The activity of D-raf in torso signal transduction is altered by serine substitution, N-terminal deletion, and membrane targeting.
The Raf family of serine/threonine kinases are essential components in many receptor tyrosine kinase-mediated signal transduction pathways. Here, we analyze the function of D-raf in the Torso (Tor) pathway required to specify cellular fates at the embryonic poles. Using mutant embryos lacking endogenous D-raf protein, we show that D-raf's serine/threonine kinase activity is essential for its role in Tor signal transduction and that human Raf-1 will substitute for D-raf in this pathway. After Tor activation, D-raf becomes hyperphosphorylated. We identified two putative serine phosphorylation sites (S388 and S743) in SF9 cells and demonstrate that S743 or its phosphorylation is essential for D-raf function in embryos. Alanine substitution at S388, N-terminal truncation, or targeted membrane association permits transmission of the Torso signal by D-raf, but these D-raf molecules differ in their rescuing potential and relative biological activity. Membrane-targeted D-raftor4021 showed the highest level of activity, followed by alanine-substituted D-rafS388A and N-terminal-truncated D-raf delta 445. Since the activity profiles for these altered forms of D-raf are distinct, these findings indicate that each structural modification differentially affects the regulation and/or propagation of the Tor signal by these mutant D-raf proteins.
Baek
K H
KH
Department of Zoology and Genetics, Iowa State University, Ames 50011, USA.
Fabian
J R
JR
Sprenger
F
F
Morrison
D K
DK
Ambrosio
L
L
D000409:Alanine; D000818:Animals; D001483:Base Sequence; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology* / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000648:ultrastructure; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D009024:Morphogenesis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf; D020794:Receptor Protein-Tyrosine Kinases / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D012694:Serine; D015398:Signal Transduction / Q000235:genetics*; D018411:Spodoptera
D029721:Drosophila Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012694:Serine; D020794:Receptor Protein-Tyrosine Kinases; C080148:tor protein, Drosophila; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D000409:Alanine