title: Screening of cell cycle fusion proteins to identify kinase signaling networks.
abstract: Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27(Kip1). We generated a p27(Kip1)-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27(Kip1)-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27(Kip1) turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.
authors.affiliation: a From the Center for Therapeutic Innovation; Department of Psychiatry and Behavioral Sciences ; University of Miami ; Miami , FL USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Trojanowsky
authors.forename: Dusica
authors.initials: D
authors.lastname: Vidovic
authors.forename: Scott
authors.initials: S
authors.lastname: Simanski
authors.forename: Clara
authors.initials: C
authors.lastname: Penas
authors.forename: Stephan
authors.initials: S
authors.lastname: Schurer
authors.forename: Nagi G
authors.initials: NG
authors.lastname: Ayad
mesh_terms: D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D007976:Leupeptins; D011993:Recombinant Fusion Proteins; D044786:S-Phase Kinase-Associated Proteins; D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D008156:Luciferases; D011494:Protein Kinases; D046988:Proteasome Endopeptidase Complex; D013755:Tetradecanoylphorbol Acetate; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: PMA, Phorbol 12-myristate 13-acetate; S/B, Signal to background ratio; Z′, Z factor; cell cycle; degradation; kinases; signaling networks; ubiquitin