title: Post-translational dual regulation of cytochrome P450 aromatase at the catalytic and protein levels by phosphorylation/dephosphorylation.
abstract: The post-translational regulation of aromatase has not been well characterized as compared with transcriptional regulation. Several studies of post-translational regulation have focused on decreases in catalytic activity following phosphorylation. We report here dual post-translational regulation of aromatase, at the catalytic activity and protein levels. Microsomal aromatase prepared from JEG-3 cells was rapidly inactivated and subsequently degraded in the presence of a cytosolic fraction with calcium, magnesium, and ATP. In a reconstituted system consisting of microsomal and cytosolic fractions, aromatase was protected from protein degradation by treatment with alkaline phosphatase, whereas degradation was enhanced by treatment with calcineurin inhibitors (FK506 and cyclosporin A). Furthermore, aromatase was protected from degradation by treatment with kinase inhibitors, especially the calcium/calmodulin kinase inhibitors KN62 and KN93. Similarly to the reconstituted system, aromatase in cultured JEG-3 cells was protected from degradation by KN93, whereas FK503 increased degradation in the presence of cycloheximide, although cellular aromatase mRNA levels were unchanged by these reagents. Knockdown of calcineurin and calcium/calmodulin kinase II (CaMKII) with small interfering RNAs resulted in a dose-dependent increase in aromatase degradation and protection from degradation, respectively. The cytosol fraction-dependent phosphorylation of microsomal aromatase was inhibited by calcineurin, KN62, and KN93, and promoted by CaMKII and FK506. These results indicate that aromatase is regulated acutely at the catalytic activity level and subsequently at the enzyme content level by CaMKII/calcineurin-dependent phosphorylation/dephosphorylation.
authors.affiliation: Department of Biochemistry, School of Medicine, Fujita Health University, Toyoake, Aichi, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Hayashi
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Harada
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000494:pharmacology; D001141:Aromatase / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D002384:Catalysis / Q000187:drug effects; D045744:Cell Line, Tumor; D002474:Cell-Free System; D002822:Choriocarcinoma / Q000473:pathology; D016572:Cyclosporine / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D005260:Female; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology
chemical_list: D009363:Neoplasm Proteins; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D001141:Aromatase; C561410:CYP19A1 protein, human; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D000469:Alkaline Phosphatase; D008274:Magnesium; D002118:Calcium; D016559:Tacrolimus
keywords: aromatase; calcineurin; calcium/calmodulin kinase II; phosphorylation; post-translational regulation