Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation.

Eukaryotic cells rely on a surveillance mechanism known as the spindle checkpoint to ensure accurate chromosome segregation. The spindle checkpoint prevents sister chromatids from separating until all kinetochores achieve bipolar attachments to the mitotic spindle. Checkpoint proteins tightly inhibit the anaphase-promoting complex (APC), a ubiquitin ligase required for chromosome segregation and progression to anaphase. Unattached kinetochores promote the binding of checkpoint proteins Mad2 and BubR1 to the APC-activator Cdc20, rendering it unable to activate APC. Once all kinetochores are properly attached, however, cells inactivate the checkpoint within minutes, allowing for the rapid and synchronous segregation of chromosomes. How cells switch from strong APC inhibition before kinetochore attachment to rapid APC activation once attachment is complete remains a mystery. Here we show that checkpoint inactivation is an energy-consuming process involving APC-dependent multi-ubiquitination. Multi-ubiquitination by APC leads to the dissociation of Mad2 and BubR1 from Cdc20, a process that is reversed by a Cdc20-directed de-ubiquitinating enzyme. The mutual regulation between checkpoint proteins and APC leaves the cell poised for rapid checkpoint inactivation and ensures that chromosome segregation promptly follows the completion of kinetochore attachment. In addition, our results suggest a mechanistic basis for how cancer cells can have a compromised spindle checkpoint without corresponding mutations in checkpoint genes.

Intracellular signal propagation in a two-dimensional autocatalytic reaction model.

We study a simple reaction scheme in a two-dimensional lattice of particles or molecules with a refractory state. We analyze the dynamics of the propagating front as a function of physical-chemical properties of the host medium. The anisotropy of the medium significantly affects the smoothness of the wave front. Similarly, if particles or molecules may diffuse slowly to neighboring sites, then the front wave is more likely to be irregular. Both situations affect the ability of the whole system to relax to the original state, which is a required feature in the biological cells. Attempts to map this simple reaction scheme to reactions involved in the intracellular pathways suggest that, in some cases, signal transduction might take both connotation of a random walk and a propagating wave, depending on the local density of the medium. In particular, a sufficient condition for the appearance of waves in high-density regions of the media, is the existence of at least one autocatalytic reaction in the chain of reactions characterizing the pathway.

Dual inhibition of sister chromatid separation at metaphase.

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.

The anaphase-promoting complex mediates TGF-beta signaling by targeting SnoN for destruction.

Degradation of SnoN is thought to play an important role in the transactivation of TGF-beta responsive genes. We demonstrate that the anaphase-promoting complex (APC) is a ubiquitin ligase required for the destruction of SnoN and that the APC pathway is regulated by TGF-beta. The destruction box of SnoN is required for its degradation in response to TGF-beta signaling. Furthermore, the APC activator CDH1 and Smad3 synergistically regulate SnoN degradation. Under these circumstances, CDH1 forms a quaternary complex with SnoN, Smad3, and APC. These results suggest that APC(CDH1) and SnoN play central roles in regulating growth through the TGF-beta signaling system.

Molecular targets of vertebrate segmentation: two mechanisms control segmental expression of Xenopus hairy2 during somite formation.

Vertebrate hairy genes are expressed in patterns thought to be readouts of a "segmentation clock" in the presomitic mesoderm. Here we use transgenic Xenopus embryos to show that two types of regulatory elements are required to reconstitute the segmental pattern of Xenopus hairy2. The first is a promoter element containing two binding sites for Xenopus Su(H), a transcriptional activator of Notch target genes. The second is a short sequence in the hairy2 3' untranslated region (UTR), which most likely functions posttranscriptionally to modulate hairy2 RNA levels. 3' UTRs of other hairy-related, segmentally expressed genes can substitute for that of hairy2. Our results demonstrate a novel mechanism regulating the segmental patterns of Notch target genes and suggest that vertebrate segmentation requires the intersection of two regulatory pathways.

Identification of multiple CDH1 homologues in vertebrates conferring different substrate specificities.

CDC20/CDH1 activates the anaphase-promoting complex (APC) and targets various substrates for degradation, thereby allowing the ordered progression through mitosis and G(1). We have found multiple functional CDH1 homologues in the chick. The transcripts of these novel genes are differentially localized to proliferating, differentiated, and postmitotic tissues. All four proteins bind and form a complex with APC in vitro and in cultural cells and have quantitatively different activities in mediating ubiquitination of various APC substrates. Our results suggest that multiple CDH1s may temporally and spatially regulate APC activity both within and outside of the cell cycle.

CR16 forms a complex with N-WASP in brain and is a novel member of a conserved proline-rich actin-binding protein family.

The Neuronal Wiskott-Aldrich syndrome protein (N-WASP) has emerged as a central regulator of the actin cytoskeleton with abilities to integrate multiple upstream signal inputs and transmit them to the Arp2/3 complex. Here, we demonstrate that native N-WASP is present in a tight complex with a proline-rich protein, CR16, which shares approximately 25% identity with WASP interacting protein. CR16 is encoded by a gene previously cloned as a glucocorticoid-regulated mRNA from a rat hippocampal cDNA library. Although N-WASP is expressed ubiquitously, full-length CR16 protein is found predominately in the brain. CR16 and N-WASP colocalize in primary hippocampal neurons and at the tips of their growth cone filopodia. In vitro, CR16 directly binds both monomeric and filamentous actin but does not affect the kinetics of actin polymerization mediated by N-WASP and the Arp2/3 complex. Sequence homologues of CR16 are found not only in other vertebrates but also in the invertebrate Caenorhabditis elegans and in yeast. Thus, CR16 and WASP interacting protein belong to a family of N-WASP-binding proteins.

A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions.

Cell morphology and motility are governed largely by complex signaling networks that ultimately engage the actin cytoskeleton. Understanding how individual circuits contribute to the process of forming cellular structures would be aided greatly by the availability of specific chemical inhibitors. We have used a novel chemical screen in Xenopus cell-free extracts to identify compounds that inhibit signaling pathways regulating actin polymerization. Here we report the results of a high-throughput screen for compounds that inhibit phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin assembly and the identification of the first compound, a cyclic peptide, known to block actin assembly by inhibiting an upstream signaling component. We identify the target of this compound as N-WASP, a protein that has been investigated for its role as a node interconnecting various actin signaling networks. We show that this compound prevents activation of the Arp2/3 complex by N-WASP by allosterically stabilizing the autoinhibited conformation of N-WASP.

Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex.

The specificity of ubiquitin-mediated protein degradation with regards to the selection of substrates to be polyubiquitinated has only been determined rather recently. Substrate targeting by the N-end rule and HECT (homology to E6AP carboxyl terminus) domain ubiquitin ligases occurs through substrate-specific binding domains. In contrast, the SCF complex recruits substrates through a substrate adaptor protein, the F-box subunit. Despite evidence showing that Cdc20 and Cdh1 bind and activate the anaphase-promoting complex (APC) in a substrate-specific manner, there is no evidence that the activating protein and substrate interact directly; hence, no clear model exists for the mechanism of APC activation or recruitment of substrates. We show here that the activators Cdc20 and Cdh1 can associate with substrates via their N termini. In the absence of APC, Cdc20 and Cdh1 bind substrates reflecting Cdc20-APC and Cdh1-APC specificity. The N termini of Cdc20 and Cdh1 provide specificity functionally, as demonstrated by the generation of active chimeras that display the specificity corresponding to their N termini. Thus, Cdc20 and Cdh1 act as both substrate recognition and activating modules for APC.

Physiological regulation of [beta]-catenin stability by Tcf3 and CK1epsilon.

The wnt pathway regulates the steady state level of beta-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit beta-catenin turnover via its competition with axin and adenomatous polyposis for beta-catenin binding. A mutant of beta-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of beta-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between beta-catenin and Tcf3 stimulate beta-catenin degradation, indicating this interaction normally plays an important role in regulating beta-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1epsilon, and phosphorylation of Tcf3 by CKIepsilon stimulates its binding to beta-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1epsilon to inhibit beta-catenin degradation, whereas CKI-7, an inhibitor of CK1epsilon, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1epsilon stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1epsilon can modulate wnt signaling in vivo by regulating both the beta-catenin-Tcf3 and the GBP-dsh interfaces.

Inhibition of Cdh1-APC by the MAD2-related protein MAD2L2: a novel mechanism for regulating Cdh1.

Exit from mitosis requires the degradation of regulatory proteins including the mitotic cyclins and securin through ubiquitination by the anaphase promoting complex (APC) bound to Cdc20 or Cdh1. Cdc20-APC is regulated through inhibition by the spindle assembly checkpoint protein MAD2. Knowledge of Cdh1-APC regulation is limited to the phosphorylation-dependent dissociation of Cdh1 from APC. We report a novel means of regulating Cdh1 by the MAD2-related gene, MAD2L2. MAD2L2 specifically binds and inhibits Cdh1-APC, paralleling the effect of MAD2 on Cdc20. We suggest that MAD2L2 and MAD2 inhibit the release of substrates from APC and propose a mechanism of inhibition.

Pin1 acts catalytically to promote a conformational change in Cdc25.

Pin1 is an essential protein that can peptidyl-prolyl-isomerize small phosphopeptides. It has been suggested that Pin1 regulates entry into mitosis by catalyzing the cis/trans-isomerization of prolines on critical protein substrates in response to phosphorylation. We show that Pin1 catalytically generates a conformational change on the mitotic phosphatase Cdc25, as assayed by limited protease digestion, differential reactivity to a phosphoserine-proline-directed monoclonal antibody (MPM-2), and by changes in Cdc25 enzymatic activity. Pin1 catalytically modifies the conformation of Cdc25 at stoichiometries less than 0.0005, and mutants of Pin1 in the prolyl isomerase domain are not active. We suggest that, although difficult to detect, phosphorylation-dependent conformational changes mediated by prolyl isomerization may play an important regulatory role in the cell cycle.

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway.

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.

Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP.

All WASP family proteins share a common C terminus that consists of the verprolin homology domain (V), cofilin homology domain (C), and acidic region (A), through which they activate Arp2/3 complex-induced actin polymerization. In this study, we characterized the Arp2/3 complex-mediated actin polymerization activity of VCA fragments of all of the WASP family proteins: WASP, N-WASP, WAVE1, WAVE2, and WAVE3. All of the VCA fragments stimulated the nucleating activity of Arp2/3 complex. Among them, N-WASP VCA, which possesses two tandem V motifs, had a more potent activity than other VCA proteins. The chimeric protein experiments revealed that the V motif was more important to the activation potency than the CA region; two V motifs were required for full activity of N-WASP. COS7 cells overexpressing N-WASP form microspikes in response to epidermal growth factor. However, when a chimeric protein in which the VCA region of N-WASP is replaced with WAVE1 VCA was overexpressed, microspike formation was suppressed. Interestingly, when the N-WASP VCA region was replaced with WAVE1 VCA, having two V motifs, this chimeric protein could induce microspike formation. These results indicate that strong activation of Arp2/3 complex by N-WASP is mainly caused by its two tandem V motifs, which are essential for actin microspike formation.

Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate.

Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) transmits signals from Cdc42 to the nucleation of actin filaments by Arp2/3 complex. Although full-length N-WASP is a weak activator of Arp2/3 complex, its activity can be enhanced by upstream regulators such as Cdc42 and PI(4,5)P(2). We dissected this activation reaction and found that the previously described physical interaction between the NH(2)-terminal domain and the COOH-terminal effector domain of N-WASP is a regulatory interaction because it can inhibit the actin nucleation activity of the effector domain by occluding the Arp2/3 binding site. This interaction between the NH(2)- and COOH termini must be intramolecular because in solution N-WASP is a monomer. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) influences the activity of N-WASP through a conserved basic sequence element located near the Cdc42 binding site rather than through the WASp homology domain 1. Like Cdc42, PI(4,5)P(2) reduces the affinity between the NH(2)- and COOH termini of the molecule. The use of a mutant N-WASP molecule lacking this basic stretch allowed us to delineate a signaling pathway in Xenopus extracts leading from PI(4, 5)P(2) to actin nucleation through Cdc42, N-WASP, and Arp2/3 complex. In this pathway, PI(4,5)P(2) serves two functions: first, as an activator of N-WASP; and second, as an indirect activator of Cdc42.

Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability.

BACKGROUND: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed. RESULTS: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization. CONCLUSIONS: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.

Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts.

Regulation of beta-catenin degradation by intracellular components of the wnt pathway was reconstituted in cytoplasmic extracts of Xenopus eggs and embryos. The ubiquitin-dependent beta-catenin degradation in extracts displays a biochemical requirement for axin, GSK3, and APC. Axin dramatically accelerates while dishevelled inhibits beta-catenin turnover. Through another domain, dishevelled recruits GBP/Frat1 to the APC-axin-GSK3 complex. Our results confirm and extend models in which inhibition of GSK3 has two synergistic effects: (1) reduction of APC phosphorylation and loss of affinity for beta-catenin and (2) reduction of beta-catenin phosphorylation and consequent loss of its affinity for the SCF ubiquitin ligase complex. Dishevelled thus stabilizes beta-catenin, which can dissociate from the APC/axin complex and participate in transcriptional activation.

Structure of the Mad2 spindle assembly checkpoint protein and its interaction with Cdc20.

The checkpoint protein Mad2 inhibits the activity of the anaphase promoting complex by sequestering Cdc20 until all chromosomes are aligned at the metaphase plate. We report the solution structure of human Mad2 and its interaction with Cdc20. Mad2 possesses a novel three-layered alpha/beta fold with three alpha-helices packed between two beta-sheets. Using deletion mutants we identified the minimal Mad2-binding region of human Cdc20 as a 40-residue segment immediately N-terminal to the WD40 repeats. Mutagenesis and NMR titration experiments show that a C-terminal flexible region of Mad2 is required for binding to Cdc20. Mad2 and Cdc20 form a tight 1:1 heterodimeric complex in which the C-terminal segment of Mad2 becomes folded. These results provide the first structural insight into mechanisms of the spindle assembly checkpoint.

The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1.

The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G(1), by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20-APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specificity. We have determined the sequence specificity for Cdh1-APC using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1-APC in Xenopus extracts. Cdc20 is itself a Cdh1-APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1-APC through a different sequence. By analysis of Cdc20 as a substrate, we have identified a new recognition signal. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1-APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, we have identified cell cycle genes Nek2 and B99 as additional Cdh1-APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation.

LIS1 regulates CNS lamination by interacting with mNudE, a central component of the centrosome.

LIS1, a microtubule-associated protein, is required for neuronal migration, but the precise mechanism of LIS1 function is unknown. We identified a LIS1 interacting protein encoded by a mouse homolog of NUDE, a nuclear distribution gene in A. nidulans and a multicopy suppressor of the LIS1 homolog, NUDF. mNudE is located in the centrosome or microtubule organizing center (MTOC), and interacts with six different centrosomal proteins. Overexpression of mNudE dissociates gamma-tubulin from the centrosome and disrupts microtubule organization. Missense mutations that disrupt LIS1 function block LIS1-mNudE binding. Moreover, misexpression of the LIS1 binding domain of mNudE in Xenopus embryos disrupts the architecture and lamination of the CNS. Thus, LIS1-mNudE interactions may regulate neuronal migration through dynamic reorganization of the MTOC.