Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles

Methods Mol Biol. 2016;1355:71-84. doi: 10.1007/978-1-4939-3049-4_5.

Rothenberg DA1,2, Gordon EA1,2, White FM1,2, Lourido S3.

Author information

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. 

KEYWORDS: 

AS kinase; IMAC; LC MS/MS; Quantitative analysis; SILAC; Toxoplasmosis

PMID:

 

26584919

 

[PubMed - in process]