HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 46, 45519-45527, November 14, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/46/45519    most recent M303106200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Harding, A. Articles by Hancock, J. F. Search for Related Content PubMed PubMed Citation Articles by Harding, A. Articles by Hancock, J. F. Social Bookmarking                      What's this?

Identification of Residues and Domains of Raf Important for Function in Vivo and in Vitro^*

Angus Harding, Virginia Hsu, Kerry Kornfeld¶, and John F. Hancock||

From the Institute for Molecular Bioscience and Department of Molecular and Cellular Pathology, University of Queensland, Brisbane 4072, Australia and the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri, 63110

Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.

Received for publication, March 26, 2003 , and in revised form, August 15, 2003.

^* This work was supported by grants from the National Health and Medical Research Council of Australia (to J. F. H.) and the National Institutes of Health (to K. K.). The IMB is a Special Research Centre of the Australian Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of the Royal Children's Hospital Foundation Scholarship Award.

^¶ Recipient of a Burroughs Welcome Foundation New Investigator Award in the Pharmacological Sciences and a Leukemia and Lymphoma Society Scholar Award.

^|| To whom correspondence should be addressed. Tel.: 61-7-3346-2033; Fax: 61-7-3346-2101; E-mail: j.hancock{at}imb.uq.edu.au.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?