Abstract
The evolutionarily conserved Ras/mitogen-activated protein kinase (MAPK) cascade is an integral part of the processes of cell division, differentiation, movement and death. Signals received at the cell surface are relayed into the nucleus, where MAPK phosphorylates and thereby modulates the activities of a subset of transcription factors1,2. Here we report the cloning and characterization of a new component of this signal transduction pathway called Mae (for modulator of the activity of Ets). Mae is a signalling intermediate that directly links the MAPK signalling pathway to its downstream transcription factor targets. Phosphorylation by MAPK of the critical serine residue (Ser 127) of the Drosophila transcription factor Yan depends on Mae, and is mediated by the binding of Yan to Mae through their Pointed domains. This phosphorylation is both necessary and sufficient to abrogate transcriptional repression by Yan. Mae also regulates the activity of the transcriptional activator Pointed-P2 by a similar mechanism. Mae is essential for the normal development and viability of Drosophila, and is required in vivo for normal signalling of the epidermal growth factor receptor. Our study indicates that MAPK signalling specificity may depend on proteins that couple specific substrates to the kinase.
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Acknowledgements
We thank S. Bullock, R. Winston, H. McNeill, P. Verrijzer, C. Hill, A. Maata, P. Mason, J. White, J. Brossens, M. Freeman, G. Rubin, T. Laverty and P. Soccorso for advice, discussions and gifts of materials. This work was supported by the Imperial Cancer Research Fund and BBSRC.
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Figure 1 (JPG 14.56 KB)
a) Gel mobility shift assay. 10 ng of GST Yan was mixed with the indicated amounts of Mae protein, immediately incubated with the ets DNA-binding site and resolved on a 5% acrylamide gel. b) All of the constructs that were used in the luciferase reporter assays were fused in-frame with the flag epitope, and equivalent expression in Cos-7 cells was shown by Western blotting with an a-flag antibody. The presence of the flag tag had no effect upon the activity of any of the constructs in the assay (data not shown). c) Luciferase reporter assay of the Yan constructs used in Fig 3d.
TCGACGGATCGAAAAAAATGTTAATCCAACAATAGTTTGTGTTGCGAATCGTCGTCGTCGAGGTTGAGTTGTTGGAAAAT TTGTTTTTTTGGGTGTATTCGAATTTTTCAGCCTTTATTATTTGTTTGGTTTCGGTTGGGTTATAAATATGTTTAAATGGTATC TGCTTCTGGAAACTGCACTTTGCTTGGCTCTCGCTCTCCCAATGGGATTGTATCTCTCGGATACGTTTCGTTTCACAACTT TTGCAGCATATAACGTATTTTTCACGCTCTGAACCATGATGAAATAACATAAGGTGGTCCCGTCGGCAAGAGACATCCAC TTAACGTATGCTTGCAATAAG
Figure 2. Sequence of the P-element insertion position in the fly line l(2)k12907. Genomic DNA was isolated from l(2)k12907 flies and following digestion with Sau 3A was ligated overnight. Inverse PCR was performed and the resultant fragment was directly sequenced (see Berkeley Fly Database). The first 10 bases are derived from the P-element and the following 336 bases are the genomic sequence at the point of insertion.
Figure 3 (JPG 6.66 KB)
a) GST pulldown assay. Erk physically associates with Yan, Pnt-P2 and Mae. 50 ng of either GST Yan (lane 2), GST Pnt-P2 (lane 3), GST Mae (lane 4) or GST alone (lane 5) were incubated with 35S-labelled Erk. Bound Erk was eluted and run on a 12% denaturing polyacrylamide gel. b) Mae but not Erk inhibits Yan binding to DNA. The ets DNA-binding site was incubated for 20 minutes with 10ng of each of the indicated purified GST-fusion proteins and the DNA-protein complexes resolved on a 5% acrylamide gel.
Details of constructs described in figures 2 and 3
In GST Yan, the full-length Yan protein is fused in-frame with Glutathione-S-transferase. GST Yan(G84>P) is identical to GST Yan except that an invariant glycine residue at position 84 and in the Pnt-domain has been mutated to proline. In GST Yan (?46-107) the entire Pnt-domain of Yan is deleted. In Yan *ets 2 invariant arginine residues R455 and R458 in the ets domain of Yan that are indispensable for binding to DNA 12,13, are mutated to glycine. In Yan ACT all of the consensus MAPK phosphorylation sites have been mutated to alanine 6. In Mae(G139>P) , glycine 139 in the Pnt-domain of Mae, and in an equivalent position to glycine 84 of Yan, is mutated to proline.
In Yan 4(S/T), a common Bsm I site has been utilised to construct a chimera in which the N-terminus of Yan is fused to the C-terminus of Yan ACT. This construct retains the 4 most N-terminal consensus sites of Erk phosphorylation. Yan 3(S/T) contains the 3 most N-terminal consensus phosphorylation sites. Yan S127, retains only the most N-terminal consensus phosphorylation site. Yan S127 (G84>P) is identical to Yan S127 except that glycine 84 in the Pnt-domain of Yan, has been mutated to a proline.
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Baker, D., Mille-Baker, B., Wainwright, S. et al. Mae mediates MAP kinase phosphorylation of Ets transcription factors in Drosophila. Nature 411, 330–334 (2001). https://doi.org/10.1038/35077122
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DOI: https://doi.org/10.1038/35077122
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