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Engineering the serine/threonine protein kinase Raf‐1 to utilise an orthogonal analogue of ATP substituted at the N 6 position
Author(s) -
Hindley Alison D,
Park Sungdae,
Wang Lily,
Shah Kavita,
Wang Yanli,
Hu Xiche,
Shokat Kevan M,
Kolch Walter,
Sedivy John M,
Yeung Kam C
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01352-8
Subject(s) - kinase , biochemistry , c raf , mitogen activated protein kinase kinase , threonine , protein kinase a , map2k7 , serine , map kinase kinase kinase , mutagenesis , phosphorylation , biology , chemistry , microbiology and biotechnology , cyclin dependent kinase 2 , mutant , gene
One key area of protein kinase research is the identification of cognate substrates. The search for substrates is hampered by problems in unambiguously assigning substrates to a particular kinase in vitro and in vivo. One solution to this impasse is to engineer the kinase of interest to accept an ATP analogue which is orthogonal (unable to fit into the ATP binding site) for the wild‐type enzyme and the majority of other kinases. The acceptance of structurally modified, γ‐ 32 P‐labelled, nucleotide analogue by active site‐modified kinase can provide a unique handle by which the direct substrates of any particular kinase can be displayed in crude mixtures or cell lysates. We have taken this approach with the serine/threonine kinase Raf‐1, which plays an essential role in the transduction of stimuli through the Ras→Raf→MEK→ERK/MAP kinase cascade. This cascade plays essential roles in proliferation, differentiation and apoptosis. Here we detail the mutagenesis strategy for the ATP binding pocket of Raf‐1, such that it can utilise an N 6 ‐substituted ATP analogue. We show that these mutations do not alter the substrate specificity and signal transduction through Raf‐1. We screen a library of analogues to identify which are orthogonal for Raf‐1, and show that mutant Raf‐1 can utilise the orthogonal analogue N 6 (2‐phenethyl) ATP in vitro to phosphorylate its currently only accepted substrate MEK. Importantly we show that our approach can be used to tag putative direct substrates of Raf‐1 kinase with 32 P‐ N 6 (2‐phenethyl) ATP in cell lysates.