Both the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways can protect cells from apoptosis following withdrawal of survival factors. We have previously shown that the ERK1/2 pathway acts independently of PI3K to block expression of the BH3-only protein, Bim EL , and prevent serum withdrawal-induced cell death, although the precise mechanism by which ERK reduced Bim EL levels was unclear. By comparing Bim mRNA and Bim protein, expression we now show that the rapid expression of Bim EL following serum withdrawal cannot be accounted for simply by increases in mRNA following inhibition of PI3K. In cells maintained in serum Bim EL is a phosphoprotein. We show that activation of the ERK1/2 pathway is both necessary and sufficient to promote Bim EL phosphorylation and that this leads to a substantial increase in turnover of the Bim EL protein. ERK1/2-dependent degradation of Bim EL proceeds via the proteasome pathway because it is blocked by proteasome inhibitors and is defective at the restrictive temperature in cells with a temperaturesensitive mutation in the E1 component of the ubiquitinconjugating system. Finally, co-transfection of Bim EL and FLAG-ubiquitin causes the accumulation of polyubiquitinated forms of Bim, and this requires the ERK1/2 pathway. Our findings provide new insights into the regulation of Bim and the role of the ERK pathway in cell survival.

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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 Bim EL , targeting it for degradation via the proteasome. However, the nature of the kinase responsible for Bim EL phosphorylation remained unclear. We now show that Bim EL 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 Bim EL occurs at (S/T)P motifs. ERK1/2 phosphorylates Bim EL , but not Bim S or Bim L , in vitro, and mutation of Ser 65 to alanine blocks the phosphorylation of Bim EL 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 GSTBim EL , but not GST-Bim L or GST-Bim S , in vitro. ERK1/2 also binds to full-length Bim EL in vivo, and we have localized a potential ERK1/2 "docking domain" lying within a 27-amino acid stretch of the Bim EL protein. Our findings provide new insights into the post-translational regulation of Bim EL and the role of the ERK1/2 pathway in cell survival signaling.

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