Dynamic visualization of signaling activities in living cells.
The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation and dynamic modulation of the activities of protein kinases, phosphatases, and second messengers. However, traditional methodologies for detecting signaling events, such as activation of kinases and second-messenger production and degradation, are limited in their spatiotemporal resolution and do not allow one to follow these events within the live-cell context. To achieve dynamic tracking of signaling activities in living cells, we have engineered genetically encoded fluorescent reporters for protein kinases and second messengers, such as cyclic adenosine monophosphate (cAMP) and phosphoinositides. Their development and specific examples of their application are discussed. In addition, a live-cell, high-throughput screening method has been developed for identification of new modulators that affect the dynamic activity of kinases and second messengers. Together, these reporters have the potential to provide important spatiotemporal information about the circuitry governing specific signaling events in living cells.
Allen
Michael D
MD
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
DiPilato
Lisa M
LM
Ananthanarayanan
Bharath
B
Newman
Robert H
RH
Ni
Qiang
Q
Zhang
Jin
J
D000818:Animals; D002451:Cell Compartmentation; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008954:Models, Biological; D010716:Phosphatidylinositols / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000502:physiology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D008164:Luminescent Proteins; D010716:Phosphatidylinositols; D000242:Cyclic AMP; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases