Abstract
The serine/threonine kinase Raf-1 acts downstream of Ras in the MAPK pathway leading to ERK activation in response to mitogens. Raf-1 has oncogenic potential, but is normally controlled by a complex interplay of inhibitory and activating mechanisms. Although Raf-1 is phosphorylated in unstimulated cells, mitogens cause its membrane recruitment by Ras and subsequent phosphorylation on additional sites. Some of these events modulate Raf-1 kinase activity while others determine interactions with other proteins. These changes regulate the ability of Raf-1 to phosphorylate its downstream targets MEK1 and MEK2. Rho family small G proteins act synergistically with Raf-1 to stimulate the ERK pathway by a cross-cascade mechanism that enhances MEK phosphorylation by Raf-1. Here we show that both Raf-1 and MEK1 are phosphorylated by PAK1 and that mutations at PAK1 phosphorylation sites in either protein prevent cross-cascade activation. In contrast, MEK1 activation by constitutively-active Raf-1 is refractory to mutations at PAK1 phosphorylation sites. Phosphorylation of MEK1 on serine 298 does not appear to regulate the interaction between Raf-1 and MEK1, but rather the ability of Raf-1 to phosphorylate MEK1 with which it is complexed in vivo. Our findings indicate that PAK1 primes MEK1 for activation by Raf-1 and imply another level of regulation in the ERK cascade.
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Acknowledgements
We thank Drs Natalie Ahn, Melanie Cobb, Kun-Liang Guan, Silvio Gutkind and Walter Kolch for the provision of plasmids and other reagents used in the course of this work. We are grateful to Neil Portman for sub-cloning and preparing the GST–MEK1 mutants. We also thank John Keyte and Ingrid Davies-Gibbs for peptide synthesis, oligonucleotide synthesis and DNA sequencing, members of the group for discussions and Tahir Pillay and Walter Kolch for reading and commenting on the manuscript. This work was supported by grants to PES from the Wellcome Trust, the Association for International Cancer Research and the Mildred-Scheel-Stiftung für Krebsforschung.
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Coles, L., Shaw, P. PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway. Oncogene 21, 2236–2244 (2002). https://doi.org/10.1038/sj.onc.1205302
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DOI: https://doi.org/10.1038/sj.onc.1205302
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