PUBLICATION
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
- Authors
- Ghosh, S., Moore, S., Bell, R.M., and Dush, M.
- ID
- ZDB-PUB-030826-14
- Date
- 2003
- Source
- The Journal of biological chemistry 278(46): 45690-45696 (Journal)
- Registered Authors
- Bell, Robert
- Keywords
- none
- MeSH Terms
-
- Alanine/chemistry
- Amino Acid Motifs
- Amino Acid Sequence
- Animals
- Binding Sites
- Dose-Response Relationship, Drug
- Gene Deletion
- Glutathione Transferase/metabolism
- Humans
- In Situ Hybridization
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- Mutation
- Phosphatidic Acids/chemistry*
- Phosphatidic Acids/metabolism
- Protein Binding
- Protein Structure, Tertiary
- Protein Transport
- Proto-Oncogene Proteins c-raf/chemistry*
- Proto-Oncogene Proteins c-raf/genetics*
- Proto-Oncogene Proteins c-raf/physiology
- RNA/metabolism
- Recombinant Fusion Proteins/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Sequence Homology, Amino Acid
- Tail/embryology
- Transcription, Genetic
- Zebrafish/embryology*
- PubMed
- 12925535 Full text @ J. Biol. Chem.
Citation
Ghosh, S., Moore, S., Bell, R.M., and Dush, M. (2003) 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. The Journal of biological chemistry. 278(46):45690-45696.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping