Affinity methods for phosphorylation-dependent interactions.
14-3-3s are a highly conserved protein family that exert many regulatory roles in eukaryotic cells by binding to phosphopeptide motifs in diverse target proteins. Here, we describe 14-3-3 affinity binding procedures that can be used to purify and identify 14-3-3-binding phosphoproteins; monitor how their phosphorylation and 14-3-3 binding is regulated by extracellular stimuli; define the functional effects of 14-3-3s on individual targets; and identify relevant protein phosphatases and kinases. In principle, these methods could be adapted to characterize other types of protein-protein interaction that depend on covalent modification of target sites.
Moorhead
Greg
G
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
MacKintosh
Carol
C
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D010748:Phosphopeptides; D014446:Tyrosine 3-Monooxygenase