Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles.
Identifying the substrates of protein kinases remains a major obstacle in the elucidation of eukaryotic signaling pathways. Promiscuity among kinases and their substrates coupled with the extraordinary plasticity of phosphorylation networks renders traditional genetic approaches or small-molecule inhibitors problematic when trying to determine the direct substrates of an individual kinase. Here we describe methods to label, enrich, and identify the direct substrates of analogue-sensitive kinases by exploiting their steric complementarity to artificial ATP analogues. Using calcium-dependent protein kinases of Toxoplasma gondii as a model for these approaches, this protocol brings together numerous advances that enable labeling of kinase targets in semi-permeabilized cells, quantification of direct labeling over background, and highly specific enrichment of targeted phosphopeptides.
['Rothenberg|Daniel A|DA|', 'Gordon|Elizabeth A|EA|', 'White|Forest M|FM|', 'Lourido|Sebastian|S|']
D000255:Adenosine Triphosphate D002135:Calcium-Binding Proteins D002460:Cell Line D002851:Chromatography, High Pressure Liquid D056148:Chromatography, Reverse-Phase D006801:Humans D010750:Phosphoproteins D010766:Phosphorylation D011494:Protein Kinases D011499:Protein Processing, Post-Translational D040901:Proteomics D015800:Protozoan Proteins D013379:Substrate Specificity D053719:Tandem Mass Spectrometry D014122:Toxoplasma D057188:Workflow
AS kinase IMAC LC MS/MS Quantitative analysis SILAC Toxoplasmosis