Comprehensive insulin receptor phosphorylation dynamics profiled by mass spectrometry. (13th December 2021)

• Record Type: Journal Article
• Authors: Liao, Zhongping; Zhang, Chen; Ding, Liyun; Moyers, Julie S.; Tang, Jason X.; Beals, John M.
• Is Part Of: FEBS journal. Volume 289:Number 9(2022)
• Page Start: 2657
• Page End: 2671
• Publication Date: 2021-12-13
• Subjects: insulin receptor endocytosis -- insulin receptor phosphorylation -- mass spectrometry; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.16299
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : Insulin receptor (IR) phosphorylation is critical for the assessment of the extent of IR agonism and nuances in the downstream signaling cascade. A thorough identification and monitoring of the phosphorylation events is important for understanding the process of insulin signaling transduction and regulation. Although IR phosphorylation has been studied extensively in the past decades, only a handful of phosphorylation sites can be identified by either traditional antibody‐based assays or recent large‐scale mass spectrometry‐based phosphoproteomics approaches. In the present study, the most exhaustive assessment of the IR phosphorylation was conducted using nano‐liquid chromatography‐tandem mass spectrometry, in which 13 IR phosphorylation sites and 22 combinations thereof were analyzed. The kinetic analysis included Y965, Y972, S968/969, and S974/976 in the juxtamembrane region; Y1158, Y1162, and Y1163 in the kinase domain; and Y1328, Y1334, S1278, S1320, S1321, and T1348 in the C‐terminal region. Employing two different receptor agonists (i.e. insulin and an IR peptide agonist), the data revealed contrasting phosphorylation kinetics across these sites with dynamics far more diverse than expected for known IR agonists. Notably, cell trafficking experiments revealed that the IR peptide agonist was incapable of inducing IR to the early endosome, which is probably linked to a difference in IR phosphorylation. The present study provides a powerful tool for investigating IR signaling and trafficking that will benefit the design of IR agonists with improved therapeutic utility. Abstract : We present a comprehensive assessment of insulin receptor phosphorylation kinetics across 13 phosphorylation sites and 22 combinations thereof using mass spectrometry. This analysis revealed contrasting phosphorylation kinetics between insulin and insulin receptor peptide agonist, probably linked to differential intracellular trafficking pathways of insulin receptor. The present study provides a powerful tool for investigating insulin receptor signaling and trafficking that will benefit the design of insulin receptor agonists with improved therapeutic utility.