Identification of a tissue-non-specific homologue of axonal fasciculation and elongation protein zeta-1

Fasciculation and elongation protein zeta-1 (FEZ1) is a mammalian orthologue of the Caenorhabditis elegans UNC-76 protein involved in the axonal outgrowth and fasciculation and promotes neurite extension of PC12 cells through interaction with protein kinase C zeta (PKCzeta). The gene coding for FEZ2, a homologue of FEZ1, has also been reported in rat and human. In this study, we compared mRNA expression of FEZ1 and FEZ2 in adult rat tissues and mouse embryos by Northern blot and in situ hybridization analyses. In contrast to FEZ1 whose mRNA is expressed almost exclusively in rat brain and temporarily around the neurogenesis stage of mouse embryos, the message for FEZ2 is detected weakly in most tissues and abundantly throughout the mouse embryonic stages. Similar to FEZ1, FEZ2 interacted with PKCzeta and induced neurite extension of PC12 cells when coexpressed with a constitutively active mutant of PKCzeta. These results suggest that FEZ2 plays an important role in the morphological changes of various cells by associating with PKCzeta in a tissue-non-specific manner.     

Properties and regulation of glutamine transporter SN1 by protein kinases SGK and PKB

The amino acid transporter SN1 with substrate specificity identical to the amino acid transport system N is expressed mainly in astrocytes and hepatocytes where it accomplishes Na(+)-coupled glutamine uptake and efflux. To characterize properties and regulation of SN1, substrate-induced currents and/or radioactive glutamine uptake were determined in Xenopus oocytes injected with cRNA encoding SN1, the ubiquitin ligase Nedd4-2, and/or the constitutively active serum and glucocorticoid inducible kinase S422DSGK1, its isoform SGK3, and the constitutively active protein kinase B T308D,S473DPKB. The substrate-induced currents were enhanced by increasing glutamine and/or Na(+) concentrations, hyperpolarization, and alkalinization (pH 8.0). They were inhibited by acidification (pH 6.0). Coexpression of Nedd4-2 downregulated SN1-mediated transport, an effect reversed by coexpression of S422DSGK1, SGK3, and T308D,S473DPKB. It is concluded that SN1 is a target for the ubiquitin ligase Nedd4-2, which is inactivated by the serum and glucocorticoid inducible kinase SGK1, its isoform SGK3, and protein kinase B.     

Protein kinase C zeta. A novel regulator of both phosphorylation and de-phosphorylation of cardiac sarcomeric proteins

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.     

Negative regulation of EGFR-Vav2 signaling axis by Cbl ubiquitin ligase controls EGF receptor-mediated epithelial cell adherens junction dynamics and cell migration

The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.     

The poxvirus p28 virulence factor is an E3 ubiquitin ligase

A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.     

CRL4-DDB1-VPRBP ubiquitin ligase mediates the stress triggered proteolysis of Mcm10

When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of Mcm10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that Mcm10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered Mcm10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates Mcm10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates Mcm10 in vitro, proving that Mcm10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets Mcm10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, Mcm10.     

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.     

c-MIR,a human E3 ubiquitin ligase,is a functional homolog of herpesvirus proteins MIR1 and MIR2 and has similar activity

Kaposi's sarcoma associated-herpes virus encodes two proteins, MIR (modulator of immune recognition) 1 and 2, which are involved in the evasion of host immunity. MIR1 and 2 have been shown to function as an E3 ubiquitin ligase for immune recognition-related molecules (e.g. major histocompatibility complex class I, B7-2, and ICAM-1) through the BKS (bovine herpesvirus 4, Kaposi's sarcoma associated-herpes virus, and Swinepox virus) subclass of plant homeodomain (PHD) domain, termed the BKS-PHD domain. Here we show that the human genome also encodes a novel BKS-PHD domain-containing protein that functions as an E3 ubiquitin ligase and whose putative substrate is the B7-2 co-stimulatory molecule. This novel E3 ubiquitin ligase was designated as c-MIR (cellular MIR) based on its functional and structural similarity to MIR1 and 2. Forced expression of c-MIR induced specific down-regulation of B7-2 surface expression through ubiquitination, rapid endocytosis, and lysosomal degradation of the target molecule. This specific targeting was dependent upon the binding of c-MIR to B7-2. Replacing the BKS-PHD domain of MIR1 with the corresponding domain of c-MIR did not alter MIR1 function. The discovery of c-MIR, a novel E3 ubiquitin ligase, highlights the possibility that viral immune regulatory proteins originated in the host genome and presents unique functions of BKS-PHD domain-containing proteins in mammals.     

Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.     

The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.     

ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites.