共 31 条
Time-lapse imaging of a dynamic phosphorylation dependent protein-protein interaction in mammalian cells
被引:55
作者:
Spotts, JM
Dolmetscht, RE
Greenberg, ME
机构:
[1] Childrens Hosp, John F Enders Pediat Labs, Div Neurosci, Boston, MA 02115 USA
[2] Harvard Univ, Sch Med, Dept Neurobiol, Boston, MA 02115 USA
来源:
关键词:
D O I:
10.1073/pnas.232565699
中图分类号:
O [数理科学和化学];
P [天文学、地球科学];
Q [生物科学];
N [自然科学总论];
学科分类号:
07 ;
0710 ;
09 ;
摘要:
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.
引用
收藏
页码:15142 / 15147
页数:6
相关论文