Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1.

53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-ß interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-ß pathway and implicate this mechanism in the maintenance of genome integrity.

Systematic computational prediction of protein interaction networks.

Determining the network of physical protein associations is an important first step in developing mechanistic evidence for elucidating biological pathways. Despite rapid advances in the field of high throughput experiments to determine protein interactions, the majority of associations remain unknown. Here we describe computational methods for significantly expanding protein association networks. We describe methods for integrating multiple independent sources of evidence to obtain higher quality predictions and we compare the major publicly available resources available for experimentalists to use.

Protein kinase CK2alpha is a target for the Abl and Bcr-Abl tyrosine kinases.

Protein kinase CK2 is a ubiquitous serine-threonine kinase in which a catalytic alpha subunit often associates with a beta subunit. CK2alpha is required for cell survival in yeast and has been proposed to be involved in cell growth control; however, its regulation in cells remains unclear. Here we present evidence that CK2alpha may be an associated substrate for the normal and oncogenic forms of the Abl tyrosine kinase. By probing CK2alpha with anti-phosphotyrosine antibodies, we found that CK2alpha can be phosphorylated on tyrosine in quiescent cells. In vitro phosphorylation of CK2alpha-containing immunoprecipitates showed that CK2alpha is substrate of an associated tyrosine kinase activity. Immunoprecipitation experiments revealed that CK2alpha is associated with normal c-Abl in mouse NIH3T3 fibroblasts and with the Bcr-Abl fusion protein in K562 human myeloid leukemia cells. Coexpression of Bcr-Abl and CK2alpha in NIH3T3 cells also leads to the formation of a Bcr-Abl/CK2alpha complex and to the inhibition of CK2alpha activity. Bcr-Abl-induced inhibition of CK2alpha could be reverted by incubating CK2alpha with a tyrosine phosphatase. These observations clearly support the idea that a signal transduction pathway contributes to CK2 regulation and point to CK2alpha as a possible mediator of Bcr-Abl effects.

Binding of polyamines to an autonomous domain of the regulatory subunit of protein kinase CK2 induces a conformational change in the holoenzyme. A proposed role for the kinase stimulation.

The means by which the cell regulates protein kinase CK2 remain obscure. However, natural polyamines, cellular compounds required for cell proliferation, have been reported to strongly stimulate CK2-mediated phosphorylation of a number of substrates. Using spermine analogs, we have shown that polyamines directly interact with the CK2 beta subunit, and the chemical features of the highly acidic binding site (Asp51-Tyr80) have been determined. In the present study, we show that the isolated beta subunit region extending from residue Asp51 to Pro110 exhibits a specific and efficient polyamine binding activity similar to that of the entire beta subunit. Moreover, the replacement of Glu60, Glu61, and Glu63 of the beta subunit by 3 alanine residues leads to a loss of the spermine-induced stimulation of CK2 activity which correlates with a decrease in spermine binding affinity. Thermal stability studies indicate that the binding of spermine induces a 4 degrees C decrease of the Tm value for the holoenzyme. This was confirmed by circular dichroism analyses, which show that the 6 degrees C negative shift of the CK2 Tm value provoked by spermine binding, reflects a conformational change in the kinase. Together, these observations strongly suggest that this newly defined polyamine-binding domain is involved in the intrasteric regulation of CK2 activity.

Regulation of protein phosphatase 2A by direct interaction with casein kinase 2alpha.

Timely deactivation of kinase cascades is crucial to the normal control of cell signaling and is partly accomplished by protein phosphatase 2A (PP2A). The catalytic (alpha) subunit of the serine-threonine kinase casein kinase 2 (CK2) bound to PP2A in vitro and in mitogen-starved cells; binding required the integrity of a sequence motif common to CK2alpha and SV40 small t antigen. Overexpression of CK2alpha resulted in deactivation of mitogen-activated protein kinase kinase (MEK) and suppression of cell growth. Moreover, CK2alpha inhibited the transforming activity of oncogenic Ras, but not that of constitutively activated MEK. Thus, CK2alpha may regulate the deactivation of the mitogen-activated protein kinase pathway.

[Has protein kinase CK2 a role in the intracellular mitogenic signalling?]. / La protéine kinase CK2 a-t-elle une place dans la messagerie intracellulaire mitogène?

The protein kinase CK2 is an ubiquitous serine-threonine kinase found in all eukaryotic cells. Although well characterized on a biochemical ground, its role and regulation in the intact cell are not clearly understood. Its possible implication in the control of cell proliferation has been examined by several different approaches. (i) Immunocytochemical detection of CK2 revealed that whereas the signal was evenly distributed throughout cycle arrested cells in primary culture, it accumulates rapidly (30-90 min) in the nuclear compartment in cells stimulated to grow. (ii) CK2 biosynthesis is activated as an early response to growth factors in quiescent cells. The neo-synthesized kinase accumulates as the cells progress through the G1 phase. This growth factor-activated biosynthesis concerns in parallel the two kinase subunits. (iii) The kinase is activated in vitro by polyamines, which are increased in cells challenged by growth factors. Spermine binds to a specific domain of the beta subunit of CK2. (iv) In addition to phosphorylation CK2 forms a molecular complex with p53, a major negative regulator of the cell cycle. The complex was demonstrated in intact cells and reconstituted in vitro (Kd 70 nM) with purified components and shown to require the beta subunit and to result in the inhibition of p53 DNA-annealing activity. These observations suggest that CK2 and p53 may play a coordinated role in the cell response to mitogenic stimuli.

Modulation of the molecular organization and activity of casein kinase 2 by naturally occurring polyamines.

Polyamines have been reported to stimulate casein kinase 2 (CK2) in vitro. We have shown that the phosphorylation of different substrates is diversely stimulated by basic effectors and that the responsiveness of CK2 activity may be influenced by the overall conformation of the protein substrate but also by a specific interaction with the enzyme itself. Our data show that native hetero tetrameric CK2 is a spermine binding protein and a spermine binding site was identified in the N-terminal region of the beta subunit of CK2. We found that recombinant CK2 undergoes a progressive polymerization in low salt conditions, giving rise to three different polymer structures. A ring-like structure formed by the association of four protomers alpha 2 beta 2 was characterized by gel filtration, sucrose density gradient analysis and electron microscopy. Polyamines like spermine, when added under conditions where the enzyme preparation contains a mixture of various oligomers, could trigger their dissociation and their interconversion in the fully active ring structure. These results suggest that in the presence of positive effectors like polyamines, CK2 adopts a ring-like structural organization which may represent the active state of this kinase.