Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function.
Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.
Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Anhco
A
Nguyen
Deborah M
DM
Rothman
Justine
J
Stehn
Barbara
B
Imperiali
Michael B
MB
Yaffe
D048948:14-3-3 Proteins / Q000378:metabolism*; D017209:Apoptosis; D045744:Cell Line, Tumor; D002470:Cell Survival; D016193:G1 Phase; D006801:Humans; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology*; D010748:Phosphopeptides / Q000378:metabolism* / Q000528:radiation effects; D010777:Photochemistry / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism / Q000528:radiation effects; D016196:S Phase; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013997:Time Factors; D014466:Ultraviolet Rays
D048948:14-3-3 Proteins; D010748:Phosphopeptides; D011994:Recombinant Proteins