Unit

UNIT 18.11 Design and Use of Analog-Sensitive Protein Kinases

  1. Justin Blethrow1,
  2. Chao Zhang1,
  3. Kevan M. Shokat1,
  4. Eric L. Weiss2

Published Online: 1 MAY 2004

DOI: 10.1002/0471142727.mb1811s66

Current Protocols in Molecular Biology

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.

Author Information

  1. 1

    University of California at San Francisco, San Francisco, California

  2. 2

    Northwestern University, Evanston, Illinois

Publication History

  1. Published Online: 1 MAY 2004
  2. Published Print: APR 2004

Abstract

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.