PubMed Health⌕ Search

Biomedical subjects

James E Ferrell

Publications and source records attributed to James E Ferrell.

At least 19 recordsLinked to original sources
PubMed2007

Cyclin A2 regulates nuclear-envelope breakdown and the nuclear accumulation of cyclin B1.

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.

Cell Nucleus↗
PubMed2006

Mechanistic studies of the mitotic activation of Mos.

The protein kinase Mos is responsible for the activation of MEK1 and p42 mitogen-activated protein kinase during Xenopus oocyte maturation and during mitosis in Xenopus egg extracts. Here we show that the activation of Mos depends upon the phosphorylation of Ser 3, a residue previously implicated in the regulation of Mos stability; the dephosphorylation of Ser 105, a previously unidentified phosphorylation site conserved in Mos proteins; and the regulated dissociation of Mos from CK2beta. Mutation of Ser 3 to alanine and/or mutation of Ser 105 to glutamate produces a Mos protein that is defective for M-phase activation, as assessed by in vitro kinase assays, and defective for induction of oocyte maturation and maintenance of the spindle assembly checkpoint in extracts. Interestingly, Ser 105 is situated at the beginning of helix alphaC in the N-terminal lobe of the Mos kinase domain. Changes in the orientation of this helix have been previously implicated in the activation of Cdk2 and Src family tyrosine kinases. Our work suggests that Ser 105 dephosphorylation represents a novel mechanism for reorienting helix alphaC.

Animals↗
PubMed2005

Interlinked fast and slow positive feedback loops drive reliable cell decisions.

Positive feedback is a ubiquitous signal transduction motif that allows systems to convert graded inputs into decisive, all-or-none outputs. Here we investigate why the positive feedback switches that regulate polarization of budding yeast, calcium signaling, Xenopus oocyte maturation, and various other processes use multiple interlinked loops rather than single positive feedback loops. Mathematical simulations revealed that linking fast and slow positive feedback loops creates a "dual-time" switch that is both rapidly inducible and resistant to noise in the upstream signaling system.

Animals↗
PubMed2005

Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations.

The cell-cycle oscillator includes an essential negative-feedback loop: Cdc2 activates the anaphase-promoting complex (APC), which leads to cyclin destruction and Cdc2 inactivation. Under some circumstances, a negative-feedback loop is sufficient to generate sustained oscillations. However, the Cdc2/APC system also includes positive-feedback loops, whose functional importance we now assess. We show that short-circuiting positive feedback makes the oscillations in Cdc2 activity faster, less temporally abrupt, and damped. This compromises the activation of cyclin destruction and interferes with mitotic exit and DNA replication. This work demonstrates a systems-level role for positive-feedback loops in the embryonic cell cycle and provides an example of how oscillations can emerge out of combinations of subcircuits whose individual behaviors are not oscillatory. This work also underscores the fundamental similarity of cell-cycle oscillations in embryos to repetitive action potentials in pacemaker neurons, with both systems relying on a combination of negative and positive-feedback loops.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx.

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.

Calcium↗
PubMed2005

Identification and comparative analysis of multiple mammalian Speedy/Ringo proteins.

In addition to their activation via binding to cyclins, cyclin-dependent kinases (CDKs) can be activated via binding to a novel cell cycle regulator termed Speedy/Ringo, which shows no apparent similarity to cyclins. The first Speedy/Ringo protein was found to be essential for Xenopus oocyte maturation and a human homolog (Spy1, herein called Speedy/ Ringo A1) regulates S-phase entry and cell survival after DNA damage in cultured somatic cells. We have identified a Speedy/Ringo-like gene in the most primitive branching clade of chordates (Ciona intestinalis), as well as four mammalian homologs. Of the mammalian proteins, two, Speedy/Ringo A and C, bind to Cdc2 and Cdk2, whereas Speedy/Ringo B binds preferentially to Cdc2. Despite their distinct CDK-binding preferences, both Speedy/Ringo A and B can promote the maturation of Xenopus oocytes and all three Speedy/Ringo proteins can bind to and activate CDKs in vivo. These mammalian Speedy/Ringo proteins exhibit distinct tissue expression patterns, though all three are enriched in testis, consistent with the initial observation that Xenopus Speedy/Ringo functions during meiosis. Speedy/Ringo A is widely expressed in tissues and cell lines. Speedy/Ringo B expression appears to be testis-specific. Speedy/Ringo C is expressed in diverse tissues, particularly those that undergo polyploidization. All Speedy/Ringo proteins share a highly conserved approximately 140-aa domain we term the Speedy/Ringo box that is essential for CDK binding. Point mutations in this domain abolish CDK binding. Besides the central Speedy/Ringo box, Speedy/Ringo A contains a C-terminal portion, which promotes CDK activation, and an N-terminal portion, which is dispersible for both CDK binding and activation but that influences protein expression. The existence of this growing family of CDK activators suggests that Speedy/Ringo proteins may play as complex a role in cell cycle control as the diverse family of cyclins.

Amino Acid Sequence↗
PubMed2005

Multisite M-phase phosphorylation of Xenopus Wee1A.

The Cdk1 inhibitor Wee1 is inactivated during mitotic entry by proteolysis, translational regulation, and transcriptional regulation. Wee1 is also regulated by posttranslational modifications, and here we have identified five phosphorylation sites in the N-terminal domain of embryonic Xenopus Wee1A through a combination of mutagenesis studies and matrix-assisted laser desorption ionization-time of flight mass spectrometry. All five sites conform to the Ser-Pro/Thr-Pro consensus for proline-directed kinases like Cdks. Three of the sites (Ser 38, Thr 53, and Ser 62) are required for the mitotic gel shift, and at least two of these sites (Ser 38 and Thr 53) regulate the proteolysis of Wee1A during interphase. The other two sites (Thr 104 and Thr 150) are primarily responsible for the mitotic inactivation of Wee1A. Alanine mutants of Thr 150 or Thr 104 had an increased capacity to inhibit mitotic entry in cyclin B-treated interphase extracts, and Thr 150 was found to be transiently phosphorylated just prior to nuclear envelope breakdown in cycling egg extracts. These findings establish the phosphorylation-dependent direct inactivation of Wee1A as a critical mechanism for the promotion of M-phase entry. These results also show that multisite phosphorylation cooperatively inactivates Wee1A and cooperatively promotes Wee1A proteolysis.

Amino Acid Sequence↗
PubMed2004

Mos mediates the mitotic activation of p42 MAPK in Xenopus egg extracts.

The ERK1/ERK2 MAP kinases (MAPKs) are transiently activated during mitosis, and MAPK activation has been implicated in the spindle assembly checkpoint and in establishing the timing of an unperturbed mitosis. The MAPK activator MEK1 is required for mitotic activation of p42 MAPK in Xenopus egg extracts; however, the identity of the kinase that activates MEK1 is unknown. Here we have partially purified a Cdc2-cyclin B-induced MEK-activating protein kinase from mitotic Xenopus egg extracts and identified it as the Mos protooncoprotein, a MAP kinase kinase kinase present at low levels in mitotic egg extracts, early embryos, and somatic cells. Immunodepletion of Mos from interphase egg extracts was found to abolish Delta90 cyclin B-Cdc2-stimulated p42 MAPK activation. In contrast, immunodepletion of Raf-1 and B-Raf, two other MEK-activating kinases present in Xenopus egg extracts, had little effect on cyclin-stimulated p42 MAPK activation. Immunodepletion of Mos also abolished the transient activation of p42 MAPK in cycling egg extracts. Taken together, these data demonstrate that Mos is responsible for the mitotic activation of the p42 MAPK pathway in Xenopus egg extracts.

Animals↗
PubMed2004

Probing the precision of the mitotic clock with a live-cell fluorescent biosensor.

Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements of the mitotic clock have been challenging. Here we present a fluorescent mitosis biosensor that monitors the time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision with 80% of cells completing mitosis in 32 +/- 6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.

Animals↗
PubMed2004

Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems.

It is becoming increasingly clear that bistability (or, more generally, multistability) is an important recurring theme in cell signaling. Bistability may be of particular relevance to biological systems that switch between discrete states, generate oscillatory responses, or "remember" transitory stimuli. Standard mathematical methods allow the detection of bistability in some very simple feedback systems (systems with one or two proteins or genes that either activate each other or inhibit each other), but realistic depictions of signal transduction networks are invariably much more complex. Here, we show that for a class of feedback systems of arbitrary order the stability properties of the system can be deduced mathematically from how the system behaves when feedback is blocked. Provided that this open-loop, feedback-blocked system is monotone and possesses a sigmoidal characteristic, the system is guaranteed to be bistable for some range of feedback strengths. We present a simple graphical method for deducing the stability behavior and bifurcation diagrams for such systems and illustrate the method with two examples taken from recent experimental studies of bistable systems: a two-variable Cdc2/Wee1 system and a more complicated five-variable mitogen-activated protein kinase cascade.

CDC2 Protein Kinase↗
PubMed2004

Picking a winner: new mechanistic insights into the design of effective siRNAs.

Recent work has shown that the efficacy of a small interfering RNA (siRNA) for silencing gene expression is a function of how easy it is to unwind the siRNA from the 5'-antisense end. Based on these insights, one group has designed an algorithm that substantially improves the odds of picking an effective siRNA, and two groups have shown that 'forked' or 'frayed' siRNAs, which should be easier to unwind from the 5'-antisense end, are more effective than conventional siRNAs. These strategies represent important steps towards the rational design of effective siRNAs.

Algorithms↗
PubMed2003

A positive-feedback-based bistable 'memory module' that governs a cell fate decision.

The maturation of Xenopus oocytes can be thought of as a process of cell fate induction, with the immature oocyte representing the default fate and the mature oocyte representing the induced fate. Crucial mediators of Xenopus oocyte maturation, including the p42 mitogen-activated protein kinase (MAPK) and the cell-division cycle protein kinase Cdc2, are known to be organized into positive feedback loops. In principle, such positive feedback loops could produce an actively maintained 'memory' of a transient inductive stimulus and could explain the irreversibility of maturation. Here we show that the p42 MAPK and Cdc2 system normally generates an irreversible biochemical response from a transient stimulus, but the response becomes transient when positive feedback is blocked. Our results explain how a group of intrinsically reversible signal transducers can generate an irreversible response at a systems level, and show how a cell fate can be maintained by a self-sustaining pattern of protein kinase activation.

Animals↗
PubMed2003

Selective regulation of neurite extension and synapse formation by the beta but not the alpha isoform of CaMKII.

Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.

Actins↗
PubMed2003

The JNK cascade as a biochemical switch in mammalian cells: ultrasensitive and all-or-none responses.

JNK proteins are ubiquitously expressed, evolutionarily conserved MAP kinases that are involved in stress responses. Recently, it was shown that the JNK cascade in Xenopus oocytes exhibits sustained, all-or-none responses to graded, transient stimuli. Here, we have examined the character of the JNK cascade's response in mammalian cells. The steady-state responses of JNK to sorbitol and anisomycin were found to be highly ultrasensitive in HeLa cells, HEK 293 cells, and Jurkat T cells. The JNK responses were also reversible, not sustained, as was the case in oocytes. Jurkat cells activated their JNK in response to phorbol myristate acetate (PMA), and the response of the entire population of Jurkat cells was graded. However, analysis of subpopulations of the PMA-treated Jurkat cells revealed that the steady-state responses of both JNK and CD69, a T cell surface activation marker, were essentially all-or-none in character. These studies show that the JNK cascade commonly exhibits switch-like responses to a variety of stimuli.

Animals↗
PubMed2003

Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing.

RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.

Cell Line↗
PubMed2003

Enforced proximity in the function of a famous scaffold.

Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore the fundamental importance of proximity in enzyme regulation and of keeping a proper distance for maintaining signaling specificity.

Adaptor Proteins, Signal Transducing↗