Harnessing the power of chemical genetics: A measure of cellular EGFR inhibition with tyrosine kinase affinity probes

BIOL 208

Jimmy A. Blair, blairj@berkeley.edu, Department of Chemistry, University of California, Berkeley, 600 16th Street, MC 2280, Genentech Hall, Room N508, San Francisco, CA 94158-2280 and Kevan M. Shokat, shokat@cmp.ucsf.edu, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 600 16th Street, Box 2280, Genentech Hall, Room N512D, San Francisco, CA 94143-2280.
While small-molecule kinase inhibitors offer a powerful method for interrogating the contributions of protein kinases to cellular signaling events, the structural conservation among kinase active sites and the dynamic nature of kinase signaling cascades presents practical challenges for cellular signaling studies. Leveraging the tools of chemical genetics, we developed a tyrosine kinase affinity probe capable of reporting the in vivo activity of EGFR using allele-specific irreversible inhibitors that target rationally designed kinases bearing two selectivity elements not found together in any wild-type kinase: an electrophile-targeted cysteine residue and a glycine gatekeeper residue. Cocrystal structures of two inhibitors with both EGFR and an engineered c-Src kinase confirmed this design strategy. Based on these structures, we developed a fluorescent affinity probe to report the fraction of kinase necessary for cellular signaling and utilized these reagents to quantitate the relationship between EGFR stimulation by EGF and its downstream outputs, Akt and Erk1/2.

 

Frontiers in Chemical Biology
5:00 PM-7:00 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Biological Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007