Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction in mammalian cells.
The ability to make sensitive measurements of protein-protein interaction kinetics in single neurons is critical for understanding the molecular and cellular basis of neuronal function. We have developed a reporter technology based on the differential induction of Escherichia coli TEM-1 beta-lactamase (Bla) enzymatic activity that can function as a sensor of the interaction state of two target proteins within single neurons in vivo. To modulate Bla enzymatic activity, we first split the enzyme into two separate, complementary protein fragments that we identified by using a functional screening approach based on circular permutation of the Bla enzyme. The split enzyme was then brought together by the phosphorylation-dependent association of the kinase inducible domain of the cAMP response element binding protein (CREB) and the KIX domain of the CREB binding protein. Using an intracellular substrate whose fluorescence spectrum changes after hydrolysis by Bla, we performed time-lapse ratiometric imaging measurements of Bla enzymatic induction after association of the CREB and CREB binding protein interaction domains. This approach permits direct imaging of protein-protein interactions in single cells with high signal discrimination.
Spotts
James M
JM
Division of Neuroscience, John F. Enders Pediatric Laboratories, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
Dolmetsch
Ricardo E
RE
Greenberg
Michael E
ME
D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000502:physiology; D004798:Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D018715:Microscopy, Video; D008959:Models, Neurological; D009474:Neurons / Q000382:microbiology* / Q000502:physiology*; D010766:Phosphorylation; D013997:Time Factors; D014162:Transfection; D001618:beta-Lactamases / Q000378:metabolism
D004798:Enzymes; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1