PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2.

PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.

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

The intracellular signaling protein regulator of presynaptic morphology 1 (RPM-1) is a conserved regulator of synapse formation and axon termination in Caenorhabditis elegans RPM-1 functions in a ubiquitin ligase complex with the F-box protein FSN-1 and functions through the microtubule binding protein RAE-1. Using a structure-function approach and positive selection for transgenic C. elegans, we explored the biochemical relationship between RPM-1, FSN-1, and RAE-1. This led to the identification of two new domains in RPM-1 that are sufficient for binding to FSN-1, called FSN-1 binding domain 2 (FBD2) and FBD3. Furthermore, we map the RAE-1 binding domain to a much smaller region of RPM-1. Point mutations in RPM-1 that reduce binding to RAE-1 did not affect FSN-1 binding, indicating that RPM-1 utilizes different biochemical mechanisms to bind these molecules. Analysis of RPM-1 protein complexes in the neurons of C. elegans elucidated two further discoveries: FSN-1 binds to RAE-1, and this interaction is not mediated by RPM-1, and RPM-1 binding to FSN-1 and RAE-1 reduces FSN-1·RAE-1 complex formation. These results indicate that RPM-1 uses different mechanisms to recruit FSN-1 and RAE-1 into independent signaling complexes in neurons.

Identification of a peptide inhibitor of the RPM-1 · FSN-1 ubiquitin ligase complex.

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.