Fbxo45 is an F-box protein that is restricted to the nervous system. Unlike other F-box proteins, Fbxo45 was found not to form an SCF complex as a result of an amino acid substitution in the consensus sequence for Cul1 binding. Proteomics analysis revealed that Fbxo45 specifically associates with PAM (protein associated with Myc), a RING finger-type ubiquitin ligase. Mice deficient in Fbxo45 were generated and found to die soon after birth as a result of respiratory distress. Fbxo45؊/؊ embryos show abnormal innervation of the diaphragm, impaired synapse formation at neuromuscular junctions, and aberrant development of axon fiber tracts in the brain. Similar defects are also observed in mice lacking Phr1 (mouse ortholog of PAM), suggesting that Fbxo45 and Phr1 function in the same pathway. In addition, neuronal migration was impaired in Fbxo45 ؊/؊ mice. These results suggest that Fbxo45 forms a novel Fbxo45-PAM ubiquitin ligase complex that plays an important role in neural development.Ubiquitin-dependent proteolysis is indispensable for various biological processes (3,40). Protein ubiquitylation is mediated by several enzymes that act in concert, with a ubiquitin ligase (E3) playing a key role in substrate recognition (14). E3 enzymes contain specific structural motifs that mediate recruitment of a ubiquitin-conjugating enzyme (E2), with these motifs including HECT, RING finger, U-box, and PHD finger domains (30). The SCF complex consists of Skp1 (adaptor subunit), Cul1 (scaffold subunit), an F-box protein (substrate recognition subunit), and Rbx1 (also known as Roc1 or Hrt1; RING finger-containing subunit). Whereas Skp1, Cul1, and Rbx1 are common to all SCF complexes, the F-box protein is variable (with ϳ70 such proteins having been identified in humans) and confers substrate specificity.Fbxo45 is an F-box protein that was originally isolated as an estrogen-induced protein (47). Human and mouse Fbxo45 genes comprise three exons and possess several consensus binding sequences for the estrogen receptor in the promoter region. Fbxo45 mRNA is rapidly induced on exposure of MCF-7 cells to 17␤-estradiol (47). FSN-1, the Caenorhabditis elegans ortholog of Fbxo45, binds to RPM-1 (regulator of presynaptic morphology 1) together with CUL-1 and SKR-1, the C. elegans orthologs of mammalian Cul1 and Skp1, respectively (21, 46). RPM-1 belongs to an evolutionarily conserved family of proteins (the PHR family) that include Highwire (HIW) (Drosophila melanogaster), Esrom (Danio rerio), Phr1 (Mus musculus), and protein associated with Myc (PAM) (Homo sapiens), each of which contains a RING-finger domain that is required for its E3 activity (7,20,21,27,44). Complete loss of function of fsn-1 in C. elegans results in defects that are characterized by the simultaneous presence of overdeveloped and underdeveloped neuromuscular junctions (NMJs) and which are similar to, but not as pronounced as, those observed in rpm-1 Ϫ/Ϫ mutants. These genetic findings support the notion that the functions of FSN-1 and RPM-1 are partially overlappi...

Neurons communicate with each other through synapses. To establish the precise yet flexible connections that make up neural networks in the brain, continuous synaptic modulation is required. The ubiquitin-proteasome system of protein degradation is one of the critical mechanisms that underlie this process, playing crucial roles in the regulation of synaptic structure and function. We identified a novel ubiquitin ligase, Fbxo45, that functions at synapses. Fbxo45 is evolutionarily conserved and selectively expressed in the nervous system. We demonstrated that the knockdown of Fbxo45 in primary cultured hippocampal neurons resulted in a greater frequency of miniature excitatory postsynaptic currents. We also found that Fbxo45 induces the degradation of a synaptic vesicle-priming factor, Munc13-1. We propose that Fbxo45 plays an important role in the regulation of neurotransmission by modulating Munc13-1 at the synapse.The nervous system stores and retrieves information via synapses, which are its primary means of communication. Synapses are specialized intercellular junctions dedicated to the transfer of information from a neuron to another neuron. Synaptic transmission is rapidly, dynamically, efficiently, and tightly regulated by several molecular mechanisms (1-4). Among these mechanisms, recent studies have shown that synaptic components are modified by protein activation (5) and degradation (6 -9).Protein degradation can be mediated by the ubiquitin-proteasome system (UPS) 4 (10 -12). The modification of proteins by the ligation of ubiquitin molecules is critical role in regulating the degradation of specific proteins, thereby controlling protein turnover. This control mechanism is extremely effective because it allows the rapid elimination of particular regulatory proteins, ensuring that the biological process regulated by the proteins can be shut down immediately. Protein ubiquitination is catalyzed by a cascade of reactions involving three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin-protein ligase). E1 activates ubiquitin in an ATP-dependent reaction and transfers it to E2 with the formation of a thiol ester bond between the C terminus of ubiquitin and a cysteine residue of E2. Next, E2, either by itself or together with E3, transfers the ubiquitin moiety to a lysine residue of the substrate protein. Various E3s have been reported, and the action of each is substrate-specific.

Cullin-based ubiquitin ligases (E3s) constitute one of the largest E3 families. Fbxw8 (also known as Fbw6 or Fbx29) is an F-box protein that is assembled with Cul7 in an SCF-like E3 complex. Here we show that Cul7 forms a heterodimeric complex with Cul1 in a manner dependent on Fbxw8. We generated mice deficient in Fbxw8 and found that Cul7 did not associate with Cul1 in cells of these mice. Two-thirds of Fbxw8 ؊/؊ embryos die in utero, whereas the remaining one-third are born alive and grow to adulthood. Fbxw8 ؊/؊ embryos show intrauterine growth retardation and abnormal development of the placenta, characterized by both a reduced thickness of the spongiotrophoblast layer and abnormal vessel structure in the labyrinth layer. Although the placental phenotype of Fbxw8 ؊/؊ mice resembles that of Cul7 ؊/؊ mice, other abnormalities of Cul7 ؊/؊ mice are not apparent in Fbxw8 ؊/؊ mice. These results suggest that the Cul7-based SCF-like E3 complex has both Fbxw8-dependent and Fbxw8-independent functions.Ubiquitin-dependent proteolysis plays indispensable roles in various biological processes (6,17,38). Protein ubiquitylation is mediated by several enzymes that act in concert. A ubiquitinactivating enzyme (E1), with ATP as a substrate, catalyzes the formation of a thioester bond between itself and ubiquitin, and it then transfers the activated ubiquitin to a ubiquitin-conjugating enzyme (E2). Certain E2 enzymes transfer ubiquitin directly to the protein substrate, whereas others require the participation of a third component, a ubiquitin ligase (E3), to achieve this effect (16). Hundreds of E3s have been identified in eukaryotes and classified into three groups: HECT-type, U-box-type, and RING-type E3s. Cullin-based E3s, a subtype of RING-type E3s, are multisubunit complexes (13,30). To date, seven mammalian proteins, Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7, have been identified as members of the cullin family. These proteins interact via their COOH-terminal cullin homology domain with Rbx1 or Rbx2, each of which contains a RING finger domain that interacts with E2. They also interact via their NH 2 -terminal cullin repeat (with or without the participation of an adapter molecule such as Skp1 or Elongin C) with substrate recognition molecules, such as F-box proteins in the Cul1-based SCF complex (7,20,23,28,39), VHL-box proteins in the Cul2-based ECV complex (22), BTB domain proteins in the 31,40), and SOCS-box proteins in the Cul5-based ECS complex (22). At least two other molecules, the APC2 subunit of the anaphase-promoting complex/cyclosome and the Cul7-related protein Parc, also possess a cullin homology domain (30, 34).Cul7 was originally identified as p185, a protein that binds the large T antigen of simian virus 40 (24). The interaction of p185 with large T antigen is important for the cellular transformation activity of the latter (2), and the BH3 domain identified in the COOH-terminal region of p185 is thought to function in the promotion of apoptosis (36).

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