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 o f cholesterol to the inner mitochondrial membrane where P450scc reside s, Tropic hormones acting through the intermediacy of cAMP rapidly inc rease pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action, The StAR protein contains two co nsensus sequences for phosphorylation catalyzed by protein kinase A th at are conserved across all species in which the amino acid sequence o f the StAR protein has been determined, We demonstrated that human StA R expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting, T he two more acidic species disappeared after treatment of the cell ext racts with alkaline phosphatase, P-32 was incorporated into StAR prote in immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased P-32 incorporation into the StAR preprote in, StAR protein generated by in vitro transcription/translation was p hosphorylated by the protein kinase A catalytic subunit in the presenc e of [gamma-P-32]ATP. Mutation of potential sites for protein kinase A -mediated phosphorylation at serine 57 and serine 195 to alanines, ind ividually, reduced P-32 incorporation from labeled ATP into StAR prepr otein produced by in vitro transcription/translation when incubated wi th protein kinase A catalytic subunit, P-32 labeling of StAR protein e xpressed 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 P-32 in corporation, To determine the functional significance of StAR phosphor ylation, we tested the steroidogenic activity of the wild-type StAR an d mutated StAR proteins in COS-1 cells expressing the human cholestero l side chain cleavage enzyme system, Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenol one synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production, The S19 5A mutant construct did not yield the more acidic species of StAR dete cted in two-dimensional Western blots, indicating that the mutation af fected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser(56) and Ser(194)) 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 huma n S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea tha t a negative charge at this site modulates StAR function, Our observat ions suggest that phosphorylation of serine 194/195 increases the biol ogical activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid produc tion.