Abstract
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.
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Acknowledgements
E Cano and FH Cruzalegui contributed equally to this study. The work was funded by the Imperial Cancer Research Fund. FH Cruzalegui was the recipient of a fellowship from the HFSP; E Cano is a postdoctoral fellow of the Howard Hughes Medical Institute; R Treisman was an International Research Scholar of the HHMI. We thank Peter Shaw for the T353A, T368A and T417A mutants; Pablo Rodriguez-Viciana for baculoviruses; Al Stewart and Neil McDonald for activated recombinant ERK2; and Ross Thomas and Caroline Hill for helpful discussions and comments on the manuscript.
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Cruzalegui, F., Cano, E. & Treisman, R. ERK activation induces phosphorylation of Elk-1 at multiple S/T-P motifs to high stoichiometry. Oncogene 18, 7948–7957 (1999). https://doi.org/10.1038/sj.onc.1203362
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DOI: https://doi.org/10.1038/sj.onc.1203362
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