Structures of PHR domains from Mus musculus Phr1 (Mycbp2) explain the loss of function mutation (Gly1092→Glu) of the C. elegans ortholog RPM-1

Targeting proteins for proteasome mediated degradation through poly-ubiquitination (ubiquitin proteasome system, UPS) represents an important mechanism controlling various aspects of cellular life.1 Dysfunction of the neuronal UPS has been implicated in neurodegenerative diseases.2 PHR proteins are one of several E3 ubiquitin ligases that function within the nervous system. In recent years, numerous publications have illustrated the critical roles PHR proteins play in proper formation and function of synapses at neuron muscular junctions and in axon guidance during embryonic development.3-5 PHR (protein associated with Myc (PAM) from human; Highwire (HIW) from Drosophila; Regulator of Presynaptic Morphology 1 (RPM-1) of C. elegans) proteins are huge, multi-domain E3 ubiquitin ligases.6-9 Phr1 and Esrom are well characterized, conserved PHR orthologs from mouse and zebrafish, respectively.10,11 Impairment of RPM-1 activity in C. elegans leads to decreased number of synapses and improper organization of presynaptic terminals. RPM-1 also postsynaptically regulates endocytosis of the AMPA-type glutamate receptor.12 hiw mutants of Drosophila, in contrast to rpm-1 mutants, show increased numbers of synaptic boutons, albeit smaller in size, and more extensive synaptic branches. In vertebrates, Phr1 and Esrom play critical roles in both nerve and synapse development and in axon growth and guidance.13-15 Phr1 associates with the microtubule cytoskeleton and regulates microtubule stability, which is necessary for appropriate neurite outgrowth.13 More recently, differential purification of neurite and soma using transwells containing 3.0μm porous filters combined with quantitative proteome analysis revealed that Phr1 is 12 fold enriched within the neurite,16 underscoring its functional importance in neurite outgrowth and axon guidance. Interestingly, Phr1 is also localized in the pseudopodium, but not the cell body of the migrating Cos-7 cell.17 Pseudopodial targeting of Phr1 and its interaction with the microtubule cytoskeleton suggest that Phr1 plays a role in regulating pseudopodium formation, which is necessary for proper cell migration. PHR proteins regulate different MAP kinase signaling pathways in various organisms.13,18,19 RPM-1 and HIW have been shown to function in an SCF-like (Skp1, Cullin, and FBox) E3 ubiquitin ligase complex. RPM-1 interacts with FSN-1 in the SKR-1:CUL-1:FSN-1 complex20 and negatively regulates the PMK-3 pathway (a C. elegans ortholog of p38 kinase) by targeting the upstream dual leucine-zipper bearing kinase-1 (DLK-1), a MAPKKK, for degradation.18 Similarly, HIW interacts with the F-Box protein DFsn to target Wallenda for degradation, the closest Drosophila ortholog of DLK-1,21 although the downstream substrate of Wallenda is JNK kinase.19 Human PAM plays a role in regulating mTOR (mammalian target of rapamycin) signaling through its interaction with tuberin, a component of Tuberous Sclerosis Complex.22 Other targets of PHR proteins include ALK,20 a C. elegans ortholog of mammalian receptor tyrosine kinase, Myc,6 Medea,23 and cyclic AMP metabolism.24 RPM-1 also positively regulates the Rab GTPase GLO-1 via a ubiquitin ligase independent mechanism.25 PHR proteins show similarity both in their modular architecture and individual domain sequences. They contain a RHD (regulator of chromosome condensation 1, RCC1-homology) domain and two PHR repeats within the N-terminal portion, a Myc-binding region, an HHD (histone-binding protein homology domain) in the middle portion, and a RING finger domain and two C2H2-type Zinc binding motifs within the C-terminal portion.6 All PHR proteins known to date are characterized by the presence of two PHR domains. In addition to the PHR family proteins, BTBD1 and BTBD2 proteins contain “PHR-like domains”.26 Mechanistic insights into the role of PHR domains have been obtained only recently. Abrams et al. (2008)27 showed that C. elegans RPM-1 mutants lacking PHR domains accumulate in cell bodies, and that the PHR domains are both necessary and sufficient for proper localization of RPM-1 in synapses, which is essential for function. Structures of the first PHR (MmPHR1, residues 1229-1390) and the second PHR (MmPHR2, residues 1723-1883) domains were determined at 1.3A and 2.25A resolution, respectively. These structures provide the first atomic views of PHR domains, which share a novel β sandwich architecture. Mapping of sequence conservation revealed unique surface features that could be important for the function of PHR proteins. Moreover, structural insights into the Gly1092→Glu loss of function mutation of RPM-1, the C. elegans homologue of MmPhr1, have been obtained.