UNIT 18.11 Design and Use of Analog-Sensitive Protein Kinases
Published Online: 1 MAY 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Blethrow, J., Zhang, C., Shokat, K. M. and Weiss, E. L. 2004. Design and Use of Analog-Sensitive Protein Kinases. Current Protocols in Molecular Biology. 66:18.11:18.11.1–18.11.19.
- Published Online: 1 MAY 2004
- Published Print: APR 2004
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.