Phosphoproteomics unveils the signaling dynamics in neuronal cells stimulated with insulin and insulin-like growth factors.

backgroundGiven the role of metabolism in brain health and disease, the investigation of the role of insulin (INS) and insulin-like growth factors (IGFs) as potential therapeutic strategies for neurodegenerative diseases is currently underway. Yet, the signaling pathways associated with INS and IGFs in the brain remain elusive, particularly for the human brain. Unraveling these pathways is critical for harnessing their therapeutic potential in metabolism-associated brain disorders.

methodsThis study employed phosphoproteomics using a human neuroblastoma cell line, SH-SY5Y, to unravel the signaling networks of INS, IGF-1, and IGF-2. Briefly, cells were stimulated for 10 and 60-minutes with the ligands, followed by protein extraction, trypsin digestion, tandem mass tag (TMT) labelling and phosphopeptide enrichment using an immobilized metal affinity chromatography (IMAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The data were processed using R statistical software. Protein annotations were obtained from the UniprotKB database, and pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA).

resultsPhosphoproteomics performed at 10 and 60 min identified 34,358 phosphosites, of which 3,284 were significant at 10 min and 2,374 at 60 min (p.adj < 0.05) across all three ligands. Ligand stimulation induced modulation in phosphorylation at both the receptor level and downstream targets at serine (S), threonine (T), and tyrosine (Y) residues. LIMA1-Y229, a regulator of actin-cytoskeletal function, was the most prominent Y phosphosite across all ligands. IPA identified Rho GTPase as the most significantly enriched pathway, with IGF-1 predominantly driving phosphorylation of Rho GTPase effectors such as Rho guanine nucleotide exchange factors (ARHGEFs), Rho GTPase activating proteins (ARHGAPs) and CDC42. Myocardin-related transcription factor A (MRTFA), a transcriptional target of Rho GTPase, was increased in ligand-stimulated cells at 10 min, and inhibition of the Rho/SRF pathway and PI3K by CCG1423 and wortmannin, respectively, prevented nuclear localization of IGF-1-induced MRTFA.

conclusionsThis study demonstrates that INS, IGF-1, and IGF-2 regulate Rho GTPase and MRTFA activation, thereby contributing to the control of actin cytoskeletal dynamics in neuronal cells. Given the role of INS and IGFs in neuronal survival and neurodegenerative conditions, elucidating these mechanisms is of critical importance, as it offers insights into disease pathogenesis and potential therapeutic targets.