Chemoselective One-Step Alkylation and Isolation of Thiophosphorylated Cdk2 Substrates in the Presence of Native Cysteine

• Published in: Chembiochem : a European journal of chemical biology Vol. 12; no. 4; pp. 633 - 640
• Main Authors: Lee, Sarah E, Elphick, Lucy M, Kramer, Holger B, Jones, Alexandra M.E, Child, Emma S, Anderson, Alexandra A, Bonnac, Laurent, Suwaki, Natsuko, Kessler, Benedikt M, Gouverneur, Véronique, Mann, David J
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 07-03-2011; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Alkylation; ATP; cdk2; chemical genetics; Chlorides - chemistry; cyclin; Cyclin-Dependent Kinase 2 - chemistry; Cyclin-Dependent Kinase 2 - genetics; Cysteine - chemistry; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; kinases; Peptides - chemistry; Peptides - genetics; Phosphorus Compounds - chemistry; Signal Transduction; substrate isolation; Substrate Specificity; thiophosphorylation
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201000528

The elucidation of signalling pathways relies heavily upon the identification of protein kinase substrates. Recent investigations have demonstrated the efficacy of chemical genetics using ATP analogues and modified protein kinases for specific substrate labelling. Here we combine N⁶-(cyclohexyl)ATPγS with an analogue-sensitive cdk2 variant to thiophosphorylate its substrates and demonstrate a pH-dependent, chemoselective, one-step alkylation to facilitate the detection or isolation of thiophosphorylated peptides.