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 (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.
Dubois
Fanny
F
Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Vandermoere
Franck
F
Gernez
Aurélie
A
Murphy
Jane
J
Toth
Rachel
R
Chen
Shuai
S
Geraghty
Kathryn M
KM
Morrice
Nick A
NA
MacKintosh
Carol
C
D048948:14-3-3 Proteins / Q000378:metabolism*; D000199:Actins / Q000737:chemistry; D017209:Apoptosis; D001665:Binding Sites; D002853:Chromatography, Liquid / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D014357:Trypsin / Q000737:chemistry
D048948:14-3-3 Proteins; D000199:Actins; D010455:Peptides; D020543:Proteome; D019869:Phosphatidylinositol 3-Kinases; D014357:Trypsin