Microtubule affinity regulating kinase activity in living neurons was examined by a genetically encoded fluorescence resonance energy transfer/fluorescence lifetime imaging-based biosensor: inhibitors with therapeutic potential
| Autor: | Timm, T., von Kries, J., Li, X., Zempel, H., Mandelkow, E. |
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| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
metabolism [Growth Cones]
metabolism [Microtubules] Green Fluorescent Proteins Growth Cones Protein Kinase Inhibitors Tau Cell Differentiation Biosensor Alzheimer Disease Protein Kinases Fluorescence Resonance Energy Transfer (FRET) Cell Polarity Fluorescence Lifetime Imaging (FLIM) genetics [Protein Serine-Threonine Kinases] Biosensing Techniques CHO Cells methods [Fluorescence Resonance Energy Transfer] Protein Serine-Threonine Kinases genetics [Protein-Serine-Threonine Kinases] Microtubules PC12 Cells genetics [Microtubules] Cricetulus metabolism [Protein Serine-Threonine Kinases] Cricetinae Fluorescence Resonance Energy Transfer genetics [Green Fluorescent Proteins] Animals Humans metabolism [14-3-3 Proteins] metabolism [Protein-Serine-Threonine Kinases] Cell Biology Protein-Serine-Threonine Kinases Rats physiology [Cell Differentiation] methods [Biosensing Techniques] 14-3-3 Proteins metabolism [Green Fluorescent Proteins] ddc:540 genetics [14-3-3 Proteins] |
| Zdroj: | The journal of biological chemistry, 286(48): 41711–41722 The journal of biological chemistry 286(48), 41711-41722 (2011). doi:10.1074/jbc.M111.257865 Journal of Biological Chemistry |
| DOI: | 10.1074/jbc.M111.257865 |
| Popis: | BACKGROUND: Deregulation of the protein kinase MARK has been linked to Alzheimer disease. - RESULTS: Mark-specific inhibitors and a biosensor are identified. - CONCLUSION: The inhibitors and the biosensor are tools to provide new insights into the role of MARK during polarity establishment and maintenance of neurons. - SIGNIFICANCE: The inhibitors might possess therapeutic potential by interfering with abnormal Tau phosphorylation in Alzheimer disease. Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ∼18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons. |
| Databáze: | OpenAIRE |
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