title: Identification of a peptide inhibitor of the RPM-1 · FSN-1 ubiquitin ligase complex.
abstract: The Pam/Highwire/RPM-1 (PHR) proteins include: Caenorhabditis elegans RPM-1 (Regulator of Presynaptic Morphology 1), Drosophila Highwire, and murine Phr1. These important regulators of neuronal development function in synapse formation, axon guidance, and axon termination. In mature neurons the PHR proteins also regulate axon degeneration and regeneration. PHR proteins function, in part, through an ubiquitin ligase complex that includes the F-box protein FSN-1 in C. elegans and Fbxo45 in mammals. At present, the structure-function relationships that govern formation of this complex are poorly understood. We cloned 9 individual domains that compose the entire RPM-1 protein sequence and found a single domain centrally located in RPM-1 that is sufficient for binding to FSN-1. Deletion analysis further refined FSN-1 binding to a conserved 97-amino acid region of RPM-1. Mutagenesis identified several conserved motifs and individual amino acids that mediate this interaction. Transgenic overexpression of this recombinant peptide, which we refer to as the RPM-1·FSN-1 complex inhibitory peptide (RIP), yields similar phenotypes and enhancer effects to loss of function in fsn-1. Defects caused by transgenic RIP were suppressed by loss of function in the dlk-1 MAP3K and were alleviated by point mutations that reduce binding to FSN-1. These findings suggest that RIP specifically inhibits the interaction between RPM-1 and FSN-1 in vivo, thereby blocking formation of a functional ubiquitin ligase complex. Our results are consistent with the FSN-1 binding domain of RPM-1 recruiting FSN-1 and a target protein, such as DLK-1, whereas the RING-H2 domain of RPM-1 ubiquitinates the target.
authors.affiliation: From the Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, 33458 and.
authors.forename: Jaiprakash
authors.initials: J
authors.lastname: Sharma
authors.affiliation: From the Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, 33458 and Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Baker
authors.affiliation: From the Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, 33458 and.
authors.forename: Shane M
authors.initials: SM
authors.lastname: Turgeon
authors.affiliation: Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Allison M
authors.initials: AM
authors.lastname: Gurney
authors.affiliation: From the Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, 33458 and.
authors.forename: Karla J
authors.initials: KJ
authors.lastname: Opperman
authors.affiliation: From the Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, 33458 and.
authors.forename: Brock
authors.initials: B
authors.lastname: Grill
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000201:enzymology; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D044783:F-Box Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D013569:Synapses / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D029742:Caenorhabditis elegans Proteins; D044783:F-Box Proteins; C530278:FSN-1 protein, C elegans; D020662:Guanine Nucleotide Exchange Factors; D010446:Peptide Fragments; C409634:RPM-1 protein, C elegans; D043743:Ubiquitin-Protein Ligase Complexes
keywords: Caenorhabditis elegans (C. elegans); Cell Signaling; F-box; FSN-1; Neurodegeneration; Neuronal Development; Ubiquitin Ligase; axon