Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling

• DOI: 10.1074/mcp.m800544-mcp200
• Publication Date: 2009-08-02T00:33:50Z
• Authors: Fanny Dubois; Franck Vandermoere; Aurélie Gernez; Jane Murphy; Rachel Toth; Shuai Chen; Kathryn M. Geraghty; Nick A. Morrice; Carol MacKintosh
• Keywords: Proteomics; Proteome; [SDV]Life Sciences [q-bio]; 610; Apoptosis; Models, Biological; Mass Spectrometry; Phosphatidylinositol 3-Kinases; 03 medical and health sciences; [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN]; [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology; Humans; [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology; Trypsin; 0303 health sciences; Binding Sites; Research; Actins; [SDV] Life Sciences [q-bio]; 14-3-3 Proteins; [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN]; Peptides; Chromatography, Liquid; HeLa Cells; Signal Transduction

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d(0)/d(4)) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d(0)/d(4) values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d(0)/d(4) scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser(19) of ZNRF2 (RTRAYpS(19)GS), phospho-Ser(90) of SASH1 (RKRRVpS(90)QD), and phospho- Ser(493) of lipolysis-stimulated lipoprotein receptor (RPRARpS(493)LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.