Kathryn Hadfield

Extracellular signal-regulated kinases 1/2 are serum-stimulated "Bim(EL) kinases" that bind to the BH3-only protein Bim(EL) causing its phosphorylation and turnover.

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 ERK1/2 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 ERK1/2 pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK1/2-dependent phosphorylation of BimEL occurs at (S/T)P motifs. ERK1/2 phosphorylates BimEL, but not BimS or BimL, in vitro, and mutation of Ser65 to alanine blocks the phosphorylation of BimEL by ERK1/2 in vitro and in vivo and prevents the degradation of the protein following activation of the ERK1/2 pathway. We also find that ERK1/2, but not JNK, can physically associate with GST-BimEL, but not GST-BimL or GST-BimS, in vitro. ERK1/2 also binds to full-length BimEL in vivo, and we have localized a potential ERK1/2 “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 ERK1/2 pathway in cell survival signaling.

Activation of ERK1/2 by ΔRaf-1 : ER* represses Bim expression independently of the JNK or PI3K pathways

CC139 fibroblasts are one of several model systems in which the Raf→MEK→ERK1/2 pathway can inhibit apoptosis independently of the PI3K pathway; however, the precise mechanism for this protective effect is not known. Serum withdrawal from CC139 fibroblasts resulted in the rapid onset of apoptosis, which was prevented by actinomycin D or cycloheximide. Serum withdrawal promoted the rapid, de novo accumulation of BimEL, a proapoptotic ‘BH3-only’ member of the Bcl-2 protein family. BimEL expression was an early event, occurring several hours prior to caspase activation. In contrast to studies in neurons, activation of the JNK→c-Jun pathway was neither necessary nor sufficient to induce BimEL expression. Selective inhibition of either the ERK pathway (with U0126) or the PI3K pathway (with LY294002) caused an increase in the expression of BimEL. Furthermore, selective activation of the ERK1/2 pathway by ΔRaf-1:ER* substantially reduced BimEL expression, abolished conformational changes in Bax and blocked the appearance of apoptotic cells. The ability of ΔRaf-1:ER* to repress BimEL expression required the ERK pathway but was independent of the PI3K→PDK→PKB pathway. Thus, serum withdrawal-induced expression of BimEL occurs independently of the JNK→c-Jun pathway and can be repressed by the ERK pathway independently of the PI3K pathway. This may contribute to Raf- and Ras-induced cell survival at low serum concentrations.

Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades

Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1 h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury.