title: Identification of a novel Bcl-2-interacting mediator of cell death (Bim) E3 ligase, tripartite motif-containing protein 2 (TRIM2), and its role in rapid ischemic tolerance-induced neuroprotection.
abstract: We have previously shown that the cell death-promoting protein Bcl-2-interacting mediator of cell death (Bim) is ubiquitinated and degraded following a neuroprotection-conferring episode of brief ischemia (preconditioning). Here, we identify the E3 ligase that ubiquitinates Bim in this model, using a proteomics approach. Using phosphorylated GST-Bim as bait, we precipitated and identified by mass spectrometry tripartite motif protein 2 (TRIM2), a RING (really interesting new gene) domain-containing protein. The reaction between TRIM2 and Bim was confirmed using co-immunoprecipitation followed by immunoblotting. We show that TRIM2 binds to Bim when it is phosphorylated by p42/p44 MAPK but does not interact with a nonphosphorylatable Bim mutant (3ABim). 12-O-tetradecanoylphorbol-13-acetate activation of p42/p44 MAPK drives Bim ubiquitination in mouse embryonic fibroblast cells and is associated with an increased interaction between TRIM2 and Bim. One hour following preconditioning ischemia, the binding of Bim to TRIM2 increased, consistent with the time window of enhanced Bim degradation. Blocking p42/p44 MAPK activation following preconditioning ischemia with U0126 or using the nonphosphorylatable 3ABim reduced the binding between Bim and TRIM2. Immunodepletion of TRIM2 from cell lysates prepared from preconditioned cells reduced Bim ubiquitination. Finally, suppression of TRIM2 expression, using lentivirus transduction of shRNAmir, stabilized Bim protein levels and blocked neuroprotection observed in rapid ischemic tolerance. Taken together, these data support a role for TRIM2 in mediating the p42/p44 MAPK-dependent ubiquitination of Bim in rapid ischemic tolerance.
authors.affiliation: Neuroscience Institute, Morehouse School of Medicine, Atlanta, Georgia 30310-1495, USA.
authors.forename: Simon
authors.initials: S
authors.lastname: Thompson
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Pearson
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Ashley
authors.forename: Veronica
authors.initials: V
authors.lastname: Jessick
authors.forename: Brona M
authors.initials: BM
authors.lastname: Murphy
authors.forename: Philip
authors.initials: P
authors.lastname: Gafken
authors.forename: David C
authors.initials: DC
authors.lastname: Henshall
authors.forename: Kate T
authors.initials: KT
authors.lastname: Morris
authors.forename: Roger P
authors.initials: RP
authors.lastname: Simon
authors.forename: Robert
authors.initials: R
authors.lastname: Meller
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D002545:Brain Ischemia / Q000235:genetics / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D019194:Ischemic Preconditioning; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; C000606778:Bcl2l11 protein, rat; D002273:Carcinogens; D008565:Membrane Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D000070584:Tripartite Motif Proteins; C485687:Trim2 protein, mouse; D044767:Ubiquitin-Protein Ligases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D013755:Tetradecanoylphorbol Acetate