14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.
14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3-Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-D-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.
Pozuelo Rubio
Mercedes
M
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Geraghty
Kathryn M
KM
Wong
Barry H C
BH
Wood
Nicola T
NT
Campbell
David G
DG
Morrice
Nick
N
Mackintosh
Carol
C
D048948:14-3-3 Proteins; D001667:Binding, Competitive; D015153:Blotting, Western; D002455:Cell Division; D002846:Chromatography, Affinity; D004798:Enzymes / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D008387:Marine Toxins; D008660:Metabolism; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification; D010080:Oxazoles / Q000494:pharmacology; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification*; D021381:Protein Transport; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism* / Q000502:physiology
D048948:14-3-3 Proteins; D004798:Enzymes; D008387:Marine Toxins; D008869:Microtubule-Associated Proteins; D010080:Oxazoles; D010748:Phosphopeptides; D010750:Phosphoproteins; C059041:calyculin A; D014446:Tyrosine 3-Monooxygenase