Ras-independent activation of ERK signaling via the torso receptor tyrosine kinase is mediated by Rap1.
In Drosophila embryos, the Torso receptor tyrosine kinase (RTK) activates the small G protein Ras (D-Ras1) and the protein kinase Raf (D-Raf) to activate ERK to direct differentiation of terminal structures . However, genetic studies have demonstrated that Torso, and by extension other RTKs, can activate Raf and ERK independently of Ras . In mammalian cells, the small G protein Rap1 has been proposed to couple RTKs to ERKs. However, the ability of Rap1 to activate ERKs remains controversial, in part because direct genetic evidence supporting this hypothesis is lacking. Here, we present biochemical and genetic evidence that D-Rap1, the Drosophila homolog of Rap1, can activate D-Raf and ERK. We show that D-Rap1 binds D-Raf and activates ERKs in a GTP- and D-Raf-dependent manner. Targeted disruption of D-Rap1 expression decreased both Torso-dependent ERK activation and the ERK-dependent expression of the zygotic genes tailless and huckebein to levels similar to those achieved in D-Ras1 null embryos. Furthermore, combined deficiencies of D-Ras1 and D-Rap1 completely abolished expression of these genes, mimicking the phenotype observed in embryos lacking D-Raf. These studies provide the first direct genetic evidence of Rap1-mediated activation of the MAP kinase cascade in eukaryotic organisms.
Mishra
Snigdha
S
Vollum Institute, Oregon Health and Science University, Portland, OR 97201, USA.
Smolik
Sarah M
SM
Forte
Michael A
MA
Stork
Philip J S
PJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila / Q000196:embryology*; D029721:Drosophila Proteins / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D018014:Gene Transfer Techniques; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D048490:raf Kinases / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C087725:Hkb protein, Drosophila; D012097:Repressor Proteins; C074090:TLL protein, Drosophila; D020794:Receptor Protein-Tyrosine Kinases; C080148:tor protein, Drosophila; D048490:raf Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; C417283:Ras85D protein, Drosophila; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins