title: Identification of a DEF-type docking domain for extracellular signal-regulated kinases 1/2 that directs phosphorylation and turnover of the BH3-only protein BimEL.
abstract: The BH3-only protein, Bim, exists as three splice variants (Bim(S), Bim(L), and Bim(EL)) of differing pro-apoptotic potency. Bim(EL), the least effective killer, is degraded by the proteasome in response to phosphorylation by extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2-dependent phosphorylation correlates with the presence of a domain unique to the Bim(EL) splice variant that includes the major ERK1/2 phosphorylation site Ser(65). However, efficient phosphorylation by ERK1/2, c-Jun N-terminal kinase, or p38 requires the presence in the substrate of a discrete kinase-docking domain as well as the phosphoacceptor site. Here we show that the region unique to Bim(EL) (amino acids 41-97) harbors two potential DEF-type ERK1/2 kinase-docking domains, DEF1 and DEF2. Peptide competition assays revealed that the DEF2 peptide could act autonomously to bind active ERK1/2, whereas the DEF1 peptide did not. Truncation analysis identified a minimal region, residues 80-97, containing the DEF2 motif as sufficient for ERK1/2 binding. Mutation of key residues in the DEF2 motif abolished the interaction of ERK1/2 and Bim(EL) and also abolished ERK1/2-dependent phosphorylation of Bim(EL) in vivo, thereby stabilizing the protein and enhancing cytotoxicity. Our results identify a new physiologically relevant functional motif in Bim(EL) that may account for the distinct biological properties of this splice variant.
authors.affiliation: Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge CB2 4AT, United Kingdom. becky.ley@bbsrc.ac.uk
authors.forename: Rebecca
authors.initials: R
authors.lastname: Ley
authors.forename: Kathryn
authors.initials: K
authors.lastname: Hadfield
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Howes
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Cook
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D001665:Binding Sites / Q000502:physiology; D045744:Cell Line, Tumor; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008565:Membrane Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000581:Amidohydrolases; C084507:peptide deformylase