A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases.
Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.
Zhang
Chao
C
Department of Cellular and Molecular Pharmacology, University of California San Francisco, 600 16th Street, San Francisco, California 94143-2280, USA.
Kenski
Denise M
DM
Paulson
Jennifer L
JL
Bonshtien
Arale
A
Sessa
Guido
G
Cross
Janet V
JV
Templeton
Dennis J
DJ
Shokat
Kevan M
KM
D019943:Amino Acid Substitution; D020869:Gene Expression Profiling / Q000379:methods*; D014644:Genetic Variation / Q000235:genetics; D023281:Genomics / Q000379:methods* / Q000639:trends; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D025941:Protein Interaction Mapping / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D011494:Protein Kinases