Adam G. Eldridge

The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases

Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.

The SCF ubiquitin ligase: An extended look

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCF(Skp2) ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished.

The Evi5 Oncogene Regulates Cyclin Accumulation by Stabilizing the Anaphase-Promoting Complex Inhibitor Emi1

The anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 controls progression to S phase and mitosis by stabilizing key APC/C ubiquitination substrates, including cyclin A. Examining Emi1 binding proteins, we identified the Evi5 oncogene as a regulator of Emi1 accumulation. Evi5 antagonizes SCF(betaTrCP)-dependent Emi1 ubiquitination and destruction by binding to a site adjacent to Emi1s DSGxxS degron and blocking both degron phosphorylation by Polo-like kinases and subsequent betaTrCP binding. Thus, Evi5 functions as a stabilizing factor maintaining Emi1 levels in S/G2 phase. Evi5 protein accumulates in early G1 following Plk1 destruction and is degraded in a Plk1- and ubiquitin-dependent manner in early mitosis. Ablation of Evi5 induces precocious degradation of Emi1 by the Plk/SCF(betaTrCP) pathway, causing premature APC/C activation; cyclin destruction; cell-cycle arrest; centrosome overduplication; and, finally, mitotic catastrophe. We propose that the balance of Evi5 and Polo-like kinase activities determines the timely accumulation of Emi1 and cyclin, ensuring mitotic fidelity.

Triggering ubiquitination of a CDK inhibitor at origins of DNA replication

To ensure proper timing of the G1–S transition in the cell cycle, the cyclin E–Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21Cip1/p27Kip1/p57Kip2 family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27Xic1 protein in Xenopus. Activation of cyclin E–Cdk2 is linked to the ubiquitination of human p27Kip1 or Xenopus p27Xic1 by SCF (for Skp1–Cullin–F-box protein) ubiquitin ligases. For human p27Kip1, ubiquitination requires direct phosphorylation by cyclin E–Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E–Cdk2, but it does require nuclear accumulation of the Xic1–cyclin E–Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E–Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E–Cdk2 and the preinitiation complex. Thus, activation of the cyclin E–Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.

Identification of Rab11 as a small GTPase binding protein for the Evi5 oncogene

The Evi5 oncogene has recently been shown to regulate the stability and accumulation of critical G1 cell cycle factors including Emi1, an inhibitor of the anaphase-promoting complex/cyclosome, and cyclin A. Sequence analysis of the amino terminus of Evi5 reveals a Tre-2, Bub2, Cdc16 domain, which has been shown to be a binding partner and GTPase-activating protein domain for the Rab family of small Ras-like GTPases. Here we describe the identification of Evi5 as a candidate binding protein for Rab11, a GTPase that regulates intracellular transport and has specific roles in endosome recycling and cytokinesis. By yeast two-hybrid analysis, immunoprecipitation, and Biacore analysis, we demonstrate that Evi5 binds Rab11a and Rab11b in a GTP-dependent manner. However, Evi5 displays no activation of Rab11 GTPase activity in vitro. Evi5 colocalizes with Rab11 in vivo, and overexpression of Rab11 perturbs the localization of Evi5, redistributing it into Rab11-positive recycling endosomes. Interestingly, in vitro binding studies show that Rab11 effector proteins including FIP3 compete with Evi5 for binding to Rab11, suggesting a partitioning between Rab11–Evi5 and Rab11 effector complexes. Indeed, ablation of Evi5 by RNA interference causes a mislocalization of FIP3 at the abscission site during cytokinesis. These data demonstrate that Evi5 is a Rab11 binding protein and that Evi5 may cooperate with Rab11 to coordinate vesicular trafficking, cytokinesis, and cell cycle control independent of GTPase-activating protein function.

Accessory Proteins for Melanocortin Signaling

Abstract: Switching from eumelanin to pheomelanin synthesis during hair growth is accomplished by transient synthesis of Agouti protein, an inverse agonist for the melanocortin‐1 receptor (Mc1r). The coat color mutations mahogany and mahoganoid prevent hair follicle melanocytes from responding to Agouti protein. The gene mutated in mahogany, which is also known as Attractin (Atrn), encodes a type I transmembrane protein that functions as an accessory receptor for Agouti protein. We have recently determined that the gene mutated in mahoganoid, which is also known as Mahogunin (Mgrn1), encodes an E3 ubiquitin ligase. Like Attractin, Mahogunin is conserved in invertebrate genomes, and its absence causes a pleiotropic phenotype that includes spongiform neurodegeneration.

Positive-negative selection for homologous recombination in Arabidopsis.

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct contained two flanking regions of the target alcohol dehydrogenase gene as homologous sequences, and neomycin phosphotransferase and cytosine deaminase as positive and negative markers, respectively. A root explant transformation procedure was used to obtain transgenic calli. Among 6250 transformants isolated by positive selection, 39 were found to be resistant to negative selection as well. Of these 39, at least one had undergone homologous recombination correlated with a unidirectional transfer of information. Although the ADH locus was not changed, our data demonstrate that a homologous recombination event can be selected by positive negative selection in plants.

Control of the centriole and centrosome cycles by ubiquitination enzymes.

The role of the centriole in organizing the cell’s cytoskeleton and its mechanism of duplication have been long-standing puzzles for cell biologists. Whereas the semi-conservative replication of chromosomes was established by Meselson and Stahl (1958), the likely parallels for semi-conservative duplication of the centrioles remain fuzzy. Further considering this parallel, molecular studies of chromosomal replication have begun to uncover how cell cycle regulators including cyclin-dependent kinases and ubiquitin ligases ensure that chromosomes replicate once-and-only-once per cell cycle (Blow and Hodgson, 2002; Dutta and Bell, 1997). The obvious need to maintain accurate control of centrosome number and thereby ensure spindle bipolarity would suggest that a similar once-and-only once control restricts the centrosome cycle. Studies over the last decade on the budding and fission yeast spindle pole bodies (SPB) and the animal cell centrosome have defined a growing parts list of conserved components, as well as those specific to fungi or animals. The functional connection between the centrosome duplication cycle and the regulatory mechanisms controlling the chromosome duplication cycle suggested that semi-conservative replication for both centrosomes and chromosomes might be linked by these global timing mechanisms. In 1999, a series of studies demonstrated that cyclin-dependent kinases and both the SCF and APC ubiquitin ligases – cell cycle regulators already well established in control of the chromosome replication cycle – also had fundamental roles in controlling the centrosome cycle (Freed et al., 1999; Hinchcliffe et al., 1999; Lacey et al., 1999; Meraldi et al., 1999; Vidwans et al., 1999). Since then, a number of other cell cycle regulators have been directly implicated in the centrosome cycle, many of which are described in the accompanying reviews. Here we will focus on the role of ubiquitin ligases in controlling the centrosome cycle, considering both those known or postulated core centrosomal factors that are directly ubiquitinated, as well as ubiquitination of specific cell cycle regulators – including kinases and ubiquitin ligases themselves – that more globally control the centrosome cycle. First, we will review the biochemistry of ubiquitin ligases, and then return to the role of these enzymes in the various phases of the centrosome cycle.

The SCF Ubiquitin Ligase

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCF(Skp2) ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished.

PreviewThe SCF Ubiquitin Ligase: An Extended Look

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCF(Skp2) ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished.