TY - JOUR
T1 - Dual-color click beetle luciferase heteroprotein fragment complementation assays
AU - Villalobos, Victor
AU - Naik, Snehal
AU - Bruinsma, Monique
AU - Dothager, Robin S.
AU - Pan, Mei Hsiu
AU - Samrakandi, Mustapha
AU - Moss, Britney
AU - Elhammali, Adnan
AU - Piwnica-Worms, David
N1 - Funding Information:
The authors thank A. Pichler-Wallace for performing hydrodynamic injections, S. Gammon for assistance with spectral unmixing of multi-colored luciferases, and Scott Harpstrite for figure preparation. The study was funded by National Institutes of Health grant P50 CA94056. This work also was supported by the Medical Scientist Training Program (V.V.), the Cancer Biology Pathway training program through the Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine (S.N., M.-H.P.), a National Science Foundation Graduate Award (B.M.), and an American Cancer Society Post-doctoral Fellowship Award (R.S.D.).
PY - 2010/9/24
Y1 - 2010/9/24
N2 - Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically relevant time scales. Herein, we describe a set of reversible multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discrete pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells.
AB - Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically relevant time scales. Herein, we describe a set of reversible multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discrete pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells.
UR - http://www.scopus.com/inward/record.url?scp=77956946268&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2010.06.018
DO - 10.1016/j.chembiol.2010.06.018
M3 - Article
C2 - 20851351
AN - SCOPUS:77956946268
SN - 1074-5521
VL - 17
SP - 1018
EP - 1029
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 9
ER -