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1.
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.  相似文献   

2.
Mutations in Nesprin-1 and 2 (also called Syne-1 and 2) are associated with numerous diseases including autism, cerebellar ataxia, cancer, and Emery-Dreifuss muscular dystrophy. Nesprin-1 and 2 have conserved orthologs in flies and worms called MSP-300 and abnormal nuclear Anchorage 1 (ANC-1), respectively. The Nesprin protein family mediates nuclear and organelle anchorage and positioning. In the nervous system, the only known function of Nesprin-1 and 2 is in regulation of neurogenesis and neural migration. It remains unclear if Nesprin-1 and 2 regulate other functions in neurons. Using a proteomic approach in C. elegans, we have found that ANC-1 binds to the Regulator of Presynaptic Morphology 1 (RPM-1). RPM-1 is part of a conserved family of signaling molecules called Pam/Highwire/RPM-1 (PHR) proteins that are important regulators of neuronal development. We have found that ANC-1, like RPM-1, regulates axon termination and synapse formation. Our genetic analysis indicates that ANC-1 functions via the β-catenin BAR-1, and the ANC-1/BAR-1 pathway functions cell autonomously, downstream of RPM-1 to regulate neuronal development. Further, ANC-1 binding to the nucleus is required for its function in axon termination and synapse formation. We identify variable roles for four different Wnts (LIN-44, EGL-20, CWN-1 and CWN-2) that function through BAR-1 to regulate axon termination. Our study highlights an emerging, broad role for ANC-1 in neuronal development, and unveils a new and unexpected mechanism by which RPM-1 functions.  相似文献   

3.
Tulgren ED  Baker ST  Rapp L  Gurney AM  Grill B 《Genetics》2011,189(4):1297-1307
The PHR (Pam/Highwire/RPM-1) proteins are evolutionarily conserved ubiquitin ligases that regulate axon guidance and synapse formation in Caenorhabditis elegans, Drosophila, zebrafish, and mice. In C. elegans, RPM-1 (Regulator of Presynaptic Morphology-1) functions in synapse formation, axon guidance, axon termination, and postsynaptic GLR-1 trafficking. Acting as an E3 ubiquitin ligase, RPM-1 negatively regulates a MAP kinase pathway that includes: dlk-1, mkk-4, and the p38 MAPK, pmk-3. Here we provide evidence that ppm-1, a serine/threonine phosphatase homologous to human PP2Cα(PPM1A) and PP2Cβ(PPM1B) acts as a second negative regulatory mechanism to control the dlk-1 pathway. We show that ppm-1 functions through its phosphatase activity in a parallel genetic pathway with glo-4 and fsn-1 to regulate both synapse formation in the GABAergic motorneurons and axon termination in the mechanosensory neurons. Our transgenic analysis shows that ppm-1 acts downstream of rpm-1 to negatively regulate the DLK-1 pathway, with PPM-1 most likely acting at the level of pmk-3. Our study provides insight into the negative regulatory mechanisms that control the dlk-1 pathway in neurons and demonstrates a new role for the PP2C/PPM phosphatases as regulators of neuronal development.  相似文献   

4.
Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.  相似文献   

5.
BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.  相似文献   

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Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.  相似文献   

9.
The opportunistic intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires’ disease. L. pneumophila delivers nearly 300 effector proteins into host cells for the establishment of a replication-permissive compartment known as the Legionella-containing vacuole (LCV). SidC and its paralog SdcA are two effectors that have been shown to anchor on the LCV via binding to phosphatidylinositol-4-phosphate [PI(4)P] to facilitate the recruitment of ER proteins to the LCV. We recently reported that the N-terminal SNL (SidC N-terminal E3 Ligase) domain of SidC is a ubiquitin E3 ligase, and its activity is required for the recruitment of ER proteins to the LCV. Here we report the crystal structure of SidC (1-871). The structure reveals that SidC contains four domains that are packed into an arch-like shape. The P4C domain (PI(4)P binding of SidC) comprises a four α-helix bundle and covers the ubiquitin ligase catalytic site of the SNL domain. Strikingly, a pocket with characteristic positive electrostatic potentials is formed at one end of this bundle. Liposome binding assays of the P4C domain further identified the determinants of phosphoinositide recognition and membrane interaction. Interestingly, we also found that binding with PI(4)P stimulates the E3 ligase activity, presumably due to a conformational switch induced by PI(4)P from a closed form to an open active form. Mutations of key residues involved in PI(4)P binding significantly reduced the association of SidC with the LCV and abolished its activity in the recruitment of ER proteins and ubiquitin signals, highlighting that PI(4)P-mediated targeting of SidC is critical to its function in the remodeling of the bacterial phagosome membrane. Finally, a GFP-fusion with the P4C domain was demonstrated to be specifically localized to PI(4)P-enriched compartments in mammalian cells. This domain shows the potential to be developed into a sensitive and accurate PI(4)P probe in living cells.  相似文献   

10.
The small ubiquitin related modifier (SUMO)-mediated posttranslational protein modification is widely conserved among eukaryotes. Similar to ubiquitination, SUMO modifications are attached to the substrate protein through three reaction steps by the E1, E2 and E3 enzymes. To date, multiple families of SUMO E3 ligases have been reported in yeast and animals, but only two types of E3 ligases have been identified in Arabidopsis: SAP and Miz 1 (SIZ1) and Methyl Methanesulfonate-Sensitivity protein 21 (MMS21)/HIGH PLOIDY 2 (HPY2), hereafter referred to as HPY2. Both proteins possess characteristic motifs termed Siz/PIAS RING (SP-RING) domains, and these motifs are conserved throughout the plant kingdom. Previous studies have shown that loss-of-function mutations in HPY2 or SIZ1 cause dwarf phenotypes and that the phenotype of siz1-2 is caused by the accumulation of salicylic acid (SA). However, we demonstrate here that the phenotype of hpy2-1 does not depend on SA accumulation. Consistently, the expression of SIZ1 driven by the HPY2 promoter does not complement the hpy2-1 phenotypes, indicating that they are not functional homologs. Lastly, we show that the siz1-2 and hpy2-1 double mutant results in embryonic lethality, supporting the hypothesis that they have non-overlapping roles during embryogenesis. Together, these results suggest that SIZ1 and HPY2 function independently and that their combined SUMOylation is essential for plant development.  相似文献   

11.
The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation.  相似文献   

12.
In the developing nervous system, cohorts of events regulate the precise patterning of axons and formation of synapses between presynaptic neurons and their targets. The conserved PHR proteins play important roles in many aspects of axon and synapse development from C. elegans to mammals. The PHR proteins act as E3 ubiquitin ligases for the dual-leucine-zipper-bearing MAP kinase kinase kinase (DLK MAPKKK) to regulate the signal transduction cascade. In C. elegans, loss-of-function of the PHR protein RPM-1 (Regulator of Presynaptic Morphology-1) results in fewer synapses, disorganized presynaptic architecture, and axon overextension. Inactivation of the DLK-1 pathway suppresses these defects. By characterizing additional genetic suppressors of rpm-1, we present here a new member of the DLK-1 pathway, UEV-3, an E2 ubiquitin-conjugating enzyme variant. We show that uev-3 acts cell autonomously in neurons, despite its ubiquitous expression. Our genetic epistasis analysis supports a conclusion that uev-3 acts downstream of the MAPKK mkk-4 and upstream of the MAPKAPK mak-2. UEV-3 can interact with the p38 MAPK PMK-3. We postulate that UEV-3 may provide additional specificity in the DLK-1 pathway by contributing to activation of PMK-3 or limiting the substrates accessible to PMK-3.CHEMICAL synapses are specialized cellular junctions that enable neurons to communicate with their targets. An electrical impulse causes calcium channel opening and consequently stimulates synaptic vesicles in the presynaptic terminals to fuse at the plasma membrane. Neurotransmitter activates receptors on the postsynaptic membrane and triggers signal transduction in the target cell. For this communication to occur efficiently, the organization of the proteins within these juxtaposed pre- and postsynaptic terminals must be tightly regulated (Jin and Garner 2008). Previous studies in Caenorhabditis elegans have identified RPM-1, a member of the conserved PHR (Pam/Highwire/RPM-1) family of proteins, as an important regulator for the synapse (Schaefer et al. 2000; Zhen et al. 2000). Recent functional studies of other PHR proteins have shown that they are also required for a number of steps during nervous system development including axon guidance, growth, and termination (Wan et al. 2000; D''souza; et al. 2005; Bloom et al. 2007; Grill et al. 2007; Lewcock et al. 2007; Li et al. 2008).The signaling cascades regulated by the PHR proteins have been identified using genetic modifier screens (Diantonio et al. 2001; Liao et al. 2004; Nakata et al. 2005; Collins et al. 2006) and biochemical approaches (Grill et al. 2007; Wu et al. 2007). These studies reveal that a major function of PHR proteins is to act as ubiquitin E3 ligases (Jin and Garner 2008). In C. elegans, RPM-1 (Regulator of Presynaptic Morphology-1) regulates the abundance of its substrate, the dual-leucine-zipper-bearing MAP kinase kinase kinase (DLK MAPKKK), and controls the activity of the MAP kinase cascade composed of three additional kinases, MAPKK MKK-4, p38 MAPK PMK-3, and MAPKAPK MAK-2 (Nakata et al. 2005; Yan et al. 2009). This signaling cascade further regulates the activity of the CCAAT/enhancer binding protein (C/EBP), CEBP-1, via a mechanism involving 3′-UTR-mediated mRNA decay.Signal transduction involving MAP kinases can be fine tuned using multiple mechanisms to ensure optimal signaling outputs (Raman et al. 2007). For example, scaffold proteins for MAP kinases can provide spatial regulation of kinase activation in response to different stimuli (Remy and Michnick 2004; Whitmarsh 2006). Small protein tags such as ubiquitin have also been shown to control the activation of kinases. Specifically, in the IKK pathway ubiquitination via Lys63 chain formation catalyzed by the Ubc13/Uev1a E2 complex and TRAF6 E3 ligase is required for TAK1 kinase activation (Skaug et al. 2009).To further the understanding of the DLK-1 pathway in the development of the nervous system, we characterized a new complementation group of rpm-1(lf) suppressors. These mutations affect the gene uev-3, a ubiquitin E2 conjugating (UBC) enzyme variant (UEV). UEV proteins belong to the UBC family, but lack the catalytic active cysteine necessary for conjugating ubiquitin (Sancho et al. 1998). The best characterized UEV proteins are yeast Mms2 and mammalian Uev1A, both of which act as the obligatory partner for the active E2 Ubc13 and function in DNA repair and IKB pathways, respectively (Deng et al. 2000; Hurley et al. 2006). In addition, UEV proteins, such as Tsg101, can also regulate endosomal trafficking (Babst et al. 2000). We find that similar to other members of the DLK-1 pathway, uev-3 functions cell autonomously in neurons. uev-3 genetically acts downstream of mkk-4 and upstream of mak-2. UEV-3 can bind PMK-3 in heterologous protein interaction assays. We hypothesize that UEV-3 may add specificity to the DLK-1 pathway by binding to PMK-3 for its activation or for selecting specific downstream targets.  相似文献   

13.
E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity.  相似文献   

14.
PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr805, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.  相似文献   

15.
The molecular mechanisms regulating smooth muscle-specific gene expression during smooth muscle development are poorly understood. Myocardin is an extraordinarily powerful cofactor of serum response factor (SRF) that stimulates expression of smooth muscle-specific genes. In an effort to search for proteins that regulate myocardin function, we identified a novel HMG box-containing protein HMG2L1 (high mobility group 2 like 1). We found that HMG2L1 expression is correlated with the smooth muscle cell (SMC) synthetic phenotype. Overexpression of HMG2L1 in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous HMG2L1 in SMCs increases smooth muscle-specific gene expression. Furthermore, we found HMG2L1 specifically abrogates myocardin-induced activation of smooth muscle-specific genes. By GST pulldown assays, the interaction domains between HMG2L1 and myocardin were mapped to the N termini of each of the proteins. Finally, we demonstrated that HMG2L1 abrogates myocardin function through disrupting its binding to SRF and abolishing SRF-myocardin complex binding to the promoters of smooth muscle-specific genes. This study provides the first evidence of this novel HMG2L1 molecule playing an important role in attenuating smooth muscle differentiation.  相似文献   

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Loss of function mutations in the hERG (human ether-a-go-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.  相似文献   

19.
Endosomal trafficking is a key mechanism to modulate signal propagation and cross talk. Ubiquitin adaptors, along with endosomal sorting complex required for transport (ESCRT) complexes, are also integrated to terminate ligand-receptor activation in late endosomes and multivesicular bodies (MVBs). Within these pathways, we recently demonstrated that the protein SIMPLE is a novel player in MVB regulation. SIMPLE is also clinically important and its mutation accounts for the Charcot-Marie-Tooth type 1C (CMT1C) disease. MVB defects of mutation and deletion of SIMPLE, however, are distinct. Here, we show that MVB defects found in mutation but not deletion of SIMPLE lead to impaired turnover and accumulation of ESCRT-0 protein Hrs puncta in late endosomes. We further uncover increased colocalization of ubiquitin ligase TRAF6 and Hrs in late endosomes. Upon stimulation with interkeukin-1 or transforming growth factor β, prolonged activation of p38 kinase/JNK is detected, while nuclear accumulation of NF-κB and phosphorylation of SMAD2 is reduced with CMT1C mutation. The aberrant kinetics we observed in inflammatory signaling may contribute to increased tumor susceptibility and changes in the levels of chemokines/cytokines that result from CMT1C mutation. We propose that altered endosomal trafficking due to malformations of MVBs and subsequent atypical signaling kinetic may account for a toxic gain of function in CMT1C pathogenesis.  相似文献   

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