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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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PKA and ERK1/2 are strictly required to achieve maximal progesterone production by isolated mitochondria.MA-10 cells were transiently transfected by electroporation with an empty vector or with a vector containing StAR cDNA (sense or antisense orientations). Mitochondria were incubated in the presence of cholesterol as substrate (a) in the presence of wild type ERK1-GST protein alone (b) or together with PKA catalytic subunit (d). The mutated inactive form of ERK1-GST (K71A) was also used (c and e). After the indicated incubations, mitochondria were pelleted and subjected to SDS-PAGE and Western blot (A). Specific antibodies that recognize StAR protein, the catalytic subunit of PKA, pMEK1/2, pERK1/2 and total ERK1/2 were used. The panel shows representative Western blots of three independently performed experiments. P4 production is shown in (B). Data are expressed as means±SD (n = 3). * p<0.05 bar b vs. bar a and § p<0.05 bar d vs. bar b. (C) MA-10 cells were treated with or without 0.5 mM of 8Br-cAMP for 3 hours; cytosolic and mitochondrial subcellular fractions were obtained and incubated in the presence or absence of human pERK1-GST bound to agarose beads. Input and pelleted proteins bound to pERK1-GST (complexes) were analyzed by SDS-PAGE and Western blot. The immunoblots show the bands corresponding to StAR and pERK1/2, as loading control. Data are representative of three independently performed experiments.
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pone-0001443-g004: PKA and ERK1/2 are strictly required to achieve maximal progesterone production by isolated mitochondria.MA-10 cells were transiently transfected by electroporation with an empty vector or with a vector containing StAR cDNA (sense or antisense orientations). Mitochondria were incubated in the presence of cholesterol as substrate (a) in the presence of wild type ERK1-GST protein alone (b) or together with PKA catalytic subunit (d). The mutated inactive form of ERK1-GST (K71A) was also used (c and e). After the indicated incubations, mitochondria were pelleted and subjected to SDS-PAGE and Western blot (A). Specific antibodies that recognize StAR protein, the catalytic subunit of PKA, pMEK1/2, pERK1/2 and total ERK1/2 were used. The panel shows representative Western blots of three independently performed experiments. P4 production is shown in (B). Data are expressed as means±SD (n = 3). * p<0.05 bar b vs. bar a and § p<0.05 bar d vs. bar b. (C) MA-10 cells were treated with or without 0.5 mM of 8Br-cAMP for 3 hours; cytosolic and mitochondrial subcellular fractions were obtained and incubated in the presence or absence of human pERK1-GST bound to agarose beads. Input and pelleted proteins bound to pERK1-GST (complexes) were analyzed by SDS-PAGE and Western blot. The immunoblots show the bands corresponding to StAR and pERK1/2, as loading control. Data are representative of three independently performed experiments.

Mentions: To test the direct effect of active ERK1/2 on cholesterol import and progesterone synthesis, we performed a cell-free assay [22], determining progesterone production by isolated mitochondria. To enrich non-stimulated mitochondria with StAR, we transfected StAR cDNA to MA-10 cells. Immunoblot showed that StAR expression was efficiently increased and processed in mitochondria (Fig. 4A). Transfection of full-length StAR cDNA in the sense orientation produced a small increase of P4 by mitochondria, as compared to the transfection with the empty vector or the antisense sequence (Fig. 4B, bar a vs. antisense and pRc/CMVi alone). After supplementation of isolated mitochondria with recombinant ERK1, the P4 yield increased (Fig. 4B, bar b vs. a). Addition of an inactive ERK1 mutant (K71A) did not affect steroid production (Fig. 4B, bar c vs. a). K71A lacks the capacity of autophosphorylation and has a reduced phosphotransferase activity [42]. Production of P4 increased more when the isolated mitochondria were supplemented with the recombinant PKA catalytic subunit and wild-type ERK1 than when they were supplemented with wild-type ERK1 alone (Fig. 4B, bar d vs. b). Again, this effect was lost when ERK1 was replaced by its K71A inactive form (Fig. 4B, bar e vs. d). Supplementation with wild-type ERK1, K71A ERK1, with or without the PKA catalytic subunit, did not affect StAR mitochondrial content (Fig. 4A). Also shown in Figure 4A is the mitochondrial content of PKA, pMEK1/2, pERK1/2 and total ERK1/2 after the corresponding incubation. It is worth to mention that in the mitochondrial samples incubated with ERK1-GST (Fig. 4A, lanes b, c, d and e) there is a strong signal of the 44 kDa ERK1, probably due to a cleavage of the fusion protein by mitochondrial proteases rendering the free protein [43]. The use of protease inhibitors is not indicated in this kind of experiment since mitochondrial steroidogenesis is dependent on StAR processing by these proteases [10], [44]. As already demonstrated in the in vivo experiments shown in Figure 3, the addition of PKA increased mitochondrial pMEK1/2 in vitro (Fig. 4A). These results support the possible formation of a large mitochondrial multi-kinase complex which leads to activation in steroid production.


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)

PKA and ERK1/2 are strictly required to achieve maximal progesterone production by isolated mitochondria.MA-10 cells were transiently transfected by electroporation with an empty vector or with a vector containing StAR cDNA (sense or antisense orientations). Mitochondria were incubated in the presence of cholesterol as substrate (a) in the presence of wild type ERK1-GST protein alone (b) or together with PKA catalytic subunit (d). The mutated inactive form of ERK1-GST (K71A) was also used (c and e). After the indicated incubations, mitochondria were pelleted and subjected to SDS-PAGE and Western blot (A). Specific antibodies that recognize StAR protein, the catalytic subunit of PKA, pMEK1/2, pERK1/2 and total ERK1/2 were used. The panel shows representative Western blots of three independently performed experiments. P4 production is shown in (B). Data are expressed as means±SD (n = 3). * p<0.05 bar b vs. bar a and § p<0.05 bar d vs. bar b. (C) MA-10 cells were treated with or without 0.5 mM of 8Br-cAMP for 3 hours; cytosolic and mitochondrial subcellular fractions were obtained and incubated in the presence or absence of human pERK1-GST bound to agarose beads. Input and pelleted proteins bound to pERK1-GST (complexes) were analyzed by SDS-PAGE and Western blot. The immunoblots show the bands corresponding to StAR and pERK1/2, as loading control. Data are representative of three independently performed experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2175533&req=5

pone-0001443-g004: PKA and ERK1/2 are strictly required to achieve maximal progesterone production by isolated mitochondria.MA-10 cells were transiently transfected by electroporation with an empty vector or with a vector containing StAR cDNA (sense or antisense orientations). Mitochondria were incubated in the presence of cholesterol as substrate (a) in the presence of wild type ERK1-GST protein alone (b) or together with PKA catalytic subunit (d). The mutated inactive form of ERK1-GST (K71A) was also used (c and e). After the indicated incubations, mitochondria were pelleted and subjected to SDS-PAGE and Western blot (A). Specific antibodies that recognize StAR protein, the catalytic subunit of PKA, pMEK1/2, pERK1/2 and total ERK1/2 were used. The panel shows representative Western blots of three independently performed experiments. P4 production is shown in (B). Data are expressed as means±SD (n = 3). * p<0.05 bar b vs. bar a and § p<0.05 bar d vs. bar b. (C) MA-10 cells were treated with or without 0.5 mM of 8Br-cAMP for 3 hours; cytosolic and mitochondrial subcellular fractions were obtained and incubated in the presence or absence of human pERK1-GST bound to agarose beads. Input and pelleted proteins bound to pERK1-GST (complexes) were analyzed by SDS-PAGE and Western blot. The immunoblots show the bands corresponding to StAR and pERK1/2, as loading control. Data are representative of three independently performed experiments.
Mentions: To test the direct effect of active ERK1/2 on cholesterol import and progesterone synthesis, we performed a cell-free assay [22], determining progesterone production by isolated mitochondria. To enrich non-stimulated mitochondria with StAR, we transfected StAR cDNA to MA-10 cells. Immunoblot showed that StAR expression was efficiently increased and processed in mitochondria (Fig. 4A). Transfection of full-length StAR cDNA in the sense orientation produced a small increase of P4 by mitochondria, as compared to the transfection with the empty vector or the antisense sequence (Fig. 4B, bar a vs. antisense and pRc/CMVi alone). After supplementation of isolated mitochondria with recombinant ERK1, the P4 yield increased (Fig. 4B, bar b vs. a). Addition of an inactive ERK1 mutant (K71A) did not affect steroid production (Fig. 4B, bar c vs. a). K71A lacks the capacity of autophosphorylation and has a reduced phosphotransferase activity [42]. Production of P4 increased more when the isolated mitochondria were supplemented with the recombinant PKA catalytic subunit and wild-type ERK1 than when they were supplemented with wild-type ERK1 alone (Fig. 4B, bar d vs. b). Again, this effect was lost when ERK1 was replaced by its K71A inactive form (Fig. 4B, bar e vs. d). Supplementation with wild-type ERK1, K71A ERK1, with or without the PKA catalytic subunit, did not affect StAR mitochondrial content (Fig. 4A). Also shown in Figure 4A is the mitochondrial content of PKA, pMEK1/2, pERK1/2 and total ERK1/2 after the corresponding incubation. It is worth to mention that in the mitochondrial samples incubated with ERK1-GST (Fig. 4A, lanes b, c, d and e) there is a strong signal of the 44 kDa ERK1, probably due to a cleavage of the fusion protein by mitochondrial proteases rendering the free protein [43]. The use of protease inhibitors is not indicated in this kind of experiment since mitochondrial steroidogenesis is dependent on StAR processing by these proteases [10], [44]. As already demonstrated in the in vivo experiments shown in Figure 3, the addition of PKA increased mitochondrial pMEK1/2 in vitro (Fig. 4A). These results support the possible formation of a large mitochondrial multi-kinase complex which leads to activation in steroid production.

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