Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase promoting complex

The anaphase promoting complex (APC) is the crucial ubiquitin ligase targeting the regulatory machinery of the cell cycle. Emi1, a major modulator of APC activity, is thought to act competitively as a pseudosubstrate. We show that the modulation of APC activity is more subtle: Emi1 inhibits ubiquitylation at both substrate binding and separately at the step of ubiquitin transfer to APC-bound substrates. The zinc-binding region of Emi1 allows multiple monoubiquitylation of substrates, but preferentially suppresses the ubiquitin chain elongation by UBCH10. Furthermore, the C-terminal tail of Emi1 antagonizes chain elongation by Ube2S, via competitively preventing its binding to APC cullin subunit through electrostatic interaction. Combinatorially, Emi1 effectively stabilizes APC substrates by suppressing ubiquitin chain extension. Deubiquitylating enzymes can then convert inhibited substrates to their basal state. Chain elongation may be a particularly sensitive step for controlling degradation and this study provides the first kinetic evidence for how it is inhibited

Evidence that fold-change, and not absolute level, of β-catenin dictates Wnt signaling

In response to Wnt stimulation, β-catenin accumulates and activates target genes. Using modeling and experimental analysis, we found that the level of β-catenin is sensitive to perturbations in the pathway, such that cellular variation would be expected to alter the signaling outcome. One unusual parameter was robust: the fold-change in β-catenin level (post-Wnt/pre-Wnt). In Xenopus, dorsal-anterior development and target gene expression are robust to perturbations that alter the final level but leave the fold-change intact. These suggest, first, that despite cellular noise, the cell responds reliably to Wnt stimulation by maintaining a robust fold-change in β-catenin. Second, the transcriptional machinery downstream of the Wnt pathway does not simply read the β-catenin level after Wnt stimulation but computes fold-changes in β-catenin. Analogous to Weber's Law in sensory physiology, some gene transcription networks must respond to fold-changes in signals, rather than absolute levels, which may buffer stochastic, genetic, and environmental variation

The surface contraction waves of Xenopus eggs reflect the metachronous cell-cycle state of the cytoplasm

AbstractActivated Xenopus laevis eggs undergo a series of surface contractions in response to cell-cycle progression but fall to cleave unless the sperm centrosome is present. These surface contraction waves (SCWs) begin at the animal pole and progress around the egg, occur every cell cycle and precede cleavage [1–3]. The SCWs are biphasic, comprising a relaxation phase (SCWa) and a contraction phase (SCWb). To investigate how these events are linked to the underlying cell cycle, we studied the temporal and spatial relationship between the SCWs and previously characterized biochemical markers of cell-cycle progression. We found that the relaxation phase was a response to activated maturation-promoting factor (MPF). In contrast, the contraction phase required inactivation of MPF and was blocked when MPF activity was maintained at elevated levels. We also found that a wave of MPF activity traveled within the cell from the animal to the vegetal hemisphere. Taken together, these experiments suggest that the SCWs are a local response to a wave of MPF activation and inactivation. The egg cytoplasm, therefore, is metachronous in terms of cell-cycle progression; multiple cell-cycle states are present and spatially distinct within the egg at the same time

The master cell cycle regulator APC-Cdc20 regulates ciliary length and disassembly of the primary cilium

The primary cilium has an important role in signaling; defects in structure are associated with a variety of human diseases. Much of the most basic biology of this organelle is poorly understood, even basic mechanisms, such as control of growth and resorption. We show that the activity of the anaphase-promoting complex (APC), an E3 that regulates the onset of anaphase, destabilizes axonemal microtubules in the primary cilium. Furthermore, the metaphase APC co-activator, Cdc20, is specifically recruited to the basal body of primary cilia. Inhibition of APC-Cdc20 activity increases the ciliary length, while overexpression of Cdc20 suppresses cilium formation. APC-Cdc20 activity is required for the timely resorption of the cilium after serum stimulation. In addition, APC regulates the stability of axonemal microtubules through targeting Nek1, the ciliary kinase, for proteolysis. These data demonstrate a novel function of APC beyond cell cycle control and implicate critical role of ubiquitin-mediated proteolysis in ciliary disassembly. DOI: http://dx.doi.org/10.7554/eLife.03083.00

A General Lack of Compensation for Gene Dosage in Yeast

Gene copy number variation has been discovered in humans, between related species, and in different cancer tissues, but it is unclear how much of this genomic-level variation leads to changes in the level of protein abundance. To address this, we eliminated one of the two genomic copies of 730 different genes in Saccharomyces cerevisiae and asked how often a 50% reduction in gene dosage leads to a 50% reduction in protein level. For at least 80% of genes tested, and under several environmental conditions, it does: protein levels in the heterozygous strain are close to 50% of wild type. For < 5% of the genes tested, the protein levels in the heterozygote are maintained at nearly wild-type levels. These experiments show that protein levels are not, in general, directly monitored and adjusted to a desired level. Combined with fitness data, this implies that proteins are expressed at levels higher than necessary for survival

Timing of Events in Mitosis

AbstractBackground: Regulation of the major transitions in the cell cycle, such as G1/S, G2/M, and metaphase to anaphase, are increasingly well understood. However, we have a poor understanding of the timing of events within each phase of the cell cycle, such as S phase or early mitosis. Two extreme models of regulation are possible. A “regulator-controlled model” in which the order of events is governed by the activation of a series of cytoplasmic regulators, such as kinases, phosphatases, or proteases; or a “substrate-controlled model” in which temporal regulation is determined by the differential responses of the cellular machinery to a common set of activators.Results: We have tried to distinguish between these two models by examining the timing of both biochemical and morphological events in Xenopus egg extracts during mitosis. Several proteins respond with different delays to the activation of Cdc2. We have found that the timing of phosphorylation is largely unchanged when these proteins are exposed to extracts that have been in mitosis for various periods of time. Similarly, when Xenopus interphase nuclei are added to extracts at different times after the G2/M transition, they undergo all the expected morphological changes in the proper sequence and with very similar kinetics.Conclusions: Our results suggest that during early mitosis (from prophase to metaphase) the timing of biochemical events (such as phosphorylation) and morphological events (such as structural changes in the nucleus) is at least partly controlled by the responses of the substrates themselves to a common set of signals

The incoherent feedforward loop can provide fold-change detection in gene regulation

Many sensory systems (e.g., vision and hearing) show a response that is proportional to the fold-change in the stimulus relative to the background, a feature related to Weber's Law. Recent experiments suggest such a fold-change detection feature in signaling systems in cells: a response that depends on the fold-change in the input signal, and not on its absolute level. It is therefore of interest to find molecular mechanisms of gene regulation that can provide such fold-change detection. Here, we demonstrate theoretically that fold-change detection can be generated by one of the most common network motifs in transcription networks, the incoherent feedforward loop (I1-FFL), in which an activator regulates both a gene and a repressor of the gene. The fold-change detection feature of the I1-FFL applies to the entire shape of the response, including its amplitude and duration, and is valid for a wide range of biochemical parameters

Microarray Discovery of New OGT Substrates: The Medulloblastoma Oncogene OTX2 Is O-GlcNAcylated

O-GlcNAc transferase (OGT) is a serine/threonine glycosyltransferase that is essential for development and continues to be critically important throughout life. Understanding OGT’s complex biology requires identifying its substrates. Here we demonstrate the utility of a microarray approach for discovering novel OGT substrates. We also report a rapid method to validate OGT substrates that combines in vitro transcription-translation with O-GlcNAc mass tagging. Among the validated new OGT targets is Orthodenticle homeobox 2 (OTX2), a transcription factor critical for brain development, which is primarily expressed only during early embryogenesis and in medulloblastomas, where it functions as an oncogene. We show that endogenous OTX2 from a medulloblastoma cell line is O-GlcNAcylated at several sites. Our results demonstrate that protein microarray technology, combined with the target validation strategy we report, is useful for identifying biologically important OGT substrates, including substrates not present in most tissue types or cell lines