Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity.
Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.
Arakane
F
F
Center for Research on Reproduction and Women's Health and the Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
King
S R
SR
Du
Y
Y
Kallen
C B
CB
Walsh
L P
LP
Watari
H
H
Stocco
D M
DM
Strauss
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D013256:Steroids / Q000096:biosynthesis*; D000098682:Steroidogenic Acute Regulatory Protein
D010750:Phosphoproteins; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases