Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function

J Biol Chem. 2006 Jan 13;281(2):813-23. doi: 10.1074/jbc.M505546200. Epub 2005 Nov 10.

Abstract

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of Bim(EL) correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked Bim(EL) phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes Bim(EL) binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 14-3-3 Proteins / metabolism
  • Alkaline Phosphatase / chemistry
  • Alkaline Phosphatase / metabolism
  • Animals
  • Apoptosis Regulatory Proteins / chemistry*
  • Apoptosis Regulatory Proteins / metabolism
  • Apoptosis*
  • Bcl-2-Like Protein 11
  • Blotting, Western
  • Carcinoma, Hepatocellular / metabolism
  • Cell Line
  • Cell Survival
  • Chromones / pharmacology
  • Culture Media, Conditioned / metabolism
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation, Enzymologic*
  • Glutathione Transferase / metabolism
  • Humans
  • Immunoprecipitation
  • Interleukin-3 / metabolism
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism
  • Morpholines / pharmacology
  • Mutation
  • Phosphorylation
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Isoforms
  • Proto-Oncogene Proteins / chemistry*
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Rats
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Proteins / chemistry
  • Serine / chemistry*
  • Time Factors
  • Transfection
  • Ubiquitin / chemistry

Substances

  • 14-3-3 Proteins
  • Apoptosis Regulatory Proteins
  • BCL2L11 protein, human
  • Bcl-2-Like Protein 11
  • Bcl2l11 protein, rat
  • Chromones
  • Culture Media, Conditioned
  • Enzyme Inhibitors
  • Interleukin-3
  • Membrane Proteins
  • Morpholines
  • Protein Isoforms
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Ubiquitin
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Serine
  • Glutathione Transferase
  • Proto-Oncogene Proteins c-akt
  • Alkaline Phosphatase
  • Proteasome Endopeptidase Complex