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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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Predictive model of the molecular interaction between ERK1/2 and StAR.The reconstruction of molecular interaction of ERK2 and StAR was performed using PyMOL (DeLano Scientific, USA; www.delanoscientific.com). ERK2 (Protein Data Bank code 2GPH) is represented in blue and StAR (START domain in StartD4 from Mus musculus) in green. In this model, StAR is located in the docking groove of ERK2. The active center of ERK2 is in dark red. The CD domain of ERK2, represented in yellow, includes Asp316 and Asp319 in contact with the D domain of StAR. Lys174 and Lys176, corresponding to Lys235 and Lys237 of StAR sequence in Mus musculus are represented in orange, and Ser171, corresponding to Ser232 of murine StAR, in dark pink.
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pone-0001443-g006: Predictive model of the molecular interaction between ERK1/2 and StAR.The reconstruction of molecular interaction of ERK2 and StAR was performed using PyMOL (DeLano Scientific, USA; www.delanoscientific.com). ERK2 (Protein Data Bank code 2GPH) is represented in blue and StAR (START domain in StartD4 from Mus musculus) in green. In this model, StAR is located in the docking groove of ERK2. The active center of ERK2 is in dark red. The CD domain of ERK2, represented in yellow, includes Asp316 and Asp319 in contact with the D domain of StAR. Lys174 and Lys176, corresponding to Lys235 and Lys237 of StAR sequence in Mus musculus are represented in orange, and Ser171, corresponding to Ser232 of murine StAR, in dark pink.

Mentions: It was confirmed here that pERK1/2 in mitochondria have a functional interaction with StAR, MEK1/2 and PKA, thus forming a mitochondrial multi-complex. On the basis of crystallographic analysis, acidic and hydrophobic patches in the ERK1/2 structure (the CD domain) were described [46]. For instance, in the model of interaction between a peptide that belongs to MKP3 (MAP kinase phosphatase 3) and ERK2 [45], Asp316 and Asp319 of the ERK2 CD domain were observed to interact with Arg20 and Arg21 of the representative MKP3 peptide (R20R21GSNVALML, the D domain), an interaction putatively attributed in the present case to Lys235 and Lys237 of StAR (K235TK237LTWLLSI). Based on a computerized model of the ERK2-StAR complex (Fig. 6), we found a possible interaction between the ε-amine group of Lys235 in StAR structure and the carboxylic group of Asp319 in ERK2 structure, separated by 11 Å. As in MKP3, other interactions of the hydrophobic motifs, underlined in the partial sequences, are expected to stabilize the StAR binding to the ERK docking groove [45].


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)

Predictive model of the molecular interaction between ERK1/2 and StAR.The reconstruction of molecular interaction of ERK2 and StAR was performed using PyMOL (DeLano Scientific, USA; www.delanoscientific.com). ERK2 (Protein Data Bank code 2GPH) is represented in blue and StAR (START domain in StartD4 from Mus musculus) in green. In this model, StAR is located in the docking groove of ERK2. The active center of ERK2 is in dark red. The CD domain of ERK2, represented in yellow, includes Asp316 and Asp319 in contact with the D domain of StAR. Lys174 and Lys176, corresponding to Lys235 and Lys237 of StAR sequence in Mus musculus are represented in orange, and Ser171, corresponding to Ser232 of murine StAR, in dark pink.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2175533&req=5

pone-0001443-g006: Predictive model of the molecular interaction between ERK1/2 and StAR.The reconstruction of molecular interaction of ERK2 and StAR was performed using PyMOL (DeLano Scientific, USA; www.delanoscientific.com). ERK2 (Protein Data Bank code 2GPH) is represented in blue and StAR (START domain in StartD4 from Mus musculus) in green. In this model, StAR is located in the docking groove of ERK2. The active center of ERK2 is in dark red. The CD domain of ERK2, represented in yellow, includes Asp316 and Asp319 in contact with the D domain of StAR. Lys174 and Lys176, corresponding to Lys235 and Lys237 of StAR sequence in Mus musculus are represented in orange, and Ser171, corresponding to Ser232 of murine StAR, in dark pink.
Mentions: It was confirmed here that pERK1/2 in mitochondria have a functional interaction with StAR, MEK1/2 and PKA, thus forming a mitochondrial multi-complex. On the basis of crystallographic analysis, acidic and hydrophobic patches in the ERK1/2 structure (the CD domain) were described [46]. For instance, in the model of interaction between a peptide that belongs to MKP3 (MAP kinase phosphatase 3) and ERK2 [45], Asp316 and Asp319 of the ERK2 CD domain were observed to interact with Arg20 and Arg21 of the representative MKP3 peptide (R20R21GSNVALML, the D domain), an interaction putatively attributed in the present case to Lys235 and Lys237 of StAR (K235TK237LTWLLSI). Based on a computerized model of the ERK2-StAR complex (Fig. 6), we found a possible interaction between the ε-amine group of Lys235 in StAR structure and the carboxylic group of Asp319 in ERK2 structure, separated by 11 Å. As in MKP3, other interactions of the hydrophobic motifs, underlined in the partial sequences, are expected to stabilize the StAR binding to the ERK docking groove [45].

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