Abstract
The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser(58) phosphorylation and 14-3-3γ Lys(49) acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser(58) or Lys(49). Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser(58) phosphorylation and Lys(49) acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser(58) phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys(49) acetylation is important for the cAMP-dependent induction of these interactions.
Highlights
The interaction between 14-3-3␥ and steroidogenic acute regulatory protein (STAR) delays maximal steroidogenesis in a pattern opposite to 14-3-3␥ homodimerization
These results indicate that Ser58 phosphorylation and Lys49 acetylation of 14-3-3␥ occur in a coordinated time-dependent manner to regulate 14-3-3␥ homodimerization. 14-3-3␥ Ser58 phosphorylation is required for STAR interactions under control conditions, and 14-3-3␥ Lys49 acetylation is important for the cAMP-dependent induction of these interactions
Dimerization Versus Target Binding State of 14-3-3␥ Regulates Steroidogenesis—To confirm the previous findings indicating that 14-3-3␥ homodimerization and interactions with STAR regulate steroidogenesis levels at 0 and 120 min or 15– 60 min post-cAMP treatment, respectively [37], dimerization or target binding sites of 14-3-3␥ were competed out using TAT14-3-3␥ 1–10 or transcription protein (TAT)-14-3-3␥ 219 –236, respectively (Fig. 1A)
Summary
The interaction between 14-3-3␥ and steroidogenic acute regulatory protein (STAR) delays maximal steroidogenesis in a pattern opposite to 14-3-3␥ homodimerization. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3␥ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation Taken together, these results indicate that Ser phosphorylation and Lys acetylation of 14-3-3␥ occur in a coordinated time-dependent manner to regulate 14-3-3␥ homodimerization. We investigated the effect of 14-3-3␥ acetylation and/or phosphorylation on STAR binding and 14-3-3␥ homodimerization during steroidogenesis
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