Defective in Mitotic Arrest 1 (Dma1) Ubiquitin Ligase Controls G1 Cyclin Degradation

Progression through the G₁ phase of the cell cycle is controlled by diverse cyclin-dependent kinases (CDKs) that might be associated to numerous cyclin isoforms. Given such complexity, regulation of cyclin degradation should be crucial for coordinating progression through the cell cycle. In Saccharomyces cerevisiae, SCF is the only E3 ligase known to date to be involved in G₁ cyclin degradation. Here, we report the design of a genetic screening that uncovered Dma1 as another E3 ligase that targets G₁ cyclins in yeast. We show that the cyclin Pcl1 is ubiquitinated in vitro and in vivo by Dma1, and accordingly, is stabilized in dma1 mutants. We demonstrate that Pcl1 must be phosphorylated by its own CDK to efficiently interact with Dma1 and undergo degradation. A nonphosphorylatable version of Pcl1 accumulates throughout the cell cycle, demonstrating the physiological relevance of the proposed mechanism. Finally, we present evidence that the levels of Pcl1 and Cln2 are independently controlled in response to nutrient availability. This new previously unknown mechanism for G₁ cyclin degradation that we report here could help elucidate the specific roles of the redundant CDK-cyclin complexes in G₁.     

RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.     

The Ubiquitin Ligase RPM-1 and the p38 MAPK PMK-3 Regulate AMPA Receptor Trafficking

Ubiquitination occurs at synapses, yet its role remains unclear. Previous studies demonstrated that the RPM-1 ubiquitin ligase organizes presynaptic boutons at neuromuscular junctions in C. elegans motorneurons. Here we find that RPM-1 has a novel postsynaptic role in interneurons, where it regulates the trafficking of the AMPA-type glutamate receptor GLR-1 from synapses into endosomes. Mutations in rpm-1 cause the aberrant accumulation of GLR-1 in neurites. Moreover, rpm-1 mutations enhance the endosomal accumulation of GLR-1 observed in mutants for lin-10, a Mint2 ortholog that promotes GLR-1 recycling from Syntaxin-13 containing endosomes. As in motorneurons, RPM-1 negatively regulates the pmk-3/p38 MAPK pathway in interneurons by repressing the protein levels of the MAPKKK DLK-1. This regulation of PMK-3 signaling is critical for RPM-1 function with respect to GLR-1 trafficking, as pmk-3 mutations suppress both lin-10 and rpm-1 mutations. Positive or negative changes in endocytosis mimic the effects of rpm-1 or pmk-3 mutations, respectively, on GLR-1 trafficking. Specifically, RAB-5(GDP), an inactive mutant of RAB-5 that reduces endocytosis, mimics the effect of pmk-3 mutations when introduced into wild-type animals, and occludes the effect of pmk-3 mutations when introduced into pmk-3 mutants. By contrast, RAB-5(GTP), which increases endocytosis, suppresses the effect of pmk-3 mutations, mimics the effect of rpm-1 mutations, and occludes the effect of rpm-1 mutations. Our findings indicate a novel specialized role for RPM-1 and PMK-3/p38 MAPK in regulating the endosomal trafficking of AMPARs at central synapses.     

Mitotic Regulation of the Stability of Microtubule Plus-end Tracking Protein EB3 by Ubiquitin Ligase SIAH-1 and Aurora Mitotic Kinases

Microtubule plus-end tracking proteins (+TIPs) control microtubule dynamics in fundamental processes such as cell cycle, intracellular transport, and cell motility, but how +TIPs are regulated during mitosis remains largely unclear. Here we show that the endogenous end-binding protein family EB3 is stable during mitosis, facilitates cell cycle progression at prometaphase, and then is down-regulated during the transition to G₁ phase. The ubiquitin-protein isopeptide ligase SIAH-1 facilitates EB3 polyubiquitination and subsequent proteasome-mediated degradation, whereas SIAH-1 knockdown increases EB3 stability and steady-state levels. Two mitotic kinases, Aurora-A and Aurora-B, phosphorylate endogenous EB3 at Ser-176, and the phosphorylation triggers disruption of the EB3-SIAH-1 complex, resulting in EB3 stabilization during mitosis. Our results provide new insight into a regulatory mechanism of +TIPs in cell cycle transition.Microtubule dynamics are essential in many cellular processes, including cell motility, intracellular transport, accurate mitosis, and cytokinesis in all eukaryotes. The regulatory factors for microtubule dynamics can be classified into two main types as follows: microtubule-destabilizing proteins, such as stathmin/Op18 (1) and the Kinesin-13 family (also known as MCAK/KIF2 family) (2), and microtubule-stabilizing proteins, the classic superfamily of microtubule-associated proteins (3). Additionally, the plus-end tracking proteins (+TIPs)³ have recently been identified; this family specifically accumulates at the ends of growing microtubules and regulates the microtubule plus-end targeting to the cell cortex or mitotic kinetochores (4, 5).The EB1 family is a member of the +TIPs family and consists of three homologs in mammals: EB1, EB2/RP1 (henceforth, EB2), and EB3 (6). As EB1 was originally identified as a protein that interacts with the well characterized tumor suppressor adenomatous polyposis coli (APC) protein (7), the function of EB1 has been investigated extensively. EB1 interacts with other +TIPs, including APC, p150^glued, CLIPs, and CLASP1/2, and the interaction network controls microtubule orientation and microtubule-cortex interaction during cell migration (5, 8, 9). EB1 functions not only in the regulation of interphase microtubule dynamics but also in mitotic spindle regulation. For accurate chromosomal segregation, sister chromatids become aligned to the metaphase plate during metaphase, and the alignment requires spindle-kinetochore attachment. Two models have been proposed; in the first, termed the “search-and-capture” model, EB1 localized at the growing microtubule plus-ends searches for binding partners located on kinetochores (10, 11). In the second model proposed recently, EB1 makes kinetochore fibers and centrosomal microtubules connect, and it is essential for the formation of a functional bipolar spindle (12). Thus, EB1 is thought to be a master controller of microtubule plus-ends; however, little is known about other EB1 family members. Given that EB3 is localized on the microtubule network and binds to APC and CLIPs identically to EB1, it is possible that EB3 acts as an EB1 analog in cells (13–15).Cell division is precisely regulated by several post-translational modifications of proteins, mainly reversible phosphorylation and ubiquitination, which is followed by degradation. Accurate mitotic phase progression requires the appropriate phosphorylation of various proteins by mitotic kinases (16, 17). One of the key mitotic kinases is the Aurora family that has been highly conserved from yeast to humans. There are three homologs (Aurora-A, -B, and -C) in human and mouse (18). Although their homology at the protein level is more than 84%, their functions and subcellular localizations are distinct. Aurora-A is located in the centrosomes and spindle and is required for mitotic entry, centrosome maturation/separation, and spindle assembly (19). Aurora-B is a chromosomal passenger protein that localizes on the inner centromere of the chromosomes until metaphase to regulate the spindle-kinetochore attachment, and from anaphase, it translocates to the central spindle and then accumulates in the midbody for cytokinesis (20, 21). The numerous substrates of the Aurora family include regulatory factors for microtubule dynamics, such as the microtubule-destabilizing proteins MCAK and stathmin, which help to establish the bipolar attachment and spindle assembly, respectively (22–24). It is possible that the Aurora family regulates the EB1 family by phosphorylation.In this study, we performed yeast two-hybrid screening and obtained the EB1 yeast homolog Bim1 as a protein that interacts with Ipl1, a yeast counterpart of Aurora. Here we demonstrate the novel regulatory mechanisms of EB3 by two cell cycle-dependent post-translational modifications, phosphorylation and ubiquitin-proteasome-mediated degradation.     

The Ubiquitin Receptor DA1 Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis

Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement.     

Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1^K29R-K31R-GFP remained. The HPG1-1 (Rsp5^P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.     

Suppression of HIV-1 Integration by Targeting HIV-1 Integrase for Degradation with A Chimeric Ubiquitin Ligase

Human immunodeficiency virus(HIV) attacks human immune system and causes life-threatening acquired immune deficiency syndrome(AIDS). Treatment with combination antiretroviral therapy(cART) could inhibit virus growth and slow progression of the disease, however, at the same time posing various adverse effects. Host ubiquitin-proteasome pathway(UPP) plays important roles in host immunity against pathogens including viruses by inducing degradation of viral proteins. Previously a series of methods for retargeting substrates for ubiquitin-proteasome degradation have been successfully established. In this study, we attempted to design and construct artificial chimeric ubiquitin ligases(E3 s) based on known human E3 s in order to manually target HIV-1 integrase for ubiquitin proteasome pathway-mediated degradation.Herein, a series of prototypical chimeric E3 s have been designed and constructed, and original substrate-binding domains of these E3 s were replaced with host protein domains which interacted with viral proteins. After functional assessment screening, 146 LI was identified as a functional chimeric E3 for HIV-1 NL4-3 integrase. 146 LI was then further optimized to generate 146 LIS(146 LI short) which has been shown to induce Lys48-specific polyubiquitination and reduce protein level of HIV-1 NL4-3 integrase more effectively in cells. Lymphocyte cells with 146 LIS knock-in generated by CRISPR/Cas-mediated homology-directed repair(HDR) showed remarkably decreased integration of HIV-1 NL4-3 viral DNAs and reduced viral replication without obvious cell cytotoxicity. Our study successfully obtained an artificial chimeric E3 which can induce Lys48-specific polyubiquitination and proteasome-mediated degradation of HIV-1 NL4-3 integrase, thus effectively inhibiting viral DNA integration and viral replication upon virus infection.     

KF-1 Ubiquitin Ligase: Anxiety Suppressor Model

Anxiety disorders are the most popular psychiatric disease in any human societies irrespective of nation, culture, religion, economics or politics. Anxiety expression mediated by the amygdala may be suppressed by signals transmitted from the prefrontal cortex and hippocampus. KF-1 is an endoplasmic reticulum (ER)-based E3-ubiquitin (Ub) ligase with a RING-H2 finger motif at the C-terminus. The kf-1 gene expression is up-regulated in the frontal cortex and hippocampus in rats after anti-depressant treatments. The kf-1 null mice show no apparent abnormalities, but exhibit selectively pronounced anxiety-like behaviors or increased timidity-like responses. The kf-1 orthologous genes had been generated after the Poriferan emergence, and are found widely in all animals except insects, arachnids and threadworms such as Drosophila, Ixodes and Caenorhabditis, respectively. This suggests that the kf-1 gene may be relevant to some biological functions characteristic to animals. Based on these observations, the Anxiety Suppressor Model has been proposed, which assumes that KF-1 Ub ligase may suppress the amygdala-mediated anxiety by degrading some anxiety promoting protein(s), such as a neurotransmitter receptor, through the ER-associated degradation pathway in the frontal cortex and hippocampus. According to this model, the emotional sensitivity to environmental stresses may be regulated by the cellular protein level of KF-1 relative to that of the putative anxiety promoter. The kf-1 null mice should be useful in elucidating the molecular mechanisms of the anxiety regulation and for screening novel anxiolytic compounds, which may block the putative anxiety promoter.     

Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrP^Sc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis.     

Herpes Simplex Virus 1 Ubiquitin Ligase ICP0 Interacts with PML Isoform I and Induces Its SUMO-Independent Degradation

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 localizes to cellular structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10 and disrupts their integrity by inducing the degradation of PML. There are six PML isoforms with different C-terminal regions in ND10, of which PML isoform I (PML.I) is the most abundant. Depletion of all PML isoforms increases the plaque formation efficiency of ICP0-null mutant HSV-1, and reconstitution of expression of PML.I and PML.II partially reverses this improved replication. ICP0 also induces widespread degradation of SUMO-conjugated proteins during HSV-1 infection, and this activity is linked to its ability to counteract cellular intrinsic antiviral resistance. All PML isoforms are highly SUMO modified, and all such modified forms are sensitive to ICP0-mediated degradation. However, in contrast to the situation with the other isoforms, ICP0 also targets PML.I that is not modified by SUMO, and PML in general is degraded more rapidly than the bulk of other SUMO-modified proteins. We report here that ICP0 interacts with PML.I in both yeast two-hybrid and coimmunoprecipitation assays. This interaction is dependent on PML.I isoform-specific sequences and the N-terminal half of ICP0 and is required for SUMO-modification-independent degradation of PML.I by ICP0. Degradation of the other PML isoforms by ICP0 was less efficient in cells specifically depleted of PML.I. Therefore, ICP0 has two distinct mechanisms of targeting PML: one dependent on SUMO modification and the other via SUMO-independent interaction with PML.I. We conclude that the ICP0-PML.I interaction reflects a countermeasure to PML-related antiviral restriction.     

Targeted Ubiquitination and Degradation of G-Protein-Coupled Receptor Kinase 5 by the DDB1-CUL4 Ubiquitin Ligase Complex

The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors (GPCRs) and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1), an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex-dependent proteolysis pathway.     

The E3 Ubiquitin Ligase HOS1 Regulates Arabidopsis Flowering by Mediating CONSTANS Degradation Under Cold Stress

The timing of flowering is coordinated by a web of gene regulatory networks that integrates developmental and environmental cues in plants. Light and temperature are two major environmental determinants that regulate flowering time. Although prolonged treatment with low nonfreezing temperatures accelerates flowering by stable repression of FLOWERING LOCUS C (FLC), repeated brief cold treatments delay flowering. Here, we report that intermittent cold treatments trigger the degradation of CONSTANS (CO), a central activator of photoperiodic flowering; daily treatments caused suppression of the floral integrator FLOWERING LOCUS T (FT) and delayed flowering. Cold-induced CO degradation is mediated via a ubiquitin/proteasome pathway that involves the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1). HOS1-mediated CO degradation occurs independently of the well established cold response pathways. It is also independent of the light signaling repressor CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase and light wavelengths. CO has been shown to play a key role in photoperiodic flowering. Here, we demonstrated that CO served as a molecular hub, integrating photoperiodic and cold stress signals into the flowering genetic pathways. We propose that the HOS1-CO module contributes to the fine-tuning of photoperiodic flowering under short term temperature fluctuations, which often occur during local weather disturbances.     

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD.     

The Ubiquitin Ligase Itch Regulates Apoptosis by Targeting Thioredoxin-interacting Protein for Ubiquitin-dependent Degradation

Thioredoxin interacting protein (TXNIP) was originally characterized as an endogenous inhibitor of thioredoxin, a key regulator in cellular redox homeostasis. TXNIP is also known to play important roles in tumor growth and metastasis, glucose and lipid metabolism. TXNIP expression is induced by various stress stimuli. However, it has been unclear how TXNIP is down-regulated. Here, we report that TXNIP undergoes proteasomal degradation in cells. We identify Itch as the E3 ubiquitin ligase for TXNIP. We demonstrate that Itch mediates polyubiquitination of TXNIP both in vitro and in vivo. Overexpression of Itch leads to TXNIP proteasomal degradation. Knockdown of Itch by small interfering RNA causes an accumulation of the steady-state level of TXNIP. We also show that the PPXY motifs of TXNIP and the WW domains of Itch mediate their interaction. Furthermore, the Itch-TXNIP interaction regulates intracellular reactive oxygen species levels and apoptosis. These findings establish a new mechanism for the negative regulation of TXNIP by Itch and shed new light on the regulation of cellular redox homeostasis.     

Activation Domain-dependent Degradation of Somatic Wee1 Kinase

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G₂ and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.