title: 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.
abstract: 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.
authors: Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.
authors: Thomas
authors: T
authors: Timm
authors: FMP, Forschungsinstitut fuer Molekulare Pharmakologie, Robert-Roessle-Strasse 10, 13125 Berlin, Germany.
authors: Jens Peter
authors: JP
authors: von Kries
authors: Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.
authors: Xiaoyu
authors: X
authors: Li
authors: Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany; DZNE (German Center for Neurodegenerative Diseases) and CAESAR (Center of Advanced European Studies and Research), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany.
authors: Hans
authors: H
authors: Zempel
authors: Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany; DZNE (German Center for Neurodegenerative Diseases) and CAESAR (Center of Advanced European Studies and Research), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany.
authors: Eckhard
authors: E
authors: Mandelkow
authors: Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany; DZNE (German Center for Neurodegenerative Diseases) and CAESAR (Center of Advanced European Studies and Research), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany. Electronic address: mandelkow@dzne.de.
authors: Eva-Maria
authors: EM
authors: Mandelkow
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D016466:CHO Cells; D002454:Cell Differentiation / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020439:Growth Cones / Q000378:metabolism*; D006801:Humans; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats
chemical_list: D048948:14-3-3 Proteins; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases