). After stripping, total ERK1/2 (indicated with ERK1/2 in A, B, and C) was detected in the same membrane. The Western blots show the results of a representative experiment performed three times. D shows the quantification, performed as described in Figure 1. **, p<0.01, *, p<0.05 vs. time 0. (E) Immunofluorescent staining for pERK1/2 (green) and mitochondria (red) in MA-10 cells after treatment with or without 0.5 mM of 8Br-cAMP or 10 ng/ml of EGF for the indicated times. Merged images are shown in the right panel.
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Related In: Results  -  Collection

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pone-0001443-g002: Comparison of subcellular distribution of hormone- or EGF-stimulated pERK1/2 activation.MA-10 cells were incubated with or without 0.5 mM 8Br-cAMP (upper panels of A, B and C), 10 ng/ml of EGF (middle panels of A, B and C) or 20 ng/ml of hCG (lower panels of A, B and C) for the indicated times. Next, subcellular fractions were obtained and 40 µg of the total protein of each fraction were subjected to SDS-PAGE and Western blot to detect pERK1/2 (indicated with pERK1/2 in A, B, and C). After stripping, total ERK1/2 (indicated with ERK1/2 in A, B, and C) was detected in the same membrane. The Western blots show the results of a representative experiment performed three times. D shows the quantification, performed as described in Figure 1. **, p<0.01, *, p<0.05 vs. time 0. (E) Immunofluorescent staining for pERK1/2 (green) and mitochondria (red) in MA-10 cells after treatment with or without 0.5 mM of 8Br-cAMP or 10 ng/ml of EGF for the indicated times. Merged images are shown in the right panel.
Mentions: The immunoblots showed that after stimulation with 8Br-cAMP, phospho-ERK1/2 (pERK1/2) was located in the cytosol and mitochondria and, to a much lesser extent, in the nuclear fraction (Fig. 2, upper panels of A, B and C). In both the mitochondria and cytosol, an early peak of ERK1/2 phosphorylation was followed by a slow progressive decrease of the signal during the first hour of 8Br-cAMP action. On the contrary, after EGF stimulation, pERK1/2 is mainly localized in the cytosol and nucleus (Fig. 2, middle panels of A, B, and C). In mitochondria, activation peaked early (5 minutes) and then decayed. hCG stimulation led to ERK1/2 activation with a similar profile to that induced by 8Br-cAMP (Fig. 2, lower panels of A, B, and C). This later result validates the use of 8Br-cAMP to replace the hormone.

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