Abstract
The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.
Highlights
Trophic hormone activation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major mediator of steroidogenesis in the adrenal and gonads
We previously reported that the Kin-8 cells express 50% of the steroidogenic acute regulatory protein (STAR) mRNA and protein compared to Y1 cells and that 8Br-cAMP-treatment resulted in a similar fold-increase in STAR
These data demonstrate for the first time that newly synthesized STAR is not phosphorylated in Kin-8 cells
Summary
Trophic hormone activation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major mediator of steroidogenesis in the adrenal and gonads. The cAMP-PKA pathway activates cholesterol delivery to the mitochondrial inner membrane for the first step in steroid biosynthesis and increases transcriptional activation of genes encoding steroidogenic enzymes (reviewed in [1]). The transport of cholesterol into mitochondria is the rate-limiting step in steroidogenesis and this step is dependent upon the expression and function of the steroidogenic acute regulatory protein (STAR, encoded by the STARD1 gene) [2,3]. Models of STAR-mediated cholesterol transport mechanisms have been reviewed and implicit in these models is the requirement for continued synthesis of precursor STAR to sustain the transient interactions of the newly synthesized protein with the mitochondria outer membrane (reviewed in [7,8,9])
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