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M W Kirschner

Publications and source records attributed to M W Kirschner.

At least 55 records · Page 3Linked to original sources
PubMed1998

The essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosis-specific phosphoproteins.

Phosphorylation of mitotic proteins on the Ser/Thr-Pro motifs has been shown to play an important role in regulating mitotic progression. Pin1 is a novel essential peptidyl-prolyl isomerase (PPIase) that inhibits entry into mitosis and is also required for proper progression through mitosis, but its substrate(s) and function(s) remain to be determined. Here we report that in both human cells and Xenopus extracts, Pin1 interacts directly with a subset of mitotic phosphoproteins on phosphorylated Ser/Thr-Pro motifs in a phosphorylation-dependent and mitosis-specific manner. Many of these Pin1-binding proteins are also recognized by the monoclonal antibody MPM-2, and they include the important mitotic regulators Cdc25, Myt1, Wee1, Plk1, and Cdc27. The importance of this Pin1 interaction was tested by constructing two Pin1 active site point mutants that fail to bind a phosphorylated Ser/Thr-Pro motif in mitotic phosphoproteins. Wild-type, but not mutant, Pin1 inhibits both mitotic division in Xenopus embryos and entry into mitosis in Xenopus extracts. We have examined the interaction between Pin1 and Cdc25 in detail. Pin1 not only binds the mitotic form of Cdc25 on the phosphorylation sites important for its activity in vitro and in vivo, but it also inhibits its activity, offering one explanation for the ability of Pin1 to inhibit mitotic entry. In a separate paper, we have shown that Pin1 is a phosphorylation-dependent PPIase that can recognize specifically the phosphorylated Ser/Thr-Pro bonds present in mitotic phosphoproteins. Thus, Pin1 likely acts as a general regulator of mitotic proteins that have been phosphorylated by Cdc2 and other mitotic kinases.

Animals↗
PubMed1998

Identification of a cullin homology region in a subunit of the anaphase-promoting complex.

The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8). The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned. APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27. Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G1 phase cyclins and cyclin-dependent kinase inhibitors. The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p. APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation.

Amino Acid Sequence↗
PubMed1998

Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1.

Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis. We show that APC is activated during mitosis and G1 by two regulatory factors, hCDC20 and hCDH1. These proteins directly bind to APC and activate its cyclin ubiquitination activity. hCDC20 confers a strict destruction-box (D-box) dependence on APC, while hCDH1 shows a much more relaxed specificity for the D-box. In HeLa cells, the protein levels of hCDC20 as well as its binding to APC peak in mitosis and decrease drastically at early G1. Thus, hCDC20 is the mitotic activator of APC and directs the degradation of substrates containing the D-box. The hCDH1 protein level remains constant during the cell cycle and may target specific substrates lacking the D-box in G1, such as polo-like kinase, for ubiquitination.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1998

Geminin, a neuralizing molecule that demarcates the future neural plate at the onset of gastrulation.

In an expression cloning screen in Xenopus embryos, we identified a gene that when overexpressed expanded the neural plate at the expense of adjacent neural crest and epidermis. This gene, which we named geminin, had no sequence similarity to known gene families. We later discovered that geminin's neuralizing domain was part of a bifunctional protein whose C-terminal coiled-coil domain may play a role in regulating DNA replication. We report here on the neuralizing function of geminin. The localization, effect of misexpression and activity of a dominant negative geminin suggest that the product of this gene has an essential early role in specifying neural cell fate in vertebrates. Maternal geminin mRNA is found throughout the animal hemisphere from oocyte through late blastula. At the early gastrula, however, expression is restricted to a dorsal ectodermal territory that prefigures the neural plate. Misexpression of geminin in gastrula ectoderm suppresses BMP4 expression and converts prospective epidermis into neural tissue. In ectodermal explants, geminin induces expression of the early proneural gene neurogenin-related 1 although not itself being induced by that gene. Later, embryos expressing geminin have an expanded dorsal neural territory and ventral ectoderm is converted to neurons. A putative dominant negative geminin lacking the neuralizing domain suppresses neural differentiation and, when misexpressed dorsally, produces islands of epidermal gene expression within the neurectodermal territory, effects that are rescued by coexpression of the full-length molecule. Taken together, these data indicate that geminin plays an early role in establishing a neural domain during gastrulation.

Amino Acid Sequence↗
PubMed1997

Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism.

Pin1 is an essential and conserved mitotic peptidyl-prolyl isomerase (PPIase) that is distinct from members of two other families of conventional PPIases, cyclophilins and FKBPs (FK-506 binding proteins). In response to their phosphorylation during mitosis, Pin1 binds and regulates members of a highly conserved set of proteins that overlaps with antigens recognized by the mitosis-specific monoclonal antibody MPM-2. Pin1 is here shown to be a phosphorylation-dependent PPIase that specifically recognizes the phosphoserine-proline or phosphothreonine-proline bonds present in mitotic phosphoproteins. Both Pin1 and MPM-2 selected similar phosphorylated serine-proline-containing peptides, providing the basis for the specific interaction between Pin1 and MPM-2 antigens. Pin1 preferentially isomerized proline residues preceded by phosphorylated serine or threonine with up to 1300-fold selectivity compared with unphosphorylated peptides. Pin1 may thus regulate mitotic progression by catalyzing sequence-specific and phosphorylation-dependent proline isomerization.

Amino Acid Isomerases↗
PubMed1997

Specific proteolysis of the kinase protein kinase C-related kinase 2 by caspase-3 during apoptosis. Identification by a novel, small pool expression cloning strategy.

The caspase family of proteases plays a critical role in the execution of apoptosis. However, efforts to decipher the molecular mechanisms by which caspases induce cell death have been greatly hindered by the lack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to rapidly isolate cDNAs encoding caspase substrates that are cleaved during apoptosis. Small cDNA pools (approximately 100 clones each) are transcribed/translated in vitro in the presence of [35S]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells. cDNA pools encoding proteins that are specifically cleaved by the apoptotic extract and whose cleavage is prevented by the caspase inhibitor acetyl-Tyr-Val-Ala-Asp chloromethylketone are subdivided and retested until a single cDNA is isolated. Using this approach, we isolated a partial cDNA encoding protein kinase C-related kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp117 and Asp700 in vitro. In addition, PRK2 is cleaved rapidly during Fas- and staurosporine-induced apoptosis in vivo by caspase-3 or a closely related caspase. Both of the major apoptotic cleavage sites of PRK2 in vivo lie within its regulatory domain, suggesting that its activity may be deregulated by proteolysis.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis.

The caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severed actin filaments in vitro in a Ca2+-independent manner. Expression of the gelsolin cleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis.

Actins↗
PubMed1997

Proteolysis and DNA replication: the CDC34 requirement in the Xenopus egg cell cycle.

The cell division cycle gene, CDC34, is required for ubiquitin-mediated degradation of G1 regulators and cell cycle progression through the transition from G1 to S phase in budding yeast. A CDC34 requirement for S phase onset in higher eukaryotes has not been established. Studies of the simple embryonic cell cycle of Xenopus laevis eggs demonstrated that Cdc34p in a large molecular size complex was required in the initiation of DNA replication. Cdc34p appears to regulate the initiation function of Cdk2-cyclin E, perhaps through the degradation of the Xenopus cdk inhibitor, Xic1.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1997

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

Activated 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] [2] [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.

Animals↗
PubMed1997

The role of cyclin-dependent kinase 5 and a novel regulatory subunit in regulating muscle differentiation and patterning.

Cyclin-dependent kinase 5, coupled with its activator p35, is required for normal neuronal differentiation and patterning. We have isolated a novel member of the p35 family, Xp35.1, from Xenopus embryos which can activate cdk5. Xp35.1 is expressed in both proliferating and differentiated neural and mesodermal cells and is particularly high in developing somites where cdk5 is also expressed. Using dominant-negative cdk5 (cdk5 DN), we show that cdk5 kinase activity is required for normal somitic muscle development; expression of cdk5 DN results in disruption of somitic muscle patterning, accompanied by stunting of the embryos. Using explants of animal pole tissue from blastula embryos, which will differentiate into mesoderm in response to activin, we show that blocking cdk5 kinase activity down-regulates the expression of the muscle marker muscle actin in response to activin, whereas the pan-mesodermal marker Xbra is unaffected. Expression of MyoD and MRF4 (master regulators of myogenesis) is suppressed in the presence of cdk5 DN, indicating that these myogenic genes may be a target for cdk5 regulation, whereas the related factor Myf5 is largely unaffected. In addition, overexpression of Xp35.1 disrupts muscle organization. Thus, we have demonstrated a novel role for cdk5 in regulating myogenesis in the early embryo.

Amino Acid Sequence↗
PubMed1997

Systematic identification of mitotic phosphoproteins.

BACKGROUND: Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified. RESULTS: Using a screen that scores for phosphorylation of proteins, which were translated from pools of cDNA plasmids in vitro, by either phosphoepitope antibody recognition or electrophoretic mobility shifts, we have identified 20 mitotically phosphorylated proteins from Xenopus embryos, 15 of which have sequence similarity to other proteins. Of these proteins, five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A), five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis. CONCLUSIONS: Examination of this broad set of mitotic phosphoproteins has allowed us to draw three conclusions about how the activation of CDKs regulates cell-cycle events. First, Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins. Second, during mitosis most of the substrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation. Third, the large fraction of mitotic phosphoproteins that are presumptive transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression.

Animals↗
PubMed1997

Sizzled: a secreted Xwnt8 antagonist expressed in the ventral marginal zone of Xenopus embryos.

An expression cloning screen was used to isolate a novel gene homologous to the extracellular cysteine-rich domain of frizzled receptors. The gene (which we called sizzled for secreted frizzled) was shown to encode a soluble secreted protein, containing a functional signal sequence but no transmembrane domains. Sizzled (szl) is capable of inhibiting Xwnt8 as assayed by (1) dose-dependent inhibition of siamois induction by Xwnt8 in animal caps, (2) rescue of embryos ventralized by Xwnt8 DNA and (3) inhibition of XmyoD expression in the marginal zone. Szl can dorsalize Xenopus embryos if expressed after the midblastula transition, strengthening the idea that zygotic expression of wnts and in particular of Xwnt8 plays a role in antagonizing dorsal signals. It also suggests that inhibiting ventralizing wnts parallels the opposition of BMPs by noggin and chordin. szl expression is restricted to a narrow domain in the ventral marginal zone of gastrulating embryos. szl thus encodes a secreted antagonist of wnt signaling likely involved in inhibiting Xwnt8 and XmyoD ventrally and whose restricted expression represents a new element in the molecular pattern of the ventral marginal zone.

Amino Acid Sequence↗
PubMed1996

Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p.

Anaphase initiation has been postulated to be controlled through the ubiquitin-dependent proteolysis of an unknown inhibitor. This process involves the anaphase promoting complex (APC), a specific ubiquitin ligase that has been shown to be involved in mitotic cyclin degradation. Previous studies demonstrated that in Saccharomyces cerevisiae, Pds1 protein is an anaphase inhibitor and suggested that it may be an APC target. Here we show that in yeast cells and in mitotic Xenopus extracts Pds1p is degraded in an APC-dependent manner. In addition, Pds1p is directly ubiquitinated by the Xenopus APC. In budding yeast Pds1p is degraded at the time of anaphase initiation and nondegradable derivatives of Pds1p inhibit the onset of anaphase. We conclude that Pds1p is an anaphase inhibitor whose APC-dependent degradation is required for the initiation of anaphase.

Anaphase↗
PubMed1996

How proteolysis drives the cell cycle.

Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity-by degrading CDK activators or inhibitors-and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

Anaphase↗
PubMed1996

Identification of BIME as a subunit of the anaphase-promoting complex.

The initiation of anaphase and exit from mitosis require the activation of a proteolytic system that ubiquitinates and degrades cyclin B. The regulated component of this system is a large ubiquitin ligase complex, termed the anaphase-promoting complex (APC) or cyclosome. Purified Xenopus laevis APC was found to be composed of eight major subunits, at least four of which became phosphorylated in mitosis. In addition to CDC27, CDC16, and CDC23, APC contained a homolog of Aspergillus nidulans BIME, a protein essential for anaphase. Because mutation of bimE can bypass the interphase arrest induced by either nimA mutation or unreplicated DNA, it appears that ubiquitination catalyzed by APC may also negatively regulate entry into mitosis.

Amino Acid Sequence↗
PubMed1996

Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation.

BACKGROUND: The destruction of cyclin B is required for exit from mitosis, and is mediated by the ubiquitin pathway. Recently, a 20S complex, termed the anaphase-promoting complex (APC) or the cyclosome, has been genetically and biochemically identified as the cyclin-specific ubiquitin ligase (E3). In addition, a ubiquitin-conjugating enzyme (E2), UBC4, was shown to be involved in cyclin ubiquitination in Xenopus egg extracts. Another E2 activity, designated UBCx, can independently support cyclin ubiquitination in Xenopus. A similar activity (E2-C) has also been observed in clams. However, the molecular identity of Xenopus UBCx or clam E2-C has not been established. RESULTS: We have purified and cloned Xenopus UBCx. Sequence comparisons with known E2s reveal that UBCx is a novel ubiquitin-conjugating enzyme. Purified recombinant UBCx is sufficient to complement purified APC and E1 in destruction box-dependent cyclin ubiquitination. UBCx and UBC4 are active in a similar concentration range and with similar kinetics. At saturating enzyme concentrations, UBCx converts twice as much substrate into ubiquitin conjugates, but generates conjugates of lower molecular mass than UBC4. CONCLUSIONS: UBCx is a novel ubiquitin-conjugating enzyme involved in cyclin ubiquitination in Xenopus. Like UBC4, ubiquitination catalyzed by UBCx is dependent on both the destruction box and the APC, suggesting that these E2s function through a similar mechanism. However, as the patterns of conjugates generated by these E2s are distinct, these enzymes may play different roles in promoting cyclin proteolysis in mitosis.

Amino Acid Sequence↗