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J. Biol. Chem., Vol. 278, Issue 46, 45690-45696, November 14, 2003

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Functional Analysis of a Phosphatidic Acid Binding Domain in Human Raf-1 Kinase

MUTATIONS IN THE PHOSPHATIDATE BINDING DOMAIN LEAD TO TAIL AND TRUNK ABNORMALITIES IN DEVELOPING ZEBRAFISH EMBRYOS^*

Sujoy Ghosh, Sean Moore¶, Robert M. Bell||, and Michael Dush

From the GlaxoSmithKline, Genetics Research, Research Triangle Park, North Carolina 27709

Previously, we and others identified a 35-amino acid segment within human Raf-1 kinase that preferentially binds phosphatidic acid. The presence of phosphatidic acid was found to be necessary for the translocation of Raf-1 to the plasma membrane. We have now employed a combination of alanine-scanning and deletion mutagenesis to identify the critical amino acid residues in Raf-1 necessary for interaction with phosphatidic acid. Progressive mutations within a tetrapeptide motif (residues 398-401 of human Raf-1) reduced and finally eliminated binding of Raf-1 to phosphatidic acid. We then injected zebrafish embryos with RNA encoding wild-type Raf-1 kinase or a mutant version with triple alanine mutations in the tetrapeptide motif and followed the morphological fate of embryonic development. Embryos with mutant but not wild-type Raf-1 exhibited defects in posterior axis formation exemplified by bent trunk and tail structures. Molecular evidence for lack of signaling through mutated Raf-1 was obtained by aberrant in situ hybridization of the ntl (no tail) gene, which functions downstream of Raf-1. Our results demonstrate that a functional phosphatidate binding site is necessary for Raf-1 function in embryonic development.

Received for publication, March 21, 2003 , and in revised form, August 14, 2003.

^* 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.

^¶ Present address: Aclara Biosciences, Mountain View, CA 94033.

^|| Present address: Intersouth Partners, Durham, NC 27707.

To whom correspondence should be addressed. Tel.: 919-483-0803; Fax: 919-315-4115; E-mail: sg45653{at}gsk.com.

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