Multisite Phosphorylation of S6K1 Directs a Kinase Phospho-code that Determines Substrate Selection.

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Signaling

Background:

• Multisite phosphorylation regulates kinase activity but its effect on substrate specificity is not fully understood.
• Ribosomal protein S6 kinase 1 (S6K1) is a key regulator in cellular signaling pathways.

Purpose of the Study:

• To investigate how multisite phosphorylation of S6K1 influences its substrate specificity.
• To elucidate the role of S6K1 phosphorylation in regulating adipocyte lipid metabolism.

Main Methods:

• Investigated S6K1 phosphorylation sites using biochemical assays.
• Analyzed S6K1 target selection through unbiased proteomic analysis.
• Studied the conformational changes in S6K1 upon multisite phosphorylation.

Main Results:

• Multisite phosphorylation of S6K1 at Ser424/Ser429 by Cdk5, in addition to Thr389 by mTORC1, is crucial for high-affinity binding and phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS).
• This specific phosphorylation pattern alters S6K1 substrate specificity, favoring EPRS over canonical targets like ribosomal protein S6.
• Identified coenzyme A synthase, lipocalin 2, and cortactin as novel targets of multisite phosphorylated S6K1 in insulin-stimulated adipocytes.

Conclusions:

• S6K1 possesses a target-selective phospho-code that integrates signals from mTORC1 and Cdk5.
• This phospho-code directs an insulin-stimulated post-translational metabolon that determines adipocyte lipid metabolism.