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.
Highlights
We and others identified a 35-amino acid segment within human Raf-1 kinase that preferentially binds phosphatidic acid
These results suggest that agonist-induced Raf-1 translocation is coupled to the generation of phospholipase D-derived phosphatidic acid
Identification of Key Residues That Mediate Raf-1-Phosphatidate Interaction—Employing deletion mutagenesis, we previously identified a 35-amino acid segment within the carboxyl terminus of Raf-1 kinase that interacts with phosphatidic acid [11]
Summary
We and others identified a 35-amino acid segment within human Raf-1 kinase that preferentially binds phosphatidic acid. A second phospholipid binding site (site II) is located between residues 390 and 423 of human Raf-1 This region displays preferential interaction with phosphatidic acid, and the binding is not dependent on ionic interactions alone. Rizzo et al [15, 16] demonstrated that in Rat-1 fibroblasts overexpressing the human insulin receptor (HIRcB cells), the stimulation of the MAP kinase pathway by insulin is dependent on phospholipase D activation and is mediated via an induction of Raf-1 translocation to the plasma membrane and early endosomes by PA. Raf-1 translocation in response to PLD-derived PA is observed in Rat-1 cells expressing constitutively activated p21Ras (Q61L mutant) suggesting that PA and Ras may act concurrently and by mutually independent pathways to promote Raf-1 translocation to the plasma membrane (reviewed in Ref. 36). Based on the results from our experiments and that of
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