title: Bioluminescent imaging of Cdk2 inhibition in vivo.
abstract: Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle-dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle-dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.
authors.affiliation: Department of Adult Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Guo-Jun
authors.initials: GJ
authors.lastname: Zhang
authors.forename: Michal
authors.initials: M
authors.lastname: Safran
authors.forename: Wenyi
authors.initials: W
authors.lastname: Wei
authors.forename: Erik
authors.initials: E
authors.lastname: Sorensen
authors.forename: Peter
authors.initials: P
authors.lastname: Lassota
authors.forename: Nikolai
authors.initials: N
authors.lastname: Zhelev
authors.forename: Donna S
authors.initials: DS
authors.lastname: Neuberg
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Shapiro
authors.forename: William G
authors.initials: WG
authors.lastname: Kaelin
mesh_terms: D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017690:Cell Transplantation; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003952:Diagnostic Imaging; D004791:Enzyme Inhibitors / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D051379:Mice; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D044786:S-Phase Kinase-Associated Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D008156:Luciferases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2