Fbxo45 Forms a Novel Ubiquitin Ligase Complex and Is Required for Neuronal Development

Abstract

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...

Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival

“…8 We confirmed that human Fbxo45 interacts with endogenous PAM and Skp1, but not with Cullin1 or Rbx1, by immunoprecipitation and western blotting (Supplementary Figure 1d). Yeast two-hybrid analyses revealed that Fbxo45 bound to Skp1 through the F-box domain, and PAM via the SPRY domain (Supplementary Figure 1e).…”

“…[1][2][3][4][5][6] Previous studies have shown that in contrast to other F-box proteins that assemble SCF ubiquitin ligase complexes, Fbxo45 forms an atypical ubiquitin ligase complex that contains Skp1 and the Ring-finger protein PAM (protein-associated with myc) also known as MYCBP2 (mycbinding protein 2). 7,8 Fbxo45 has been linked to the proteasomal degradation of a few targets including p73 9 and Munc13-1. 10 Fbxo45 is one of only six F-box proteins that are conserved in metazoans and the only F-box protein known to contain a SPRY domain, 3 which was first identified as a sequence repeat in the dual-specificity kinase splA and ryanodine receptors.…”

Fbxo45 Inhibits Calcium-sensitive Proteolysis of N-cadherin and Promotes Neuronal Differentiation

“…However, in the F-box protein family, Fbxo45 is the only member that contains a SPRY domain. Previous studies have indicated that Fbxo45 is involved in controlling synapse formation (3), neurotransmitter release (9), as well as neural differentiation (4,10). However, the molecular mechanisms leading to such diverse effects of Fbxo45 are complex and poorly understood.…”

“…However, the molecular mechanisms leading to such diverse effects of Fbxo45 are complex and poorly understood. Both Fbxo45 and MYCBP2 knock-out mice exhibit perinatal lethality and display similar neurological defects suggesting that both molecules may functionally interact and play key roles in neuronal development (4,11).…”

“…Unlike other F-box proteins that interact with CUL1 to form a SCF (Skp1, CUL1, F-box protein) complex (2). Fbxo45 along with Skp1 interacts with MYCBP2 (Myc binding protein 2) also known as protein associated with Myc (PAM) to form an atypical E3 ligase (3)(4)(5). Structurally, Fbxo45 contains an F-box motif at its N terminus that interacts with Skp1 and a SPRY (SplA and ryanodine receptor) domain at its C terminus, which has been shown to function as a substrate recognition motif in other SPRY-containing E3 ligases (6 -8).…”