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Molecular and Cellular Biology, March 2003, p. 1983-1993, Vol. 23, No. 6
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.6.1983-1993.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

A Raf-1 Mutant That Dissociates MEK/Extracellular Signal-Regulated Kinase Activation from Malignant Transformation and Differentiation but Not Proliferation

Amardeep S. Dhillon,1* Sharon Meikle,1 Carole Peyssonnaux,2 Joan Grindlay,1 Christian Kaiser,3 Helge Steen,4,{dagger} Peter E. Shaw,4 Harald Mischak,5 Alain Eychène,2 and Walter Kolch1

The Beatson Institute for Cancer Research, CR-UK Beatson Laboratories, Bearsden, Glasgow G61 1BD,1 School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom,4 GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Klinische Molekularbiologie und Tumorgenetik, D-81377 Münich,3 Department of Nephrology, Medizinische Hochschule Hannover, 30625 Hannover, Germany,5 UMR 146 CNRS, Institut Curie, Centre Universitaire, F-91405 Orsay, France2

Received 17 July 2002/ Returned for modification 4 September 2002/ Accepted 23 December 2002

It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.

* Corresponding author. Mailing address: The Beatson Institute for Cancer Research, CRC Beatson Laboratories, Garscube Estate, Glasgow G61 1BD, United Kingdom. Phone: 44-141-330 3984. Fax: 44-141-942 6521. E-mail: A.Dhillon{at}beatson.gla.ac.uk.

{dagger} Present address: BioChip Technologies GmbH, GeneScan Europe AG, D-79108 Freiburg, Germany.

Molecular and Cellular Biology, March 2003, p. 1983-1993, Vol. 23, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.6.1983-1993.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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