Many protein kinases can be engineered to accept analogs of ATP that are not efficiently used by wild-type kinases. These engineered kinases, which are referred to as "analog-sensitive" or "-as" alleles, are also often sensitive to protein kinase inhibitor variants that do not block the activity of nonmutant kinases. Selective in vitro use of radiolabeled ATP analogs by -as kinases can be exploited to identify the direct phosphorylation targets of individual kinases in complex extracts. In organisms in which it is practical to replace wild-type kinase genes with engineered alleles, the in vivo activity of a -as kinase can be reversibly blocked with an allele-specific inhibitor. Thus, analog-sensitive kinases can be effective tools for discovery of the cellular functions and phosphorylation targets of individual enzymes. A theoretical background for the design and use of these alleles is discussed, as are strategies for construction of candidate -as alleles of any kinase.
|Original language||English (US)|
|Pages (from-to)||Unit 18.11|
|Journal||Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]|
|State||Published - May 2004|
ASJC Scopus subject areas
- Molecular Biology