c-Cbl is not required for ERK1/2-dependent degradation of BimEL

Abstract

Bim(EL) the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim(EL). It has been proposed that the RING finger protein Cbl binds to Bim(EL) and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim(EL) is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim(EL) turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim(EL) is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim(EL) does not involve its tyrosine phosphorylation; indeed, Bim(EL) is not a tyrosine phosphoprotein. Furthermore, Bim(EL) fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim(EL) turnover proceeds normally in Cbl-containing or Cbl-/- fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim(EL) turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.

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

“…When TRIM2 was immunodepleted from neuronal lysates we did not observe an increase in Bim ubiquitination following preconditioning ischemia. Therefore, our identification of TRIM2 as a Bim E3 ligase is consistent with the known biology of Bim ubiquitination as reported by Cook's group (11,14).…”

“…2A). Serum starvation of MEFs followed by serum stimulation has been shown to activate p42/p44 MAPK kinase and promote Bim ubiquitination (11,14). Phospho-p42/p44 MAPK levels were increased following serum stimulation of cells, and Bim protein levels were decreased accordingly ( Fig.…”

“…To date the tyrosine kinase c-Cbl (12) and CIS, a member of the SKIP-Cullen E3 ligase family (13), have been suggested to function as a Bim E3 ligase; however, other studies suggest that c-Cbl may not mediate p42/p44 MAPK-dependent Bim ubiquitination (14). Taken together, this would suggest that, at least, an additional protein may function as a Bim E3 ligase regulating p42/p44 MAPK-dependent Bim degradation in neurons following brief ischemia.…”

Tumour cell survival signalling by the ERK1/2 pathway

“…It has been suggested that CBL is the E3 ligase responsible for poly-ubiquitination of BIM EL ; 32 however, others fail to detect binding of BIM EL to CBL and BIM EL is turned over normally in CBLÀ/À fibroblasts arguing against this. 33 Regulation of BAD binding properties and stability. BAD is inhibited by phosphorylation at three distinct sites ( Figure 2): Ser 112 , catalysed by RSK; Ser 136 , catalysed by PKB and Ser 155 catalysed by PKA.…”

Intrinsic resistance to the MEK1/2 inhibitor AZD6244 (ARRY‐142886) is associated with weak ERK1/2 signalling and/or strong PI3K signalling in colorectal cancer cell lines

“…Cells were cultured in 'complete medium', comprising [Dulbecco's modified eagle medium (DMEM) supplemented with 4.5 mg ml 21 glucose, 100 U ml 21 penicillin, 100 lg ml 21 streptomycin, 2 mM glutamine, 10% (v/v) foetal bovine serum (FBS)]. Culture of HEK293 cells expressing DRAF-1:ER* (HR1 cells) 28 or DMEKK3:ER* (HM3 cells) 30 has been described. For serum starvation, cells judged to be 50-60% confluent were washed in serum-free medium and then placed in fresh serum-free medium with 4-HT or equivalent volume of ethanol (vehicle control) for the times indicated in the figure legends.…”