ERK activation induces phosphorylation of Elk-1 at multiple S/T-P motifs to high stoichiometry.
Elk-1, a member of the TCF family of Ets domain proteins, contains a C-terminal transcriptional activation domain with multiple copies of the MAPK core consensus sequence S/T-P. This region is phosphorylated by MAP kinases in vitro and in vivo, but the extent and kinetics of phosphorylation at the different sites have not been investigated in detail. We prepared antisera against the phosphorylated forms of residues T353, T363, T368, S383, S389 and T417. The antisera specifically recognize the phosphorylated Elk-1 C terminus and are specific for their cognate sites, as assessed by peptide competition and mutagenesis experiments. Analysis of cells stably expressing Elk-1 in vivo shows that following serum or TPA stimulation, residues T353, T363, T368, S383, S389 and T417 become phosphorylated with similar kinetics. Mutation of any one site does not prevent phosphorylation of the others. Mutation to alanine of S383, F378 or W379, which virtually abolishes transcriptional activation by Elk-1, does not affect phosphorylation of any sites tested. Analysis of Elk-1 using two-dimensional gel electrophoresis shows that following ERK activation Elk-1 receives at least six phosphates in addition to those present prior to stimulation. We propose that the Elk-1 C-terminal regulatory domain becomes stoichiometrically phosphorylated following growth factor stimulation.
Cruzalegui
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Transcription Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
Cano
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Treisman
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; C494113:Elk1 protein, mouse; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D020928:Mitogen-Activated Protein Kinases