Design and use of analog-sensitive protein kinases.
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.
Blethrow
Justin
J
University of California at San Francisco, San Francisco, California, USA.
Zhang
Chao
C
Shokat
Kevan M
KM
Weiss
Eric L
EL
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000483:Alleles; D015202:Protein Engineering; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis*; D011494:Protein Kinases / Q000737:chemistry*; D011720:Pyrazoles / Q000138:chemical synthesis; D011743:Pyrimidines / Q000138:chemical synthesis; D015003:Yeasts / Q000187:drug effects
C479693:1-tert-butyl-3-naphthalen-1-ylmethyl-1H-pyrazolo(3,4-d)pyrimidin-4-ylemine; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D000255:Adenosine Triphosphate; D011494:Protein Kinases