title: Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles.
abstract: 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.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.|Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Rothenberg
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.|Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Gordon
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.|Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
authors.forename: Forest M
authors.initials: FM
authors.lastname: White
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA, 02142, USA. lourido@wi.mit.edu.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lourido
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D006801:Humans; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D014122:Toxoplasma / Q000201:enzymology*; D057188:Workflow
chemical_list: D002135:Calcium-Binding Proteins; D010750:Phosphoproteins; D015800:Protozoan Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases
keywords: AS kinase; IMAC; LC MS/MS; Quantitative analysis; SILAC; Toxoplasmosis