Proteomic data on the nuclear interactome of human MCM9

AbstractWe present data relating to the interactome of MCM9 from the nuclei of human cells. MCM9 belongs to the AAA+ superfamily, and contains an MCM domain and motifs that may confer DNA helicase activity. MCM9 has been shown to bind MCM8, and has been implicated in DNA replication and homologous recombination. However, the mechanistic basis of MCM9’s role in DNA repair is poorly understood, and proteins with which it interacts were hitherto unknown. We performed tandem affinity purification of MCM9 and its interacting proteins from nuclear extracts of human cells, followed by proteomic analysis, thereby generating a set of mass spectrometry data corresponding to the MCM9 interactome [1]. The proteomic data set comprises 29 mass spectrometry RAW files, deposited to the ProteomeXchange Consortium, and freely available from the PRIDE partner repository with the data set identifier http://www.ebi.ac.uk/pride/archive/projects/PXD000212. A set of 22 interacting proteins identified from the proteomic data was used to create an MCM9-centered interactive network diagram, using the Cytoscape program. These data allow the scientific community to access, mine and explore the human nuclear MCM9 interactome

Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis

AbstractThe small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation [1, 2]. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells [3]. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin α/β and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis

Substrate specificity determinants of the checkpoint protein kinase Chk1

AbstractThe Chk1 protein kinase plays a critical role in a DNA damage checkpoint pathway conserved between fission yeast and animals. We have developed a quantitative assay for Chk1 activity, using a peptide derived from a region of Xenopus Cdc25C containing Ser-287, a known target of Chk1. Variants of this peptide were used to determine the residues involved in substrate recognition by Chk1, revealing the phosphorylation motif Φ-X-β-X-X-(S/T)*, where * indicates the phosphorylated residue, Φ is a hydrophobic residue (M>I>L>V), β is a basic residue (R>K) and X is any amino acid. This motif suggests that Chk1 is a member of a group of stress-response protein kinases which phosphorylate target proteins with related specificities

Dephosphorylation of the inhibitory phosphorylation site S287 in Xenopus Cdc25C by protein phosphatase-2A is inhibited by 14-3-3 binding

AbstractCdc25C phosphatase induces mitosis by dephosphorylating and activating Cdc2/cyclin B protein kinase. Phosphorylation of Xenopus Cdc25C at serine 287 creates a binding site for a 14-3-3 protein and restrains activation during interphase. Here, we show that dephosphorylation of S287 is catalysed by protein phosphatase-2A in Xenopus egg extracts. 14-3-3 protein binding to Cdc25C inhibits dephosphorylation of S287, providing a mechanism to maintain phosphorylation of that site during interphase. The rate of dephosphorylation of S287 is not increased in mitotic extracts, indicating that the phosphorylation status of the site is likely to be controlled through modulation of kinases or 14-3-3 binding activity

Role of importin-β in the control of nuclear envelope assembly by Ran

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran [1, 2]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [2]. Here, we show that importin-β, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-β on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-β in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-α is functional. Thus, importin-β functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-α with karyophilic proteins carrying classical nuclear localization signals. Importin-β links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division

Democracy and education: about the future of a problem

In 20th century's European theory of education there was little interest in philosophy ofdemocracy. John Dewey'sDemocracy and Education was translated in nearly every European language but did not become the center of discussion. Even "radical education” was much more childcentered than open to radical questions of political democracy. This article discusses the problem in two respects, first the tension between neo-liberalism's concept of individuality and public education, and second the future problems of a theory of "democratic education”after Dewey. The aim is to overcome traditional European dualisms like that of "citizen”or "man” i.e. to pave the way for a post-Rousseauian theory of educatio