title: Estrogen receptor β and its domains interact with casein kinase 2, phosphokinase C, and N-myristoylation sites of mitochondrial and nuclear proteins in mouse brain.
abstract: The localization of estrogen receptor (ER)β in mitochondria suggests ERβ-dependent regulation of genes, which is poorly understood. Here, we analyzed the ERβ interacting mitochondrial as well as nuclear proteins in mouse brain using pull-down assay and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In the case of mitochondria, ERβ interacted with six proteins of 35-152 kDa, its transactivation domain (TAD) interacted with four proteins of 37-172 kDa, and ligand binding domain (LBD) interacted with six proteins of 37-161 kDa. On the other hand, in nuclei, ERβ interacted with seven proteins of 30-203 kDa, TAD with ten proteins of 31-160 kDa, and LBD with fourteen proteins of 42-179 kDa. For further identification, these proteins were cleaved by trypsin into peptides and analyzed by MALDI-MS using mascot search engine, immunoprecipitation, immunoblotting, and far-Western blotting. To find the consensus binding motifs in interacting proteins, their unique tryptic peptides were analyzed by the motif scan software. All the interacting proteins were found to contain casein kinase (CK) 2, phosphokinase (PK)C phosphorylation, and N-myristoylation sites. These were further confirmed by peptide pull-down assays using specific mutations in the interacting sites. Thus, the present findings provide evidence for the interaction of ERβ with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling.
authors.affiliation: Biochemistry and Molecular Biology Laboratory, Centre of Advanced Study, Department of Zoology, Banaras Hindu University, Varanasi-221 005, India.
authors.forename: Vijay
authors.initials: V
authors.lastname: Paramanik
authors.forename: Mahendra Kumar
authors.initials: MK
authors.lastname: Thakur
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D047390:Casein Kinase II / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D047629:Estrogen Receptor beta / Q000378:metabolism*; D005786:Gene Expression Regulation; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000737:chemistry*; D009446:Neurobiology / Q000379:methods; D011493:Protein Kinase C / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D047629:Estrogen Receptor beta; D011994:Recombinant Proteins; D019814:Myristic Acid; D047390:Casein Kinase II; D011493:Protein Kinase C