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Article Dans Une Revue Molecular and Cellular Proteomics Année : 2009

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

Aurélie Gernez
  • Fonction : Auteur
Jane Murphy
  • Fonction : Auteur
Rachel Toth
  • Fonction : Auteur
Shuai Chen
  • Fonction : Auteur
Kathryn Geraghty
  • Fonction : Auteur
Nick Morrice
  • Fonction : Auteur
Carol Mackintosh
  • Fonction : Auteur
Fanny Dubois
  • Fonction : Auteur
Jane Auré Lie Gernez
  • Fonction : Auteur
Rachel Murphy
  • Fonction : Auteur
Shuai Toth
  • Fonction : Auteur
Kathryn M Chen
  • Fonction : Auteur
Nick A Geraghty
  • Fonction : Auteur
Carol Morrice
  • Fonction : Auteur

Résumé

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.
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Dates et versions

hal-03030169 , version 1 (29-11-2020)

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Aurélie Gernez, Jane Murphy, Rachel Toth, Shuai Chen, Kathryn Geraghty, et al.. Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling. Molecular and Cellular Proteomics, 2009, 8 (11), pp.2487-2499. ⟨10.1074/mcp.M800544-MCP200⟩. ⟨hal-03030169⟩
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