To gain insight into how this pathway can be employed to induce different cellular responses, we sought to identify factors that modify specific levels of RasMAPK signaling in the genetically malleable model organism Drosophila.Montague, Sarah V.Paramore, Erez Cohen, Hamed Zaribafzadeh, Christopher M.Montague 1 Department of Pharmacology Cancer Biology, Duke University Medical Center, DUMC Box 3813, Durham, NC 27710 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sarah V.
Paramore 1 Department of Pharmacology Cancer Biology, Duke University Medical Center, DUMC Box 3813, Durham, NC 27710 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Erez Cohen 2 Department of Cell Biology, Duke University Medical Center, DUMC Box 3813, Durham, NC 27710 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hamed Zaribafzadeh 1 Department of Pharmacology Cancer Biology, Duke University Medical Center, DUMC Box 3813, Durham, NC 27710 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christopher M. Counter 1 Department of Pharmacology Cancer Biology, Duke University Medical Center, DUMC Box 3813, Durham, NC 27710 3 Duke Cancer Institute, Duke University Medical Center, DUMC Box 3813, Durham, NC 27710 Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence. Given this intensity-dependence, we hypothesized that different levels of RasMAPK signaling may be subjected to regulation by different sets of proteins. To identify such differential signaling modifiers, we turned to the Drosophila eye as a model. First, we created flies whereby mutant active Ras V12 expressed in the eyes was enriched with either rare or commonly occurring codons. We show that this codon manipulation can generate either low or high levels of Ras protein and MAPK signaling, and correspondingly a mild or severe rough-eye phenotype. We then mapped the underlying gene from one deficiency to the gene RpS21. Disrupting RpS21 expression increases MAPK signaling and enhances the rough-eye phenotype specifically when Ras V12 is encoded by rare codons (low signaling), and RpS21 negatively regulates Ras protein and MAPK signaling in several contexts. We also provide evidence that the MAPK pathway promotes expression of RpS21, providing potential negative feedback. INTRODUCTION The Rasmitogen activated protein kinase (MAPK) pathway is evolutionarily conserved throughout eukaryotes, serving to transfer external cues to the nucleus to promote a host of cellular responses such as tissue growth and fate determination. Canonical MAPK signaling initiates with the activation of a Receptor Tyrosine Kinase (RTK) by its cognate factor. In turn, this activates the Ras GTPase, by converting it from an inactive GDP-bound to an active GTP-bound conformation. Ras-GTP then activates the MAPK pathway, comprised of Raf kinases, which are activated by Ras and phosphorylateactive Mek kinases, which do the same to Erk kinases. Activation of this cascade then initiates further downstream signaling, often through activation of downstream transcription factors 1. Dysregulation of the RasMAPK pathway is well established to derail a host of biological phenotypes 2. In humans, weak mutations activating components of this signaling cascade give rise to developmental diseases termed RASopathies 3 while stronger mutations in Ras, Raf, and Mek are well established to cause cancer 4. Understanding how the RasMAPK pathway is regulated thus has important implications not only across evolution, but also with regards to human health. Recent evidence suggests that the signaling intensity of the RasMAPK pathway has biologically important implications. Specifically, expressing the same activating mutant of Mek in either Drosophila melanogaster (fruit fly) or Danio rerio (zebrafish) was recently shown to either activate or repress Erk phosphorylation depending on the cell type and gene expression environment. These results suggest that specialtemporal modifiers of specific RasMAPK signaling intensities may drive distinct cellular phenotypes 5.
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