title: A fluorescent reporter of ATP binding-competent receptor kinases.
abstract: ERBB receptor kinases play a crucial role in normal development and cancer malignancies. A broad range of modifications creates receptor subpopulations with distinct functional properties in live cells. Their apparent activation state, typically assayed by tyrosine phosphorylation of substrates, reflects a complex equilibrium of competing reactions. With the aim of developing optical tools to investigate ERBB populations and their state of activation, we have synthesized a fluorescent 'turn-on' probe, DMAQ, targeting the ERBB ATP binding pocket. Upon binding, probe emission increases due to the hydrophobic environment and restricted geometry of the ERBB2 kinase domain, facilitating the analysis of receptor states at low occupancy and without the removal of unbound probes. Cellular ERBB2 autophosphorylation is inhibited with saturation kinetics that correlate with the increase in probe fluorescence. Thus, DMAQ is an example of a new generation of 'turn-on' probes with potential applications in querying receptor kinase populations both in vitro and in live cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Miami, Miami, FL 33101-6129, United States.
authors.forename: Renaud
authors.initials: R
authors.lastname: Sicard
authors.forename: Jyothi
authors.initials: J
authors.lastname: Dhuguru
authors.forename: Wenjun
authors.initials: W
authors.lastname: Liu
authors.forename: Nirav
authors.initials: N
authors.lastname: Patel
authors.forename: Ralf
authors.initials: R
authors.lastname: Landgraf
authors.forename: James N
authors.initials: JN
authors.lastname: Wilson
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D010766:Phosphorylation; D011799:Quinazolines / Q000737:chemistry / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D000255:Adenosine Triphosphate; D005456:Fluorescent Dyes; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases