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Intrinsically disordered regions (IDRs) in proteins are often targets of combinatorial posttranslational modifications, which serve to regulate protein structure and function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits, which are essential components of heterotrimeric G proteins, are intrinsically disordered, phosphorylation-dependent determinants of G protein signaling. Here, we found that the yeast Gγ subunit Ste18 underwent combinatorial, multisite phosphorylation events within its N-terminal IDR. G protein–coupled receptor (GPCR) activation and osmotic stress induced phosphorylation at Ser7, whereas glucose and acid stress induced phosphorylation at Ser3, which was a quantitative indicator of intracellular pH. Each site was phosphorylated by a distinct set of kinases, and phosphorylation of one site affected phosphorylation of the other, as determined through exposure to serial stimuli and through phosphosite mutagenesis. Last, we showed that phosphorylation resulted in changes in IDR structure and that different combinations of phosphorylation events modulated the activation rate and amplitude of the downstream mitogen-activated protein kinase Fus3. These data place Gγ subunits among intrinsically disordered proteins that undergo combinatorial posttranslational modifications that govern signaling pathway output.
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Roles of KLF4 and AMPK in the inhibition of glycolysis by pulsatile shear stress in endothelial cells
Vascular endothelial cells (ECs) sense and respond to hemodynamic forces such as pulsatile shear stress (PS) and oscillatory shear stress (OS). Among the metabolic pathways, glycolysis is differentially regulated by atheroprone OS and atheroprotective PS. Studying the molecular mechanisms by which PS suppresses glycolytic flux at the epigenetic, transcriptomic, and kinomic levels, we have demonstrated that glucokinase regulatory protein (GCKR) was markedly induced by PS in vitro and in vivo, although PS down-regulates other glycolysis enzymes such as hexokinase (HK1). Using next-generation sequencing data, we identified the binding of PS-induced Krüppel-like factor 4 (KLF4), which functions as a pioneer transcription factor, binding to the GCKR promoter to change the chromatin structure for transactivation of GCKR. At the posttranslational level, PS-activated AMP-activated protein kinase (AMPK) phosphorylates GCKR at Ser-481, thereby enhancing the interaction between GCKR and HK1 in ECs. In vivo, the level of phosphorylated GCKR Ser-481 and the interaction between GCKR and HK1 were increased in the thoracic aorta of wild-type AMPKα2+/+mice in comparison with littermates with EC ablation of AMPKα2 (AMPKα2−/−). In addition, the level of GCKR was elevated in the aortas of mice with a high level of voluntary wheel running. The underlying mechanisms for the PS induction of GCKR involve regulation at the epigenetic level by KLF4 and at the posttranslational level by AMPK.
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- Award ID(s):
- 1655362
- PAR ID:
- 10228913
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 118
- Issue:
- 21
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- Article No. e2103982118
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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