Abstract
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, BimEL, and prevent serum withdrawal-induced cell death, although the precise mechanism by which ERK reduced BimEL levels was unclear. By comparing Bim mRNA and Bim protein, expression we now show that the rapid expression of BimEL following serum withdrawal cannot be accounted for simply by increases in mRNA following inhibition of PI3K. In cells maintained in serum BimEL is a phosphoprotein. We show that activation of the ERK1/2 pathway is both necessary and sufficient to promote BimEL phosphorylation and that this leads to a substantial increase in turnover of the BimEL protein. ERK1/2-dependent degradation of BimEL proceeds via the proteasome pathway because it is blocked by proteasome inhibitors and is defective at the restrictive temperature in cells with a temperature-sensitive mutation in the E1 component of the ubiquitin-conjugating system. Finally, co-transfection of BimEL 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.
Highlights
Higher eukaryotes possess two major pathways for initiating programmed cell death or apoptosis: the cell-extrinsic death pathway, involving death receptors, or the cell-intrinsic pathway (1)
We have previously shown that the ERK1/2 pathway acts independently of phosphatidylinositol 3-kinase (PI3K) to block expression of the BH3-only protein, BimEL, and prevent serum withdrawal-induced cell death, the precise mechanism by which ERK reduced BimEL levels was unclear
By comparing Bim mRNA and Bim protein, expression we show that the rapid expression of BimEL following serum withdrawal cannot be accounted for by increases in mRNA following inhibition of PI3K
Summary
Materials—Cell culture reagents were purchased from Invitrogen. U0126 was purchased from Promega. Phospho-ERK1/2, total ERK1/2, phospho-PKB (Ser-473), and total PKB were from Cell Signaling Technology/New England Biolabs; Bim was from Chemicon; Bad and rabbit anti-HA were from Santa Cruz Biotechnology; anti-FLAG was from Sigma; and mouse anti-HA was provided by the Babraham Institute Monoclonal Antibody Facility. Real-time RT-PCR—Preparation of total RNA was performed as described previously (11). RT-PCR was performed according to the protocol supplied with the Taqman® Reverse Transcription reagents (Applied Biosystems) as described previously (11). For hamster Bim we used 5Ј-TAAGGCAATCTCAGGAGGAACCT-3Ј as the forward primer and 5Ј-AGATTCGTTGAACTCGTCTCCAA-3Ј as the reverse primer. For treatment with calf intestinal phosphatase, cells were lysed in lysis buffer without phosphatase inhibitors and treated with 10 units of calf intestinal phosphatase in the presence of the appropriate buffer for 2 h at 37 °C. HA-tagged Bim was immunoprecipitated from cell lysates using either mouse anti-HA antibodies conjugated to protein G-Sepharose beads or rabbit anti-HA antibodies conjugated to protein A-Sepharose
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