Site-specific NMR Mapping and Time-resolved Monitoring of Serine and Threonine Phosphorylation in Reconstituted Kinase Reactions and Mammalian Cell Extracts

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2013 Jun 29

PMID 23807285

Citations 44

Authors

Francois-Xavier Theillet

Honor May Rose

Stamatios Liokatis

Andres Binolfi

Rossukon Thongwichian

Marchel Stuiver

Philipp Selenko

Affiliations

Soon will be listed here.

Abstract

We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the ¹⁵N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D ¹H-¹⁵N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.

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