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Investigates the promotion of phosphorylation and proteasome-dependent degradation of the BH-3 only protein bim by activating the extracellular signal-regulated kinase 1 and the phosphatidylinositol 3'-kinase signaling pathways. Cell protection from apoptosis following withdrawal of survival factors; Increase in the turnover rate of the Bim[sub EL] protein; Accumulation of polyubiquitinated forms of Bim.

Abstract: The pro-apoptotic BH3 only protein BIMEL is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIMEL to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIMEL, which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5−/− fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIMEL occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIMEL drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIMEL and may be relevant to the therapeutic use of agents such as paclitaxel. [Copyright &y& Elsevier]

Abstract: The conditional protein kinase ΔMEKK3:ER* allows activation of the mitogen-activated and stress-activated protein kinases (MAPKs and SAPKs) without imposing a primary cellular stress or damage. Such separation of stress from stress-induced signalling is particularly important in the analysis of apoptosis. Activation of ΔMEKK3:ER* in cycling CCl39 cells caused a rapid stimulation of the ERK1/2, JNK and p38 pathways but resulted in a slow, delayed apoptotic response. Paradoxically, activation of the same pathways inhibited the rapid expression of BimEL and apoptosis following withdrawal of serum. Inhibition of the ERK1/2 pathway prevented the down-regulation of BimEL but caused only a partial reversion of the cyto-protective effect of ΔMEKK3:ER*. In contrast, inhibition of p38 had no effect, raising the possibility that activation of JNK might also exert a protective effect. To test this we used CCl39 cells expressing ΔMEKK1:ER* which activates JNK but not ERK1/2, p38, PKB or IκB kinase. Activation of ΔMEKK1:ER* inhibited serum withdrawal-induced conformational changes in Bax and apoptosis. These results suggest that in the absence of any overt cellular damage or chemical stress activation of JNK can act independently of the ERK1/2 or PKB pathways to inhibit serum withdrawal-induced cell death. [Copyright &y& Elsevier]

Abstract: The duration of ERK1/2 activation influences the nature of the biological response to agonist. Members of the AP-1 transcription factor family are well known targets of the ERK1/2 pathway and are expressed in a temporally coordinated fashion during cell cycle re-entry. In CCl39 fibroblasts, sustained ERK1/2 activation is required for the expression of Fra-1, Fra-2, c-Jun and JunB, whereas expression of c-Fos is still strongly induced even in response to transient ERK activation. However, the significance of this pattern of expression for AP-1 activity has not been addressed. Here we show that growth factor stimulated activation of the C-terminal c-Fos transactivation domain (TAD) serves as a sensor for ERK1/2 signal duration whereas the c-JunTAD is not responsive to growth factors. In addition, sustained ERK1/2 activation determines the duration of increases in AP-1 DNA binding complexes as well as their qualitative make up. Finally, this is reflected in both the duration and quantitative transcriptional output of stably integrated AP-1 reporter constructs, indicating that AP-1 activity is finely tuned to ERK1/2 signal duration. These results provide new insights into the importance of ERK1/2 signal duration in the regulation of AP-1 and provide an explanation for how differences in signal duration can lead to both quantitative and qualitative changes in gene expression. [Copyright &y& Elsevier]

Bim, a ‘BH3-only’ protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK½ pathway promotes phosphorylation of BimEL, targeting it for degradation via the proteasome. However, the nature of the kinase responsible for BimEL phosphorylation remained unclear. We now show that BimEL is phosphorylated on at least three sites in response to activation of the ERK½ pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK½-dependent phosphorylation of BimEL occurs at (S/T)P motifs. ERK½ phosphorylates BimEL, but not BimS or BimL, in vitro, and mutation of Ser65 to alanine blocks the phosphorylation of BimEL by ERK½ in vitro and in vivo and prevents the degradation of the protein following activation of the ERK½ pathway. We also find that ERK½, but not JNK, can physically associate with GST-BimEL, but not GST-BimL or GST-BimS, in vitro. ERK½ also binds to full-length BimEL in vivo, and we have localized a potential ERK½ ‘docking domain’ lying within a 27-amino acid stretch of the BimEL protein. Our findings provide new insights into the post-translational regulation of BimEL and the role of the ERK½ pathway in cell survival signaling. [ABSTRACT FROM AUTHOR]