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

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d₀/d₄) 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 d₀/d₄ 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 d₀/d₄ scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser¹⁹ of ZNRF2 (RTRAYpS¹⁹GS), phospho-Ser⁹⁰ of SASH1 (RKRRVpS⁹⁰QD), and phospho- Ser⁴⁹³ of lipolysis-stimulated lipoprotein receptor (RPRARpS⁴⁹³LD) 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.Activated tyrosine kinase receptors generally drive cells to assimilate nutrients; regulate partitioning of the assimilate to make storage polymers and biosynthetic precursors and for energy production; and promote cellular survival, growth, division, movement, and differentiation. From this spectrum, each cell displays a specific subset of responses depending on the hormone, specific receptors, cross-talk from other signaling pathways, metabolic conditions, and cellular complement of effector proteins. For example, insulin stimulates glucose uptake and glycogen synthesis in skeletal muscle, whereas IGF1¹ promotes survival, growth, and proliferation of many cell types (1, 2).Many of these cellular responses are mediated via PI 3-kinase, which generates phosphatidylinositol 3,4,5-trisphosphate, promoting the activation of AGC protein kinases such as PKB/Akt and other signaling components (1, 3). PI 3-kinase is activated by binding to tyrosine-phosphorylated receptors such as the platelet-derived growth factor receptor or via adaptor molecules such as insulin receptor substrates, which are phosphorylated by the activated insulin receptor. Deregulated PI 3-kinase and downstream signaling has been linked to problems with wound healing, immune responses, neurodegeneration, and cardiovascular disease; decreased PI 3-kinase signaling may underlie insulin resistance and type II diabetes; and this pathway is often activated in human tumors (4, 5). To help pinpoint drug targets for these diseases we must define the mechanisms linking PI 3-kinase and other signaling pathways to downstream effectors and understand specificity with respect to different hormone/cell type combinations.Many missing substrates of PI 3-kinase/AGC kinases must be found to explain all the cellular responses to insulin and growth factors (3). Several targets of PI 3-kinase/PKB signaling, including TSC2 (6), PRAS40 (7), AS160 (8), and FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase (9) were identified using the anti-PAS antibody, which loosely recognizes the minimal phosphorylated consensus for PKB, which is RXRXX(pS/pT) where pS is phosphoserine and pT is phosphothreonine. Another helpful feature for identifying new downstream targets is that phosphorylation by PKB sometimes creates binding sites for 14-3-3s, which are dimeric proteins that bind to specific phosphorylated sites on target proteins. Thus PKB promotes the binding of 14-3-3s to proteins including PFKFB2 cardiac PFK-2 (10, 11), BimEL (12), β-catenin (13), p27(Kip1) (14), PRAS40 (7), FOXO1 (15), Miz1 (16), TBC1D4 (AS160 (17, 18), and TBC1D1 (19). Functionally 14-3-3s can trigger changes in the conformations of their targets and alter how targets interact with other proteins. Consistent with 14-3-3/target interactions being important in cellular responses to growth factors and insulin, reagents that compete with targets for binding to 14-3-3s inhibit the IGF1-stimulated increase in the glycolytic stimulator fructose-2,6-bisphosphate (10) and PKB-dependent cell survival (20).Some 14-3-3-binding sites on the above named proteins can also be phosphorylated by other basophilic protein kinases (21). For example, AS160 and TBC1D1 are two related RabGAPs (GTPase-activating protein for Rabs) regulated by multisite phosphorylation that regulate trafficking of GluT4 transporter to the plasma membrane for uptake of glucose. The two 14-3-3-binding sites on AS160 can be phosphorylated by PKB, p90RSK, serum- and glucocorticoid-inducible kinase, and other kinases, whereas one of the 14-3-3-binding sites on TBC1D1 is also a substrate of the energy-sensing kinase AMP-activated protein kinase (17–19). Thus, the relative sensitivity of glucose trafficking to insulin and AMP-activated protein kinase activators in different tissues may depend in part on the distribution of AS160 and TBC1D1. Other insulin-regulated 14-3-3 targets, such as myosin 1C (22), are also convergence points for phosphorylation by more than one AGC and/or Ca²⁺/calmodulin-dependent protein kinase.Here many more proteins than those already identified were found to display 14-3-3 and/or PAS binding signals when the PI 3-kinase pathway was activated in cells against a “background” of other proteins whose 14-3-3 and PAS binding status was unaffected by PI 3-kinase signaling. We aimed to pick out the PI 3-kinase-regulated proteins, which was challenging given the hundreds of 14-3-3 binding partners in mammalian cells (10, 23–27). We used 14-3-3 affinity capture and release, identified phosphopeptides, and devised a quantitative proteomics approach in which 14-3-3-binding proteins from insulin-stimulated versus unstimulated cells were labeled with formaldehyde containing light or heavy isotopes, respectively. Biochemical checking of candidates from these screens, which included proteins with links to diabetes, cancers, and neurodegenerative disorders, confirmed the identification of novel downstream targets of PI 3-kinase, some of which are also convergence points for regulation by MAPK/p90RSK signaling.