UBR4 (Ubiquitin Ligase E3 Component N-Recognin 4)
p600/UBR4 was initially discovered from a human brain cDNA library that was size fractionated for large cDNAs (Ohara et al. 1997). Subsequent investigations into the structure and function of p600/UBR4 identified it as a large, multifunctional protein that is ubiquitously expressed throughout the body. The name UBR4 refers to its function as an E3 ubiquitin (Ub) ligase in the N-end rule proteolytic pathway, whereas the alternative name of p600 reflects the ∼600 kDa size of this protein. Its role in the N-end rule pathway was revealed in a study that established significant retention of the pathway’s activity in fibroblasts that lacked the two known E3 Ub ligases, UBR1 and UBR2 (Tasaki et al. 2005). Further experiments showed that p600/UBR4 and UBR5 were responsible for the residual N-end rule pathway activity (Tasaki et al. 2005). Mammalian p600/UBR4 is a sequelog of Drosophila melanogaster PUSHOVER/POE (purity of essence) and Arabidopsis thaliana BIG, which share similarities in their sequence, size, and protein domains (Tasaki et al. 2005).
Structure and Expression
Molecular and Cellular Functions
Protein degradation in eukaryotes is largely mediated by the ubiquitin-proteasome system (UPS), where the polyubiquitination of target proteins is accomplished by the combined action of three enzyme complexes: the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin ligases. The polyubiquitinated protein is then recognized by the 26S proteasome and degraded (Tasaki et al. 2005). A specific recognition mode of this proteolytic system is the evolutionarily conserved N-end rule pathway. The N-end rule pathway involves the recognition of a substrate’s destabilizing N-terminal residue referred to as the N-degron (degradation signal) by a special class of E3 ubiquitin ligases called N-recognins. In mammals, p600/UBR4 is one of four identified N-recognins (Tasaki et al. 2005).
As an N-recognin, p600/UBR4 has been implicated in the selective proteasomal degradation of cellular proteins. In mouse embryonic fibroblasts (MEFs), p600/UBR4 mediates the proteasomal degradation of the PARKIN-recruiting protein, PTEN-induced putative kinase 1 (PINK1), which is involved in the elimination of damaged mitochondria (Yamano and Youle 2013). Mutations in PINK1 have been found in patients with Parkinson’s disease; however, whether p600/UBR4 plays a role in the pathogenesis of Parkinson’s disease remains unknown (Pan and Yue 2014). Moreover, in mIMCD3 renal collecting duct cells, p600/UBR4 interacts with and is responsible for the ubiquitination and proteasomal degradation of the transient receptor potential cation channel subfamily V member 5 (TRPV5) calcium-selective channel in response to inflammatory cytokines (Radhakrishnan et al. 2013). In human embryonic kidney (HEK) 293 cells and A549 lung cancer cells, p600/UBR4 plays a role in cellular metabolism by associating with and mediating the ubiquitin-dependent proteasomal degradation of ATP citrate lyase (ACLY), a protein that couples energy metabolism with fatty acid synthesis (Lin et al. 2013). The ubiquitination of ACLY occurs at lysine residues 540, 546, and 554 (Lin et al. 2013). In kidney podocytes, p600/UBR4 interacts with and ubiquitinates podocin, a membrane protein that comprises the filtering complex, at two conserved lysine residues (Rinschen et al. 2016). UBR4 regulates the unfolding, stability, and proteasomal-mediated degradation of podocin (Rinschen et al. 2016).
Furthermore, p600/UBR4 is crucial for integrin-dependent membrane ruffling and cell survival (Nakatani et al. 2005). p600/UBR4 physically associates with microtubules (MT) and the endoplasmic reticulum (ER) via its MT-binding domains and ER-binding domains, respectively (Shim et al. 2008). In cultured primary neurons, p600/UBR4 is required for neurite extension and stability (Shim et al. 2008). Loss of p600/UBR4 expression impairs neuronal migration in the neocortex, leading to mispositioning of cortical neurons in the developing brain (Shim et al. 2008). This is due partly to poor development of the leading process of migrating neurons through destabilization of microtubules and reduced localization of ER membranes in this domain (Shim et al. 2008). In apical neural progenitor cells (aNPs), p600/UBR4 promotes the proper alignment of mitotic spindle fibers, likely as a consequence of its stabilizing effect on MTs and its interaction with the neurogenic protein Ndel1, which is critical for spindle organization (Belzil et al. 2014a). The aberrant tilting of spindle fibers in aNPs upon loss of p600/UBR4 correlates with fragmentation of the ER, premature neuronal differentiation of aNPs, reduced neuronal survival, and overall reduction in neurogenesis (Belzil et al. 2014a).
p600/UBR4 has also been implicated in Ca2+ signaling, which may contribute to p600/UBR4’s effects on cytoskeletal remodeling, neuronal migration, and cell survival (Fig. 2). In mature hippocampal neurons, p600/UBR4 is required to maintain cellular homeostasis in response to glutamate-induced Ca2+ influx (Belzil et al. 2013). Glutamate induces Ca2+-dependent association of p600/UBR4 and calmodulin-bound Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα), and formation of this protein complex is essential for neuronal survival (Belzil et al. 2013). p600/UBR4 depletion leads to CaMKIIα aggregation, ER fragmentation, and neuronal degeneration, effects that are similarly triggered by disrupting direct binding between p600/UBR4 and CaM (Belzil et al. 2013). Unlike glutamate stimulation, direct depolarization of neurons suppresses self-aggregation of CaMKIIα by an indirect mechanism that involves p600/UBR4-mediated MT stabilization and recruitment of CaMKIIα to MTs (Belzil et al. 2014b). In Drosophila melanogaster, PUSHOVER has been identified as a CaM-binding protein with functions in perineural glial cell growth and synaptic transmission at neuromuscular junctions, where it affects neuronal excitation and neurotransmitter release (Richards et al. 1996; Xu et al. 1998; Yager et al. 2001). In Arabidopsis thaliana, BIG is required for vesicle-mediated transport of the plant hormone auxin and contributes to light and hormone signaling pathways (Gil et al. 2001; Kanyuka et al. 2003).
In human foreskin fibroblasts, p600/UBR4 is required for the formation of membrane ruffles and anterior lamellipodia, structures required for cellular migration and adhesion, respectively (Fig. 2) (Nakatani et al. 2005). As a result, p600/UBR4-deficient fibroblasts show defective migration, impaired cell-cell and cell-matrix interactions, and detachment-induced apoptosis (anoikis) (Nakatani et al. 2005). p600/UBR4 may inhibit anoikis through its ability to activate integrin-dependent survival pathways. The non-receptor protein tyrosine kinase, focal adhesion kinase (FAK), functions as a regulator of cellular survival in response to extracellular signals. When triggered by extracellular ligand binding, integrin clusters at the leading edge promote the recruitment and formation of the focal adhesion complex, which acts to facilitate cell-matrix interactions. A crucial step in the assembly of this complex is the activation of FAK via phosphorylation at tyrosine-397. Loss of p600/UBR4 in fibroblast cells correlates with significantly lower levels of the phospho-active form of FAK in both total cell lysate and in the poorly developed lamellipodia extensions (Nakatani et al. 2005). The anti-apoptotic role of p600/UBR4 may also be mediated by its physical interactions with the anti-apoptotic proteins, cellular inhibitors of apoptosis 1 and 2 (c-IAP1, c-IAP2) (Chu et al. 1997; Goncharov et al. 2013), as well as with etoposide-induced 2.4 kb transcript (Ei24), a pro-apoptotic factor involved in p53-mediated apoptosis (Gu et al. 2000; Bahk et al. 2010). p600/UBR4 may also influence p53-induced apoptosis through a binding interaction with the retinoblastoma (Rb) protein (Nakatani et al. 2005).
Consistent with its role in promoting cell migration and survival, p600/UBR4 expression is upregulated in gastric cancer and contributes to the increased invasiveness and ability of cancer cells to proliferate and survive under anchorage-independent conditions, enhancing their metastatic potential (Sakai et al. 2011). Similarly, analysis of HPV-16-positive CaSki cervical cancer cells and HPV-16 E7-transformed NIH-3T3 mouse fibroblasts shows that loss of p600/UBR4 expression leads to a marked reduction in anchorage-independent growth, an effect also observed in the HPV-negative U2OS osteosarcoma cells (Huh et al. 2005). Endogenous p600/UBR4 physically associates with Rb and the HPV-16 E7 oncoprotein and is suggested to be a cellular target through which HPV-16 E7 exerts its transforming effects in virally induced cancers; however, the molecular underpinnings remain to be determined (Huh et al. 2005; Nakatani et al. 2005). p600/UBR4 is also utilized by the dengue virus N5S protein (DENV N5S) to mediate the degradation of signal transducer and activator of transcription 2 (STAT2); STAT2 degradation enables the virus to evade immune detection and enhances viral replication in host cells (Morrison et al. 2013).
Developmental and Physiological Functions
The molecular and cellular functions of p600/UBR4 drive important pathways that are critical for proper organismal development and physiological function (Fig. 2). During embryogenesis, maternal proteins are digested via the autophagosome-lysosome pathway in the endodermal layer of the yolk sac. This liberates amino acids that are the building blocks for the development of new tissues, for example, vascular formation in the mesodermal layer of the yolk sac. As a result, p600/UBR4 conventional knockout (KO) mouse embryos exhibit disruptions in autophagic pathways in the yolk sac, which are partly responsible for arrest in the angiogenic remodeling of the primary capillary plexus at E9.5 (Tasaki et al. 2013; Nakaya et al. 2013). This leads to halted embryonic development and a 100% mortality rate between E11.5 and E13.5 (Tasaki et al. 2013; Nakaya et al. 2013). There are also several pleiotropic abnormalities in both the placenta and fetal organ development (Nakaya et al. 2013). The labyrinth layer of the placenta in p600/UBR4 KO embryos is thinner and contains abnormally dilated and disorganized blood vessels compared to wild-type placenta (Nakaya et al. 2013).
A conditional knockout (cKO) mouse strain of p600/UBR4 in which the gene is ablated in the embryo but left intact in the placenta exhibits defects in multiple organs (Nakaya et al. 2013). Cardiac development in p600/UBR4 cKO embryos is severely affected: ventricular walls are thinner, and the interventricular septum and the inferior atrioventricular endocardial cushions are both underdeveloped and not properly fused, creating a large gap between the left and right ventricles (Nakaya et al. 2013). Such gross structural defects in the heart can be partly explained by a significant reduction in cardiomyocyte proliferation in p600/UBR4 cKO embryos (Nakaya et al. 2013). Moreover, the activation of FAK and levels of its downstream target, myocyte enhancer factor 2 (MEF2), are significantly reduced in the heart of p600/UBR4 cKO embryos (Nakaya et al. 2013). Given that FAK-deficient embryos exhibit a similar cardiac phenotype as p600/UBR4 cKO embryos, this suggests that p600/UBR4 regulates cardiac development through FAK-dependent signaling pathways (Nakaya et al. 2013).
In terms of other organs, the liver of p600/UBR4 cKO embryos has abnormally dilated blood vessels and lacks the normal, dense packing of parenchymal hepatocytes (Nakaya et al. 2013). p600/UBR4 ablation also causes microcephaly and other structural deficits of the embryonic brain (Nakaya et al. 2013). Microcephaly is typically caused by a substantial depletion of neural progenitors (NPs) due to increased apoptosis, autophagy, and/or terminal differentiation of NPs: the overall effect is decreased production of neurons. Given that p600/UBR4 has been implicated in cellular autophagy, NP differentiation, neuronal survival, and neurogenesis, it is probable that alterations in one or several of these cellular processes contribute to microcephaly in p600/UBR4 cKO embryos (Belzil et al. 2014a).
p600/UBR4 may play a significant role in the pathogenesis of several diseases. Through its effects on autophagy, p600/UBR4 may contribute to X-linked intellectual disability (XLID). Two point mutations in RAD6 have been detected in some patients with XLID: the mutations R7W and R11Q have been mapped to the KCMF1 binding site and prevent the binding of RAD6 with both KCMF1 and p600/UBR4 but not with other RAD6-interacting proteins (Hong et al. 2015). This suggests that the p600/UBR4-KCMF1-RAD6 complex is crucial for maintaining neuronal function, which is compromised in XLID patients (Hong et al. 2015). p600/UBR4 has also been identified as a candidate gene for a novel form of autosomal dominant episodic ataxia, which is characterized by imbalance and loss of coordination (Conroy et al. 2014). Although the mechanistic details underlying the role of p600/UBR4 in episodic ataxia remain largely unknown, its function in Ca2+ signaling is a potential mechanism (Conroy et al. 2014). Notably, adult pushover mutant flies exhibit sluggishness and a lack of coordination, in part, due to impaired synaptic transmission and neuronal excitability at the neuromuscular junction (Richards et al. 1996). However, the CaM-binding activity of PUSHOVER cannot be excluded as a possible contributor.
p600/UBR4 may also be involved in osteoporosis through its positive effects on TRPV5 degradation. Impaired TRPV5 function is observed in patients suffering from inflammatory bowel disease (IBD). Loss of bone mass density, a secondary symptom of IBD, may result from impaired Ca2+ reabsorption due to reduced TRPV5 expression. By controlling levels of TRPV5 protein, p600/UBR4 may play a role in the maintenance of bone mineral density (Radhakrishnan et al. 2013).
p600/UBR4 was first identified as a novel 600 kDa protein that displays a broad pattern of expression in virtually every tissue. Further investigations unraveled its role in various molecular and cellular functions including proteasomal degradation, autophagy, Ca2+ signaling, membrane morphogenesis, cell migration, and cell survival. p600/UBR4 has also been identified as a cellular target that is exploited by viral proteins such as HPV16 E7 and DENV N5S to facilitate invasion of host cells. p600/UBR4 has been implicated in several human diseases including Parkinson’s disease, episodic ataxia, and some cancers. In addition, p600/UBR4 is indispensable for survival and fetal development as evident by its critical role in the development of the yolk sac, placenta, and various fetal organs including the heart, liver, and brain. Despite the tremendous amount of progress that has been made to explore the role of p600/UBR4 in regulating cellular, developmental, and physiological functions, the molecular mechanisms underlying the diverse actions of this novel molecule remain largely unknown and warrant further investigation.
- Hong JH, Kaustov L, Coyaud E, Srikumar T, Wan J, Arrowsmith C, et al. KCMF1 (potassium channel modulatory factor 1) Links RAD6 to UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4) and lysosome-mediated degradation. Mol Cell Proteomics. 2015;14(3):674–85.PubMedPubMedCentralCrossRefGoogle Scholar