Negative regulation of the E3 ubiquitin ligase itch via Fyn-mediated tyrosine phosphorylation

Conjugation of ubiquitin (Ub) to a protein substrate targets the substrate for degradation or functional modification, which is tightly controlled by diverse mechanisms including phosphorylation of the substrate. An emerging mechanism involves regulation of the E3 Ub ligase, for example, the JNK-dependent phosphorylation and activation of Itch E3 ligase, which controls the turnover of Jun proteins and T cell differentiation. Here we show that Itch is also modulated by an Src kinase Fyn via tyrosine phosphorylation at the Tyr371 residue. Fyn associates with Itch, and loss of Fyn results in reduced Itch phosphorylation. Importantly, tyrosine phosphorylation of Itch appears to reduce its interaction with its substrate JunB. The turnover of JunB is accelerated in Fyn-deficient T cells, which is further reconstituted by Itch Tyr371 mutation. Thus, in contrast to the activation pathway mediated by serine/threonine phosphorylation, tyrosine phosphorylation of Itch plays a negative role in modulating Itch-promoted ubiquitination.     

Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase

Ubiquitin ligases (E3) select proteins for ubiquitylation, a modification that directs altered subcellular trafficking and/or degradation of the target protein. HECT domain E3 ligases not only recognize, but also directly catalyze, ligation of ubiquitin to their protein substrates. The crystal structure of the HECT domain of the human ubiquitin ligase WWP1/AIP5 maintains a two-lobed structure like the HECT domain of the human ubiquitin ligase E6AP. While the individual N and C lobes of WWP1 possess very similar folds to those of E6AP, the organization of the two lobes relative to one another is different from E6AP due to a rotation about a polypeptide hinge linking the N and C lobes. Mutational analyses suggest that a range of conformations achieved by rotation about this hinge region is essential for catalytic activity.     

Negative regulation of Rap1 activation by the Cbl E3 ubiquitin ligase

Cbl functions as an adaptor protein by interacting with other signalling molecules to form multimolecular complexes. Previous studies have proposed that Cbl is also a positive regulator of CrkL–C3G signalling, which leads to Rap1 activation. However, there is a lack of genetic evidence for a physiological function of Cbl in regulating this pathway. Here, we show that Cbl deficiency results in enhanced activation of Rap1. Cbl was shown to promote the ubiquitylation of CrkL without any apparent effect on its stability. Remarkably, the membrane translocation of C3G, its association with CrkL, and the guanine-nucleotide exchange activity of C3G were all increased in Cbl^−/− thymocytes. Consistent with a function of Rap1 in integrin activation, enhanced integrin-mediated cell adhesion was also seen in Cbl^−/− thymocytes. Thus, Cbl negatively regulates Rap1 activation, probably through a proteolysis-independent E3-ubiquitin-ligase activity of Cbl that modulates protein–protein interactions.     

Negative regulation of AMPK signaling by high glucose via E3 ubiquitin ligase MG53

1. Download : Download high-res image (149KB)
2. Download : Download full-size image

     

PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity

PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.     

E3 ubiquitin ligase Cbl-b regulates Pten via Nedd4 in T cells independently of its ubiquitin ligase activity

E3 ubiquitin ligase Cbl-b plays a crucial role in T cell activation and tolerance induction. However, the molecular mechanism by which Cbl-b inhibits T cell activation remains unclear. Here, we report that Cbl-b does not inhibit PI3K but rather suppresses TCR/CD28-induced inactivation of Pten. The elevated Akt activity in Cbl-b(-/-) T cells is therefore due to heightened Pten inactivation. Suppression of Pten inactivation in T cells by Cbl-b is achieved by impeding the association of Pten with Nedd4, which targets Pten K13 for K63-linked polyubiquitination. Consistent with this finding, introducing Nedd4 deficiency into Cbl-b(-/-) mice abrogates hyper-T cell responses caused by the loss of Cbl-b. Hence, our data demonstrate that Cbl-b inhibits T cell activation by suppressing Pten inactivation independently of its ubiquitin ligase activity.     

CHIP functions an E3 ubiquitin ligase of Runx1

Runx1 is a key factor in the generation and maintenance of hematopoietic stem cells. Improper expression and mutations in Runx1 are frequently implicated in human leukemia. Here, we report that CHIP, the carboxyl terminus of Hsc70-interacting protein, also named Stub1, physically interacts with Runx1 through the TPR and Charged domains in the nucleus. Over-expression of CHIP directly induced Runx1 ubiquitination and degradation through the ubiquitin-proteasome pathway. Interestingly, we found that CHIP-mediated degradation of Runx1 is independent of the molecular chaperone Hsp70/90. Taken together, we propose that CHIP serves as an E3 ubiquitin ligase that regulates Runx1 protein stability via an ubiquitination and degradation mechanism that is independent of Hsp70/90.     

Functional dissection of a HECT ubiquitin E3 ligase

Ubiquitination is one of the most prevalent protein post-translational modifications in eukaryotes, and its malfunction is associated with a variety of human diseases. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitination remain largely unknown. Here we used a combination of yeast proteome chip assays, genetic screening, and in vitro/in vivo biochemical analyses to identify and characterize eight novel in vivo substrates of the ubiquitinating enzyme Rsp5, a homolog of the human ubiquitin-ligating enzyme Nedd4, in yeast. Our analysis of the effects of a deubiquitinating enzyme, Ubp2, demonstrated that an accumulation of Lys-63-linked polyubiquitin chains results in processed forms of two substrates, Sla1 and Ygr068c. Finally we showed that the localization of another newly identified substrate, Rnr2, is Rsp5-dependent. We believe that our approach constitutes a paradigm for the functional dissection of an enzyme with pleiotropic effects.     

Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 δ isoform (PP1cδ) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.     

Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast

Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.     

SCFhFBH1 can act as helicase and E3 ubiquitin ligase

In our previous study, we found that a human F-box DNA helicase, named hFBH1, interacted with SKP1 to form an SCF (SKP1–Cul1–F-box protein) complex together with CUL1 and ROC1 in an F-box-dependent manner. The complex immunoprecipitated from crude cell extracts catalyzed polyubiquitin formation in the presence of the ubiquitin-activating and ubiquitin-conjugating enzymes, E1 and E2, respectively. In this report, we characterized the enzymatic properties of the recombinant SCF^hFBH1 complex purified from insect cells expressing hFBH1, SKP1, CUL1 and ROC1. The SCF^hFBH1 complex was isolated as a single tight complex that retained DNA helicase, DNA-dependent ATPase and E3 ubiquitin ligase activities. The helicase and ATPase activities residing in the SCF^hFBH1 complex were indistinguishable from those of the hFBH1 protein alone. Moreover, the ubiquitin ligase activity of the SCF^hFBH1 complex was hardly affected by single-stranded or double-stranded DNA. The multiple activities present in this complex act independently of each other, suggesting that the SCF^hFBH1 complex can catalyze a ubiquitination reaction while acting as a DNA helicase or translocating along DNA. The potential roles of the SCF^hFBH1 complex in DNA metabolism based upon the enzymatic activities associated with this complex are discussed.     

FBXL2 is a ubiquitin E3 ligase subunit that triggers mitotic arrest

Mitotic progression is regulated by ubiquitin E3 ligase complexes to carefully orchestrate eukaryotic cell division. Here, we show that a relatively new E3 ligase component belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family, SCF^FBXL2, impairs cell proliferation by mediating cyclin D3 polyubiquitination and degradation. Both cyclin D3 and FBXL2 colocalize within the centrosome. FBXL2 overexpression led to G₂/M-phase arrest in transformed epithelia, resulting in the appearance of supernumerary centrosomes, tetraploidy and nuclei where condensed chromosomes are arranged on circular monopolar spindles typical of mitotic arrest. RNAi-mediated knockdown of cyclin D3 recapitulated effects of SCF^FBXL2 expression. SCF^FBXL2 impaired the ability of cyclin D3 to associate with centrosomal assembly proteins [Aurora A, polo-like kinase 4 (Plk4), CDK11]. Thus, these results suggest a role for SCF^FBXL2 in regulating the fidelity of cellular division.Key words: F-box protein, centrosome, mitosis, cyclin D3, Aurora A     

FBXL2 is a ubiquitin E3 ligase subunit that triggers mitotic arrest

Mitotic progression is regulated by ubiquitin E3 ligase complexes to carefully orchestrate eukaryotic cell division. Here, we show that a relatively new E3 ligase component belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family, SCF^FBXL2, impairs cell proliferation by mediating cyclin D3 polyubiquitination and degradation. Both cyclin D3 and FBXL2 colocalize within the centrosome. FBXL2 overexpression led to G₂/M-phase arrest in transformed epithelia, resulting in the appearance of supernumerary centrosomes, tetraploidy and nuclei where condensed chromosomes are arranged on circular monopolar spindles typical of mitotic arrest. RNAi-mediated knockdown of cyclin D3 recapitulated effects of SCF^FBXL2 expression. SCF^FBXL2 impaired the ability of cyclin D3 to associate with centrosomal assembly proteins [Aurora A, polo-like kinase 4 (Plk4), CDK11]. Thus, these results suggest a role for SCF^FBXL2 in regulating the fidelity of cellular division.     

The Cullin-RING E3 ubiquitin ligase CRL4-DCAF1 complex dimerizes via a short helical region in DCAF1

The cullin4A-RING E3 ubiquitin ligase (CRL4) is a multisubunit protein complex, comprising cullin4A (CUL4), RING H2 finger protein (RBX1), and DNA damage-binding protein 1 (DDB1). Proteins that recruit specific targets to CRL4 for ubiquitination (ubiquitylation) bind the DDB1 adaptor protein via WD40 domains. Such CRL4 substrate recognition modules are DDB1- and CUL4-associated factors (DCAFs). Here we show that, for DCAF1, oligomerization of the protein and the CRL4 complex occurs via a short helical region (residues 845-873) N-terminal to DACF1's own WD40 domain. This sequence was previously designated as a LIS1 homology (LisH) motif. The oligomerization helix contains a stretch of four Leu residues, which appear to be essential for α-helical structure and oligomerization. In vitro reconstituted CRL4-DCAF1 complexes (CRL4(DCAF1)) form symmetric dimers as visualized by electron microscopy (EM), and dimeric CRL4(DCAF1) is a better E3 ligase for in vitro ubiquitination of the UNG2 substrate compared to a monomeric complex.     

UHRF2, another E3 ubiquitin ligase for p53

UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.