title: Phosphorylation and function of the hamster adrenal steroidogenic acute regulatory protein (StAR).
abstract: In order to study the effect of phosphorylation on the function of the steroidogenic acute regulatory protein (StAR), 10 putative phosphorylation sites were mutated in the hamster StAR. In pcDNA3.1-StAR transfected COS-1 cells, decreases in basal activity were found for the mutants S55A, S185A and S194A. Substitution of S185 by D or E to mimic phosphorylation resulted in decreased activity for all mutants; we concluded that S185 was not a phosphorylation site and we hypothesized that mutations on S185 created StAR conformational changes resulting in a decrease in its binding affinity for cholesterol. In contrast, the mutation S194D resulted in an increase in StAR activity. We have calculated the relative rate of pregnenolone formation (App. V(max)) in transfected COS-1 cells with wild type (WT) and mutant StAR-pcDNA3.1 under control and (Bu)(2)-cAMP stimulation. The App. V(max) values refer to the rate of cholesterol transported and metabolized by the cytochrome P450scc enzyme present in the inner mitochondrial membrane. The App. V(max) was 1.61 +/- 0.28 for control (Ctr) WT StAR and this value was significantly increased to 4.72 +/- 0.09 for (Bu)(2)-cAMP stimulated preparations. App. V(max) of 5.53 (Ctr) and 4.82 ((Bu)(2)-cAMP) found for S194D StAR preparations were similar to that of the WT StAR stimulated preparations. At equal StAR quantity, an anti-phospho-(S/T) PKA substrate antibody revealed four times more phospho-(S/T) in (Bu)(2)-cAMP than in control preparations. The intensity of phosphorylated bands was decreased for the S55A, S56A and S194A mutants and it was completely abolished for the S55A/S56A/S194A mutant. StAR activity of control and stimulated preparations were diminished by 73 and 72% for the mutant S194A compared to 77 and 83% for the mutant S55A/S56A/S194A. The remaining activity appears to be independent of phosphorylation at PKA sites and could be due to the intrinsic activity of non-phosphorylated StAR or to an artefact due to the pharmacological quantity of StAR expressed in COS-1. In conclusion we have shown that (Bu)(2)-cAMP provokes an augmentation of both the quantity and activity of StAR, and that an enhancement in StAR phosphorylation increases its activity. The increased quantity of StAR upon (Bu)(2)-cAMP stimulation could be due to an augmentation of its mRNA or protein synthesis stability, or both; this is yet to be determined.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4.
authors.forename: Alain
authors.initials: A
authors.lastname: Fleury
authors.forename: Axel P
authors.initials: AP
authors.lastname: Mathieu
authors.forename: Lyne
authors.initials: L
authors.lastname: Ducharme
authors.forename: Dale Buchanan
authors.initials: DB
authors.lastname: Hales
authors.forename: Jean-Guy
authors.initials: JG
authors.lastname: LeHoux
mesh_terms: D000311:Adrenal Glands / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003994:Bucladesine / Q000494:pharmacology; D019556:COS Cells; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011284:Pregnenolone / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D014162:Transfection; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; D003994:Bucladesine; D011284:Pregnenolone; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D017868:Cyclic AMP-Dependent Protein Kinases