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

Content Provider	PubMed Central
Author	Dubois, Fanny Franck, Vandermoere Gernez, Aurélie Murphy, Jane Toth, Rachel Chen, Shuai Geraghty, Kathryn M. Morrice, Nick A. Mackintosh, Carol
Abstract	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 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.
Related Links	http://dx.doi.org/10.1074/mcp.m800544-mcp200
Ending Page	2499
Page Count	13
Starting Page	2487
File Format	PDF
ISSN	15359476
e-ISSN	15359484
Journal	Molecular & Cellular Proteomics : MCP
Issue Number	11
Volume Number	8
Language	English
Publisher	The American Society for Biochemistry and Molecular Biology
Publisher Date	2009-11-01
Access Restriction	Open
Rights Holder	The American Society for Biochemistry and Molecular Biology
Subject Keyword	Analytical Chemistry Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Medicine Analytical Chemistry Molecular Biology Biochemistry

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