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.
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.
Ghosh
Sujoy
S
GlaxoSmithKline, Genetics Research, Research Triangle Park, North Carolina 27709, USA. sg45653@gsk.com
Moore
Sean
S
Bell
Robert M
RM
Dush
Michael
M
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010712:Phosphatidic Acids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013623:Tail / Q000196:embryology; D014158:Transcription, Genetic; D015027:Zebrafish / Q000196:embryology*
D010712:Phosphatidic Acids; D011993:Recombinant Fusion Proteins; D012313:RNA; D005982:Glutathione Transferase; D019908:Proto-Oncogene Proteins c-raf; D000409:Alanine