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M W Kirschner

Publications and source records attributed to M W Kirschner.

At least 19 recordsLinked to original sources
PubMed2000

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.

Actins↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

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.

Adaptor Proteins, Signal Transducing↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

The fate of cells in the tailbud of Xenopus laevis.

The vertebrate tailbud and trunk form very similar tissues. It has been a controversial question for decades whether cell determination in the developing tail proceeds as part of early axial development or whether it proceeds by a different mechanism. To examine this question more closely, we have used photoactivation of fluorescence to mark small neighborhoods of cells in the developing tailbud of Xenopus laevis. We show that, in one region of the tailbud, very small groups of adjacent cells can contribute progeny to the neural tube, notochord and somitic muscle, as well as other identified cell types within a single embryo. Groups averaging three adjacent cells at a later stage can contribute progeny with a similar distribution. Our data suggest that the tailbud contains multipotent cells that make very late germ-layer decisions.

Animals↗
PubMed1999

Control of mitotic transitions by the anaphase-promoting complex.

Proteolysis controls key transitions at several points in the cell cycle. In mitosis, the activation of a large ubiquitin-protein ligase, the anaphase-promoting complex (APC), is required for anaphase initiation and for exit from mitosis. We show that APC is under complex control by a network of regulatory factors, CDC20, CDH1 and MAD2. CDC20 and CDH1 are activators of APC; they bind directly to APC and activate its cyclin ubiquitination activity. CDC20 activates APC at the onset of anaphase in a destruction box (DB)-dependent manner, while CDH1 activates APC from late anaphase through G1 with apparently a much relaxed specificity for the DB. Therefore, CDC20 and CDH1 control both the temporal order of activation and the substrate specificity of APC, and hence regulate different events during mitosis and G1. Counteracting the effect of CDC20, the checkpoint protein MAD2 acts as an inhibitor of APC. When the spindle-assembly checkpoint is activated, MAD2 forms a ternary complex with CDC20 and APC to prevent activation of APC, and thereby arrests cells at prometaphase. Thus, a combination of positive and negative regulators establishes a regulatory circuit of APC, ensuring an ordered progression of events through cell division.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1999

Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis.

A vertebrate securin (vSecurin) was identified on the basis of its biochemical analogy to the Pds1p protein of budding yeast and the Cut2p protein of fission yeast. The vSecurin protein bound to a vertebrate homolog of yeast separins Esp1p and Cut1p and was degraded by proteolysis mediated by an anaphase-promoting complex in a manner dependent on a destruction motif. Furthermore, expression of a stable Xenopus securin mutant protein blocked sister-chromatid separation but did not block the embryonic cell cycle. The vSecurin proteins share extensive sequence similarity with each other but show no sequence similarity to either of their yeast counterparts. Human securin is identical to the product of the gene called pituitary tumor-transforming gene (PTTG), which is overexpressed in some tumors and exhibits transforming activity in NIH 3T3 cells. The oncogenic nature of increased expression of vSecurin may result from chromosome gain or loss, produced by errors in chromatid separation.

3T3 Cells↗
PubMed1999

The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly.

Although small GTP-binding proteins of the Rho family have been implicated in signaling to the actin cytoskeleton, the exact nature of the linkage has remained obscure. We describe a novel mechanism that links one Rho family member, Cdc42, to actin polymerization. N-WASP, a ubiquitously expressed Cdc42-interacting protein, is required for Cdc42-stimulated actin polymerization in Xenopus egg extracts. The C terminus of N-WASP binds to the Arp2/3 complex and dramatically stimulates its ability to nucleate actin polymerization. Although full-length N-WASP is less effective, its activity can be greatly enhanced by Cdc42 and phosphatidylinositol (4,5) bisphosphate. Therefore, N-WASP and the Arp2/3 complex comprise a core mechanism that directly connects signal transduction pathways to the stimulation of actin polymerization.

Actin-Related Protein 2↗
PubMed1999

Neuronal differentiation and patterning in Xenopus: the role of cdk5 and a novel activator xp35.2.

Cdk5, a member of the cyclin-dependent kinase family, has been shown to play an important role in development of the central nervous system in mammals when partnered by its activator p35. Here we describe the cloning and characterization of a novel activator of cdk5 in Xenopus, Xp35.2. Xp35.2 is expressed during development initially in the earliest differentiating primary neurons in the neural plate and then later in differentiating neural tissue of the brain. This is in contrast to the previously described Xenopus cdk5 activator Xp35.1 which is expressed over the entire expanse of the neural plate in both proliferating and differentiating cells. Expression of both Xp35.1 and Xp35.2 and activation of cdk5 kinase occur when terminal neural differentiation is induced by neurogenin and neuro D overexpression but not when only early stages of neural differentiation are induced by noggin. Moreover, blocking cdk5 kinase activity specifically results in disruption and reduction of the embryonic eye where cdk5 and its Xp35 activators are expressed. Thus, cdk5/p35 complexes function in aspects of neural differentiation and patterning in the early embryo and particularly in formation of the eye.

Amino Acid Sequence↗
PubMed1999

Identification of a new uracil-DNA glycosylase family by expression cloning using synthetic inhibitors.

BACKGROUND: The cellular environment exposes DNA to a wide variety of endogenous and exogenous reactive species that can damage DNA, thereby leading to genetic mutations. DNA glycosylases protect the integrity of the genome by catalyzing the first step in the base excision-repair of lesions in DNA. RESULTS: Here, we report a strategy to conduct genome-wide screening for expressed DNA glycosylases, based on their ability to bind to a library of four synthetic inhibitors that target the enzyme's active site. These inhibitors, used in conjunction with the in vitro expression cloning procedure, led to the identification of novel Xenopus and human proteins, xSMUG1 and hSMUG1, respectively, that efficiently excise uracil residues from DNA. Despite a lack of statistically significant overall sequence similarity to the two established classes of uracil-DNA glycosylases, the SMUG1 enzymes contain motifs that are hallmarks of a shared active-site structure and overall protein architecture. The unusual preference of SMUG1 for single-stranded rather than double-stranded DNA suggests a unique biological function in ridding the genome of uracil residues, which are potent endogenous mutagens. CONCLUSIONS: The 'proteomics' approach described here has led to the isolation of a new family of uracil-DNA glycosylases. The three classes of uracil-excising enzymes (SMUG1 being the most recently discovered) represent a striking example of structural and functional conservation in the almost complete absence of sequence conservation.

Amino Acid Sequence↗
PubMed1999

Quantitative measurement of the catastrophe rate of dynamic microtubules.

Previous work has shown that catastrophe frequency is the predominant dynamic parameter of microtubules that changes dramatically during the cell cycle. As an alternative to videomicroscopy assays, we have developed a biochemical assay to measure directly the average catastrophe rate of a population of microtubules. In this assay, the growing plus end of the microtubules, polymerized off seeds, are labeled with a brief pulse of alpha-32P-GTP, followed by a cold GTP chase. The rate of loss of 32P label in microtubules measured by this method is equal to the catastrophe frequency at microtubule plus ends measured by videomicroscopy of individual microtubules. Addition of mitotic extract from Xenopus eggs increases the catastrophe rate of purified tubulin by almost 100-fold, while interphase extract alters the catastrophe rate by about 20-fold as compared to pure tubulin. Most of the catastrophe-promoting activities in both mitotic and interphase extracts is found in particulate fractions. High-speed centrifugation of extracts appears to eliminate the components required for increasing microtubule catastrophe, but does not eliminate the cell cycle difference in microtubule dynamics. This assay provides a new approach to quantitate microtubule catastrophe rates. It will be of particular interest to search for catastrophe factors associated with intracellular membranes or other insoluble components.

3T3 Cells↗
PubMed1999

Microtubule bending and breaking in living fibroblast cells.

Microtubules in living cells frequently bend and occasionally break, suggesting that relatively strong forces act on them. Bending implies an increase in microtubule lattice energy, which could in turn affect the kinetics and thermodynamics of microtubule-associated processes such as breaking. Here we show that the rate of microtubule breaking in fibroblast cells increases approximately 40-fold as the elastic energy stored in curved microtubules increases to > approximately 1 kT/tubulin dimer. In addition, the length-normalized breaking rate is sufficiently large (2.3 breaks x mm(-1) x minute(-1)) to infer that breaking is likely a major mechanism by which noncentrosomal microtubules are generated. Together the results suggest a physiologically important, microtubule-based mechanism for mechanochemical information processing in the cell.

3T3 Cells↗
PubMed1998

The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42.

The small GTP-binding protein Cdc42 is thought to induce filopodium formation by regulating actin polymerization at the cell cortex. Although several Cdc42-binding proteins have been identified and some of them have been implicated in filopodium formation, the precise role of Cdc42 in modulating actin polymerization has not been defined. To understand the biochemical pathways that link Cdc42 to the actin cytoskeleton, we have reconstituted Cdc42-induced actin polymerization in Xenopus egg extracts. Using this cell-free system, we have developed a rapid and specific assay that has allowed us to fractionate the extract and isolate factors involved in this activity. We report here that at least two biochemically distinct components are required, based on their chromatographic behavior and affinity for Cdc42. One component is purified to homogeneity and is identified as the Arp2/3 complex, a protein complex that has been shown to nucleate actin polymerization. However, the purified complex alone is not sufficient to mediate the activity; a second component that binds Cdc42 directly and mediates the interaction between Cdc42 and the complex also is required. These results establish an important link between a signaling molecule, Cdc42, and a complex that can directly modulate actin networks in vitro. We propose that activation of the Arp2/3 complex by Cdc42 and other signaling molecules plays a central role in stimulating actin polymerization at the cell surface.

Actin-Related Protein 2↗
PubMed1998

Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins.

We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA. Using this technique, we have isolated two proteins that are structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in Xenopus. The Mix family of proteins are expressed during late blastula and gastrula stages of Xenopus development. During gastrulation, these genes are expressed at high levels in distinct, yet overlapping regions in mesoderm and endoderm. The members of the Mix family heterodimerize with each other and overexpression of each results in severe axial abnormalities. Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot. Injection of Mix.3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeoproteins are downstream effectors of growth factor signaling during endoderm and ventral mesoderm formation.

Amino Acid Sequence↗
PubMed1998

Mitotic inactivation of a human SWI/SNF chromatin remodeling complex.

During mitosis, chromatin is condensed into mitotic chromosomes and transcription is inhibited, processes that might be opposed by the chromatin remodeling activity of the SWI/SNF complexes. Brg1 and hBrm, which are components of human SWI/SNF (hSWI/SNF) complexes, were recently shown to be phosphorylated during mitosis. This suggested that phosphorylation might be used as a switch to modulate SWI/SNF activity. Using an epitope-tag strategy, we have purified hSWI/SNF complexes at different stages of the cell cycle, and found that hSWI/SNF was inactive in cells blocked in G2-M. Mitotic hSWI/SNF contained Brg1 but not hBrm, and was phosphorylated on at least two subunits, hSWI3 and Brg1. In vitro, active hSWI/SNF from asynchronous cells can be phosphorylated and inactivated by ERK1, and reactivated by dephosphorylation. hSWI/SNF isolated as cells traversed mitosis regained activity when its subunits were dephosphorylated either in vitro or in vivo. We propose that this transitional inactivation and reactivation of hSWI/SNF is required for formation of a repressed chromatin structure during mitosis and reformation of an active chromatin structure as cells leave mitosis.

Base Sequence↗
PubMed1998

The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation.

The spindle assembly checkpoint mechanism delays anaphase initiation until all chromosomes are aligned at the metaphase plate. Activation of the anaphase-promoting complex (APC) by binding of CDC20 and CDH1 is required for exit from mitosis, and APC has been implicated as a target for the checkpoint intervention. We show that the human checkpoint protein hMAD2 prevents activation of APC by forming a hMAD2-CDC20-APC complex. When injected into Xenopus embryos, hMAD2 arrests cells at mitosis with an inactive APC. The recombinant hMAD2 protein exists in two-folded states: a tetramer and a monomer. Both the tetramer and the monomer bind to CDC20, but only the tetramer inhibits activation of APC and blocks cell cycle progression. Thus, hMAD2 binding is not sufficient for inhibition, and a change in hMAD2 structure may play a role in transducing the checkpoint signal. There are at least three different forms of mitotic APC that can be detected in vivo: an inactive hMAD2-CDC20-APC ternary complex present at metaphase, a CDC20-APC binary complex active in degrading specific substrates at anaphase, and a CDH1-APC complex active later in mitosis and in G1. We conclude that the checkpoint-mediated cell cycle arrest involves hMAD2 receiving an upstream signal to inhibit activation of APC.

Anaphase↗