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PMID:21478148

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Citation

Thompson, S, Pearson, AN, Ashley, MD, Jessick, V, Murphy, BM, Gafken, P, Henshall, DC, Morris, KT, Simon, RP and Meller, R (2011) 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. J. Biol. Chem. 286:19331-9

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.

Links

PubMed PMC3103311 Online version:10.1074/jbc.M110.197707

Keywords

Animals; Apoptosis Regulatory Proteins/genetics; Apoptosis Regulatory Proteins/metabolism; Brain Ischemia/genetics; Brain Ischemia/metabolism; Carcinogens/pharmacology; Cells, Cultured; Embryo, Mammalian/metabolism; Fibroblasts/metabolism; Gene Expression Regulation/drug effects; Gene Expression Regulation/genetics; Ischemic Preconditioning; Membrane Proteins/genetics; Membrane Proteins/metabolism; Mice; Mitogen-Activated Protein Kinase 1/genetics; Mitogen-Activated Protein Kinase 1/metabolism; Mitogen-Activated Protein Kinase 3/genetics; Mitogen-Activated Protein Kinase 3/metabolism; Mutation; Phosphorylation/drug effects; Protein Binding; Proteins/genetics; Proteins/metabolism; Proto-Oncogene Proteins/genetics; Proto-Oncogene Proteins/metabolism; Rats; Tetradecanoylphorbol Acetate/pharmacology; Ubiquitin-Protein Ligases/genetics; Ubiquitin-Protein Ligases/metabolism; Ubiquitination

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

RAT:TRIM2

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O54918

F

Seeded From UniProt

complete

RAT:TRIM2

involved_in

GO:0043523: regulation of neuron apoptotic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:B2L11

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P63170

F

Seeded From UniProt

complete

MOUSE:B2L11

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P63102

F

Seeded From UniProt

complete

MOUSE:B2L11

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:D3ZQG6

F

Seeded From UniProt

complete

MOUSE:B2L11

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q9ESN6

F

Seeded From UniProt

complete

RAT:B2L11

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q9C040

F

Seeded From UniProt

complete

RAT:1433Z

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O54918

F

Seeded From UniProt

complete

RAT:DYL1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O54918

F

Seeded From UniProt

complete

HUMAN:TRIM2

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O88498

F

Seeded From UniProt

complete

MOUSE:TRIM2

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O54918

F

Seeded From UniProt

complete

MOUSE:TRIM2

enables

GO:0004842: ubiquitin-protein transferase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

MOUSE:TRIM2

enables

GO:0061630: ubiquitin protein ligase activity

ECO:0000314: direct assay evidence used in manual assertion

F

  • has_input:(MGI:MGI:1197519)

Seeded From UniProt

complete


See also

References

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