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.Cerebral ischemia, the deprivation of oxygen and glucose to the brain, can result in neuronal death. However, prior exposure to a brief nonharmful dose of ischemia (preconditioning) activates an endogenous neuroprotective program, rendering the brain protected against subsequent ischemic injury (ischemic tolerance). Rapid (short term) ischemic tolerance in brain and cultured neurons occurs 1 h following a preconditioning stimulus, resulting in profound neuroprotection (1, 2). Although the exact molecular mechanisms of rapid tolerance are not fully resolved, they appear to be mediated by rapid biochemical events, including activation of adenosine receptors, ATP-activated potassium channels, multiple protein kinases, and the ubiquitin-proteasome system (1-7).The ubiquitin-proteasome system rapidly degrades short lived proteins in the cell. Ubiquitin is added to a target protein following a sequential series of reactions, whereby ubiquitin is bound first to an E1 ligase in a reaction requiring ATP. Ubiquitin is transferred to an E2 protein and then transferred via the E3 ligase to the target protein lysine residue (8). The HECT 3 (homologous to the E6-AP carboxyl terminus) class of E3 ligase binds to ubiquitin, prior to conjugating the ubiquitin to the target, whereas the RING (really interesting new gene)-containing E3 ligases appear to mediate the tran...

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“…TRIM2 is a member of the TRIM superfamily mainly expressed in the brain [18,44] containing a RING finger, B-box, and coiled-coil domains in the amino terminus. TRIM2 interacts with NF-L and regulates its metabolism by ubiquitination [8].…”

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“…Moreover, some members of the TRIM family of ubiquitin ligases have been shown to be involved in the regulation of these signaling pathways [23,28,31,32]. Thus, it is important to clarify the correlation between the functions of TRIM family ubiquitin ligases and the effects of these signaling pathways.…”

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“…A transgenic mouse study demonstrated that replacement of the ERK phosphorylation sites with Ala prolongs BIM protein half-life and increases apoptotic cell death in response to serum starvation [4]. SCF β-TrCP , c-CBL, and TRIM2 are the E3 ligases responsible for ERK-dependent BIM degradation [100–102]. Notably, SCF β-TrCP -mediated BIM ubiquitination requires phosphorylation of the degron sequence containing S93/S94/S98 by RSK (ribosomal S6 kinase), which is promoted by ERK phosphorylation at S69 [101] (Table 1).…”