Allan M. Weissman

RING Finger Proteins: Mediators of Ubiquitin Ligase Activity

Ubiquitination is a highly regulated and common cellular process with E3s as central players. RING finger proteins represent the largest class of E3s to date. Whether RING fingers are primarily modules that mediate ubiquitination and other protein modifications or if they have other discrete functions should soon become apparent. Mammalian genomes encode hundreds of RING finger proteins. Extrapolating from in vitro studies, we predict that many of these have the capacity to interact with E2s and to potentially mediate ubiquitination. It remains to be determined how many of these will be bona fide E3s for heterologous substrates, targets for ubiquitination, or both. The number of potential E3s is further enhanced by the combinatorial association of certain RING finger proteins with other proteins that provide docking sites for substrates.The field of intracellular protein degradation now leaves the era where mediators of substrate-specific ubiquitination were scarce and enters a new and exciting phase where databases provide us with a large number of candidate E3s awaiting characterization. Each has the potential to help us understand how cells orchestrate the complexities of rapidly regulating protein levels and activity.‡E-mail: cjoazeiro@hotmail.com (C. A. P. J.); amwo@nih.gov (A. M. W.).

Mind Bomb Is a Ubiquitin Ligase that Is Essential for Efficient Activation of Notch Signaling by Delta

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.

SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals

The plant hormone indole-3 acetic acid (IAA or auxin) controls many aspects of plant development, including the production of lateral roots. Ubiquitin-mediated proteolysis has a central role in this process. The genes AXR1 and TIR1 aid the assembly of an active SCF (Skp1/Cullin/F-box) complex that probably promotes degradation of the AUX/IAA transcriptional repressors in response to auxin. The transcription activator NAC1, a member of the NAM/CUC family of transcription factors, functions downstream of TIR1 to transduce the auxin signal for lateral root development. Here we show that SINAT5, an Arabidopsis homologue of the RING-finger Drosophila protein SINA, has ubiquitin protein ligase activity and can ubiquitinate NAC1. This activity is abolished by mutations in the RING motif of SINAT5. Overexpressing SINAT5 produces fewer lateral roots, whereas overexpression of a dominant-negative Cys49 → Ser mutant of SINAT5 develops more lateral roots. These lateral root phenotypes correlate with the expression of NAC1 observed in vivo. Low expression of NAC1 in roots can be increased by treatment with a proteasome inhibitor, which indicates that SINAT5 targets NAC1 for ubiquitin-mediated proteolysis to downregulate auxin signals in plant cells.

The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum

gp78, also known as the tumor autocrine motility factor receptor, is a transmembrane protein whose expression is correlated with tumor metastasis. We establish that gp78 is a RING finger-dependent ubiquitin protein ligase (E3) of the endoplasmic reticulum (ER). Consistent with this, gp78 specifically recruits MmUBC7, a ubiquitin-conjugating enzyme (E2) implicated in ER-associated degradation (ERAD), through a region distinct from the RING finger. gp78 can target itself for proteasomal degradation in a RING finger- and MmUBC7-dependent manner. Importantly, gp78 can also mediate degradation of CD3-δ, a well-characterized ERAD substrate. In contrast, gp78 lacking an intact RING finger or its multiple membrane-spanning domains stabilizes CD3-δ. gp78 has thus been found to be an example of a mammalian cellular E3 intrinsic to the ER, suggesting a potential link between ubiquitylation, ERAD, and metastasis.

HECT and RING finger families of E3 ubiquitin ligases at a glance

The post-translational attachment of ubiquitin, a highly conserved 76-amino-acid polypeptide, directs myriad eukaryotic proteins to a variety of fates and functions. Ubiquitylation is best-known for targeting proteins for degradation by the 26S proteasome. Other functions include internalization and

Inhibitors of Ubiquitin-Activating Enzyme (E1), a New Class of Potential Cancer Therapeutics

The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer. Several classes of proteasome inhibitors, which block degradation of ubiquitylated proteins, are widely used in research, and one, Bortezomib, is now in clinical use. Despite the well-defined and central role of the ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation, PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally, PYR-41 attenuates cytokine-mediated nuclear factor-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IkappaB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IkappaBalpha. Furthermore, PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus, PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as therapeutics in cancer. These inhibitors can also be valuable tools for studying ubiquitylation.

Failure to synthesize the T cell CD3-ζ chain: structure and function of a partial T cell receptor complex.

The T cell antigen receptor is composed of two variable chains (alpha and beta, termed Ti), which confer ligand specificity, and five constant chains (gamma, delta, epsilon, zeta, and p21, collectively termed CD3) whose functions are poorly understood. To explore the roles of the individual CD3 components, an antigen-specific murine T cell hybridoma was chemically mutagenized and antigen-induced growth inhibition was used to select CD3/Ti expression variants. One variant produced all CD3/Ti components except CD3-zeta and was able to express small amounts of surface CD3/Ti. This variant failed to respond normally to either antigen or a mitogenic anti-Thy-1 antibody. Surprisingly, in the absence of CD3-zeta, direct cross-linking of the partial receptor induced both phosphatidylinositol hydrolysis and interleukin 2 production. These data indicate that CD3-zeta determines the normal intracellular fate of the T cell antigen receptor and is likely to play an important role in physiologically relevant transmembrane signaling.

Antigen Activation of Murine T Cells Induces Tyrosine Phosphorylation of a Polypeptide Associated with the T Cell Antigen Receptor

The antigen receptor complex on murine MHC class II-restricted T cells consists of disulfide-linked alpha and beta chains noncovalently associated with four additional polypeptides, two that are endoglycosaminidase F-sensitive, gp26 and gp21, and two that are endoglycosaminidase F-resistant, p25 and p16. We demonstrate here that treatment of murine T cell hybridomas with phorbol 12-myristate 13-acetate results in phosphorylation of p25 and gp21 on serine residues. However, activation of cells by antigen results in the phosphorylation of the gp21 chain and a heretofore unidentified 21 kd protein. This newly defined polypeptide, p21, is specifically immunoprecipitated with the antigen receptor complex, is endoglycosaminidase F-resistant, and is itself part of a disulfide-linked molecule. Unlike antigen-induced phosphorylation of gp21, which occurs on serine residues, phosphorylation of p21 occurs uniquely on tyrosine residues.

RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination.

RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5

Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination and contains an intrinsic ubiquitin ligase activity, all of which require an N-terminal A20 zinc finger followed immediately by a helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5-Å resolution shows that Rabex-5–ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin-binding domain, an inverted ubiquitin-interacting motif, which binds with ∼29-μM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with ∼22-μM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc-finger diaromatic patch mediates ubiquitin-ligase activity by directly recruiting a ubiquitin-loaded ubiquitin-conjugating enzyme.