期刊
JOURNAL OF CELL BIOLOGY
卷 161, 期 5, 页码 899-909出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.200302125
关键词
calcium; fluorescence resonance energy transfer; oscillation; phosphatase; PKC
类别
资金
- NIDDK NIH HHS [P01 DK054441, P01 DK54441] Funding Source: Medline
- NIGMS NIH HHS [GM62114, R01 GM043154, U54 GM062114, T32 GM007752, R37 GM043154, GM07752, GM43154] Funding Source: Medline
- NINDS NIH HHS [NS27177, R01 NS027177, R37 NS027177] Funding Source: Medline
Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of similar to10 s. Novel FRET based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.
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