Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex

Abstract

Background: How RPM-1 interacts with FSN-1 remains unknown. Results: Structure-function and transgenic analysis define the biochemical relationship between RPM-1 and FSN-1. Conclusion: RPM-1 uses a conserved mechanism to bind FSN-1 that is independent of RPM-1 ubiquitin ligase activity. Significance: Our biochemical and genetic analysis has led to identification of RIP, an in vivo inhibitor of the RPM-1⅐FSN-1 ubiquitin ligase complex.

Defining Minimal Binding Regions in Regulator of Presynaptic Morphology 1 (RPM-1) Using Caenorhabditis elegans Neurons Reveals Differential Signaling Complexes

“…This approach revealed two new domains in RPM-1, FBD2 and FBD3, which are sufficient for binding to FSN-1. Because these interactions were not observed previously using 293 cells (24), our results show that binding of FSN-1 to portions of RPM-1 requires in vivo expression in worm neurons. Using a similar approach, we map the RBD to a significantly smaller region in RPM-1 and show that mutations in RPM-1 that reduce binding to RAE-1 do not affect binding to FSN-1.…”

“…However, corresponding mutations in full-length RPM-1 did not affect binding to FSN-1 in worm neurons (24). This raised two points: there were likely to be other sites in RPM-1, besides FBD1, that were sufficient for binding to FSN-1, and identification of these sites was likely to require in vivo biochemistry from transgenic C. elegans.…”

“…Multiple Domains in RPM-1 Are Sufficient for Binding to FSN-1-Our previous work showed that FBD1 of RPM-1 is sufficient for binding to FSN-1, and we identified point mutations in FBD1 that reduce binding in 293 cells (24). However, corresponding mutations in full-length RPM-1 did not affect binding to FSN-1 in worm neurons (24).…”

“…FBD1 represents a small portion of D5, and D5 is sufficient for binding to FSN-1 in 293 cells (24). We generated worms carrying transgenic extrachromosomal arrays that used a pan-neuronal promoter (Prgef-1) to simultaneously express FLAG-tagged FSN-1 (FLAG::FSN-1) and GFP fusions with different RPM-1 constructs (Fig.…”

“…We previously analyzed the biochemical mechanism underlying the interaction between RPM-1 and FSN-1 (24). Using HEK 293 cells as a heterologous expression system, we found a single, conserved domain in RPM-1 that is sufficient for binding to FSN-1.…”