Dissection of the insulin signaling pathway via quantitative phosphoproteomics

Abstract

The insulin signaling pathway is of pivotal importance in metabolic diseases, such as diabetes, and in cellular processes, such as aging. Insulin activates a tyrosine phosphorylation cascade that branches to create a complex network affecting multiple biological processes. To understand the full spectrum of the tyrosine phosphorylation cascade, we have defined the tyrosine-phosphoproteome of the insulin signaling pathway, using high resolution mass spectrometry in combination with phosphotyrosine immunoprecipitation and stable isotope labeling by amino acids in cell culture (SILAC) in differentiated brown adipocytes. Of 40 identified insulininduced effectors, 7 have not previously been described in insulin signaling, including SDR, PKC␦ binding protein, LRP-6, and PISP/ PDZK11, a potential calcium ATPase binding protein. A proteomic interaction screen with PISP/PDZK11 identified the calcium transporting ATPase SERCA2, supporting a connection to calcium signaling. The combination of quantitative phosphoproteomics with cell culture models provides a powerful strategy to dissect the insulin signaling pathways in intact cells.diabetes ͉ insulin action ͉ tyrosine phosphorylation R eversible phosphorylation is a major regulatory mechanism controlling the activity of proteins. Many signaling pathways, including the insulin/IGF-1 signaling pathway, transduce signals from the cell surface to downstream targets via tyrosine kinases and phosphatases (1). Insulin or IGF-1 binding initiates a complex cascade of events, starting with phosphorylation of specific tyrosine residues on the insulin and the IGF-1 receptors (2). Once activated, these receptors phosphorylate a number of docking proteins; the best characterized are the insulin receptor substrate (IRS) proteins 1-4 (3). IRS 1-4 interact with other intracellular signaling molecules primarily through SH2 domains leading to activation of several downstream pathways. These in turn coordinate and regulate vesicle trafficking, protein synthesis, and glucose uptake. The insulin receptor and its substrates, therefore, constitute the first critical node in the insulin signaling network (1) and thus define the full set of proteins that are tyrosine phosphorylated upon insulin stimulation and provide information that is central to determining the molecules involved in this signaling network.Mass spectrometry (MS)-based proteomics has become increasingly powerful not only to identify complex protein mixtures but also regulated protein modifications (4). We have studied tyrosine phosphorylated effectors and interactors in the EGF pathway (5, 6) and others have used MS to study tyrosine phosphorylation of the insulin pathway in white adipocytes (7). To quantify the time course of stimulation, we employ stable isotope labeling with amino acids in cell culture (SILAC) (8). Labeling three cell states by SILAC allows measuring a time course of activation of tyrosine phosphorylation by quantifying the relative protein amounts after antiphosphotyrosine immunoprecipitation. Here, we quanti...

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

“…Immunoaffinity purification based on PTMs such as phosphorylation, ubiquitylation, and acetylation allows the enrichment of proteins targeted for such modification. The combination of such purification with online LC-MS/MS analysis then allows the identification of large sets of proteins with specific PTMs [3][4][5][6][7][8][9][10][11][12]. Furthermore, methods for tagging with stable isotopes, such as stable isotope labeling in cell culture (SILAC), ICAT, or iTRAQ, provide quantitative information about proteins or peptides from MS or MS/MS spectra [13][14][15][16].…”

“…Furthermore, methods for tagging with stable isotopes, such as stable isotope labeling in cell culture (SILAC), ICAT, or iTRAQ, provide quantitative information about proteins or peptides from MS or MS/MS spectra [13][14][15][16]. Indeed, the application of these quantitative methods to studies of proteins phosphorylated on tyrosine has identified many new substrates of tyrosine kinases [11,12,17].…”

High Precision Quantitative Proteomics Using iTRAQ on an LTQ Orbitrap: A New Mass Spectrometric Method Combining the Benefits of All

“…[10][11][12] Furthermore, quantitative techniques have also been employed for quantifying post-translational modifications such as phosphorylation. 13 However, mass spectrometry is not quantitative per se. The signal strength corresponding to the individual peptide ions recorded in the mass spectrometer does not solely depend on the concentration of the peptides, but also on many other partly uncontrollable parameters such as signal suppression effects.…”

Temporal and spatial profiling of nuclei-associated proteins upon TNF-α/NF-κB signaling

“…This indicated the SILAC results could measure changes in protein levels in our study. The relative standard deviation (%SD) of all the proteins in different fractions showed a quantitation precision better than 15% (Supplementary information, Figure S2B) [23]. Therefore, 1.5 was chosen as the conservative threshold for a significant ratio change during TNF-α stimulation in the present study.…”