(P20-039-25) P300 Enhances The Phosphorylation Of IRS-1 S1101 By P70 S6K-1

Objectives: Type II diabetes, a global epidemic affecting over five hundred million people, thrives in the wake of modern sedentary lifestyles and excessive caloric intake. Characterized by obesity and insulin resistance, on the cellular level this disease presents with the dysregulation of IRS1 (Insulin Receptor Substrate 1), driven by regulatory kinases. While IRS1 dysregulation is caused by excessive serine phosphorylation, studies now show that histone acetyltransferase p300 can excessively inhibit IRS1 especially in HFD (high fat diet) fed mice leading to the overexpression and increased activity of p300 and resulting in the dysregulation of IRS1. The implication this has to the excessive serine phosphorylation is not known. The first aim narrows down which of the three regulatory phosphorylation sites (IRS1 S1101, S312, and S636/639) are specific to S6K1. The second aim explores the impact of p300-mediated acetylation on the phosphorylation status of IRS1 at regulatory sites targeted by S6K1(Ribosomal protein S6 kinase beta 1).

Methods: Insulin stimulated HepG2 cells were treated with PF4708671, a S6K1 inhibitor, or C646, a p300 inhibitor, to assess which phosphorylation sites are S6K1 targets and how the phosphorylation status of these sites changes in the absence of p300. Gel electrophoresis and western blots were run to visualize the findings. Protein assays were performed to understand how the acetylation and phosphorylation status changed when either active S6K1 or p300 were supplemented. Each experiment had an N of three.

Results: Inhibiting S6K-1 resulted in a dose-dependent reduction in the phosphorylation at S312 and S1101, but not at S636. Similarly, treatment with C646 significantly decreased phosphorylation at S312 and S1101 while upregulating insulin-stimulated ribosomal protein S6. p300 activity enhanced S1101 phosphorylation, while S6K1-mediated phosphorylation amplified IRS1 acetylation, underscoring a complex interplay between these modifications.

Conclusions: p300 inhibition reduces phosphorylation at critical negative regulatory sites, highlighting its involvement as an accomplice to inhibitory phosphorylation of IRS1. Understanding these regulatory interactions provides valuable insights into mitigating excessive IRS1 serine phosphorylation to discover new forms of treatment.

Funding Sources: Liberty University